Endothelial delivery of antioxidant enzymes loaded into non-polymeric magnetic nanoparticles.

PubMed

Chorny, Michael; Hood, Elizabeth; Levy, Robert J; Muzykantov, Vladimir R

2010-08-17

Antioxidant enzymes have shown promise as a therapy for pathological conditions involving increased production of reactive oxygen species (ROS). However the efficiency of their use for combating oxidative stress is dependent on the ability to achieve therapeutically adequate levels of active enzymes at the site of ROS-mediated injury. Thus, the implementation of antioxidant enzyme therapy requires a strategy enabling both guided delivery to the target site and effective protection of the protein in its active form. To address these requirements we developed magnetically responsive nanoparticles (MNP) formed by precipitation of calcium oleate in the presence of magnetite-based ferrofluid (controlled aggregation/precipitation) as a carrier for magnetically guided delivery of therapeutic proteins. We hypothesized that antioxidant enzymes, catalase and superoxide dismutase (SOD), can be protected from proteolytic inactivation by encapsulation in MNP. We also hypothesized that catalase-loaded MNP applied with a high-gradient magnetic field can rescue endothelial cells from hydrogen peroxide toxicity in culture. To test these hypotheses, a family of enzyme-loaded MNP formulations were prepared and characterized with respect to their magnetic properties, enzyme entrapment yields and protection capacity. SOD- and catalase-loaded MNP were formed with average sizes ranging from 300 to 400 nm, and a protein loading efficiency of 20-33%. MNP were strongly magnetically responsive (magnetic moment at saturation of 14.3 emu/g) in the absence of magnetic remanence, and exhibited a protracted release of their cargo protein in plasma. Catalase stably associated with MNP was protected from proteolysis and retained 20% of its initial enzymatic activity after 24h of exposure to pronase. Under magnetic guidance catalase-loaded MNP were rapidly taken up by cultured endothelial cells providing increased resistance to oxidative stress (62+/-12% cells rescued from hydrogen peroxide induced cell death vs. 10+/-4% under non-magnetic conditions). We conclude that non-polymeric MNP formed using the controlled aggregation/precipitation strategy are a promising carrier for targeted antioxidant enzyme therapy, and in combination with magnetic guidance can be applied to protect endothelial cells from oxidative stress mediated damage. This protective effect of magnetically targeted MNP impregnated with antioxidant enzymes can be highly relevant for the treatment of cardiovascular disease and should be further investigated in animal models. Copyright 2010 Elsevier B.V. All rights reserved.

Benefits from Tween during enzymic hydrolysis of corn stover

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kaar, W.E.; Holtzapple, M.T.

1998-08-20

Corn stover is a potential substrate for fermentation processes. Previous work with corn stover demonstrated that lime pretreatment rendered it digestible by cellulase; however, high sugar yields required very high enzyme loadings. Because cellulase is a significant cost in biomass conversion processes, the present study focused on improving the enzyme efficiency using Tween 20 and Tween 80; Tween 20 is slightly more effective than Tween 80. The recommended pretreatment conditions for the biomass remained unchanged regardless of whether Tween was added during the hydrolysis. The recommended Tween loading was 0.15 g Tween/g dry biomass. The critical relationship was the Tweenmore » loading on the biomass, not the Tween concentration in solution. The 72-h enzymic conversion of pretreated corn stover using 5 FPU cellulase/g dry biomass at 50 C with Tween 20 as part of the medium was 0.85 g/g for cellulose, 0.66 g/g for xylan, and 0.75 for total polysaccharide; addition of Tween improved the cellulose, xylan, and total polysaccharide conversions by 42, 40, and 42%, respectively. Kinetic analyses showed that Tween improved the enzymic absorption constants, which increased the effective hydrolysis rate compared to hydrolysis without Tween. Furthermore, Tween prevented thermal deactivation of the enzymes, which allows for the kinetic advantage of higher temperature hydrolysis. Ultimate digestion studies showed higher conversions for samples containing Tween, indicating a substrate effect. It appears that Tween improves corn stover hydrolysis through three effects: enzyme stabilizer, lignocellulose disrupter, and enzyme effector.« less

Immobilization and Stabilization of Acylase on Carboxylated Polyaniline Nanofibers for Highly Effective Antifouling Application via Quorum Quenching.

PubMed

Lee, Jeongjoon; Lee, Inseon; Nam, Jahyun; Hwang, Dong Soo; Yeon, Kyung-Min; Kim, Jungbae

2017-05-10

Acylase (AC) was immobilized and stabilized on carboxylated polyaniline nanofibers (cPANFs) for the development of antifouling nanobiocatalysts with high enzyme loading and stability. AC was immobilized via three different approaches: covalent attachment (CA), enzyme coating (EC), and magnetically separable enzyme precipitate coating (Mag-EPC). The enzyme activity per unit weight of cPANFs with Mag-EPC was 75 and 300 times higher than that of those with CA and EC, respectively, representing improved enzyme loading in the form of Mag-EPC. After incubation under shaking at 200 rpm for 20 days, Mag-EPC maintained 55% of its initial activity, whereas CA and EC showed 3 and 16% of their initial activities, respectively. The antifouling of highly loaded and stable Mag-EPC against the biofouling/biofilm formation of Pseudomonas aeruginosa was tested under static- and continuous-flow conditions. Biofilm formation in the presence of 40 μg/mL Mag-EPC under static condition was 5 times lower than that under control condition with no addition of Mag-EPC. Under continuous membrane filtration, Mag-EPC delayed the increase of transmembrane pressure (TMP) more effectively as the concentration of added Mag-EPC increased. When separating Mag-EPC and membranes in two different vessels under internal circulation of the culture solution, Mag-EPC maintained a higher permeability than the control with no Mag-EPC addition. It was also confirmed that the addition of Mag-EPC reduced the generation of N-acyl homoserine lactone (AHL) autoinducers. This result reveals that the inhibition of biofilm formation and biofouling in the presence of Mag-EPC is due to the hydrolysis of AHL autoinducers, catalyzed by the immobilized and stabilized AC in the form of Mag-EPC. Mag-EPC of AC with high enzyme loadings and improved stability has demonstrated its great potential as an antifouling agent by reducing biofilm formation and membrane biofouling based on "enzymatic quorum quenching" of autoinducers.

Differential reinforcement of enzymatic hydrolysis by adding chemicals and accessory proteins to high solid loading substrates with different pretreatments.

PubMed

Du, Jian; Song, Wenxia; Zhang, Xiu; Zhao, Jian; Liu, Guodong; Qu, Yinbo

2018-04-23

High dosage of enzyme is required to achieve effective lignocellulose hydrolysis, especially at high-solid loadings, which is a significant barrier to large-scale bioconversion of lignocellulose. Here, we screened four chemical additives and three accessory proteins for their effects on the enzymatic hydrolysis of various lignocellulosic materials. The effects were found to be highly dependent on the composition and solid loadings of substrates. For xylan-extracted lignin-rich corncob residue, the enhancing effect of PEG 6000 was most pronounced and negligibly affected by solid content, which reduced more than half of enzyme demand at 20% dry matter (DM). Lytic polysaccharide monooxygenase enhanced the hydrolysis of ammonium sulfite wheat straw pulp, and its addition reduced about half of protein demand at the solid loading of 20% DM. Supplementation of the additives in the hydrolysis of pure cellulose and complex lignocellulosic materials revealed that their effects are tightly linked to pretreatment strategies.

Impact of enzyme loading on the efficacy and recovery of cellulolytic enzymes immobilized on enzymogel nanoparticles.

PubMed

Samaratunga, Ashani; Kudina, Olena; Nahar, Nurun; Zakharchenko, Andrey; Minko, Sergiy; Voronov, Andriy; Pryor, Scott W

2015-03-01

Cellulase and β-glucosidase were adsorbed on a polyacrylic acid polymer brush grafted on silica nanoparticles to produce enzymogels as a form of enzyme immobilization. Enzyme loading on the enzymogels was increased to a saturation level of approximately 110 μg (protein) mg(-1) (particle) for each enzyme. Enzymogels with varied enzyme loadings were then used to determine the impact on hydrolysis rate and enzyme recovery. Soluble sugar concentrations during the hydrolysis of filter paper and Solka-Floc with the enzymogels were 45 and 53%, respectively, of concentrations when using free cellulase. β-Glucosidase enzymogels showed lower performance; hydrolyzate glucose concentrations were just 38% of those using free enzymes. Increasing enzyme loading on the enzymogels did not reduce net efficacy for cellulase and improved efficacy for β-glucosidase. The use of free cellulases and cellulase enzymogels resulted in hydrolyzates with different proportions of cellobiose and glucose, suggesting differential attachment or efficacy of endoglucanases, exoglucanases, and β-glucosidases present in cellulase mixtures. When loading β-glucosidase individually, higher enzyme loadings on the enzymogels produced higher hydrolyzate glucose concentrations. Approximately 96% of cellulase and 66 % of β-glucosidase were recovered on the enzymogels, while enzyme loading level did not impact recovery for either enzyme.

Co-immobilization of multiple enzymes by metal coordinated nucleotide hydrogel nanofibers: improved stability and an enzyme cascade for glucose detection

NASA Astrophysics Data System (ADS)

Liang, Hao; Jiang, Shuhui; Yuan, Qipeng; Li, Guofeng; Wang, Feng; Zhang, Zijie; Liu, Juewen

2016-03-01

Preserving enzyme activity and promoting synergistic activity via co-localization of multiple enzymes are key topics in bionanotechnology, materials science, and analytical chemistry. This study reports a facile method for co-immobilizing multiple enzymes in metal coordinated hydrogel nanofibers. Specifically, four types of protein enzymes, including glucose oxidase, Candida rugosa lipase, α-amylase, and horseradish peroxidase, were respectively encapsulated in a gel nanofiber made of Zn2+ and adenosine monophosphate (AMP) with a simple mixing step. Most enzymes achieved quantitative loading and retained full activity. At the same time, the entrapped enzymes were more stable against temperature variation (by 7.5 °C), protease attack, extreme pH (by 2-fold), and organic solvents. After storing for 15 days, the entrapped enzyme still retained 70% activity while the free enzyme nearly completely lost its activity. Compared to nanoparticles formed with AMP and lanthanide ions, the nanofiber gels allowed much higher enzyme activity. Finally, a highly sensitive and selective biosensor for glucose was prepared using the gel nanofiber to co-immobilize glucose oxidase and horseradish peroxidase for an enzyme cascade system. A detection limit of 0.3 μM glucose with excellent selectivity was achieved. This work indicates that metal coordinated materials using nucleotides are highly useful for interfacing with biomolecules.Preserving enzyme activity and promoting synergistic activity via co-localization of multiple enzymes are key topics in bionanotechnology, materials science, and analytical chemistry. This study reports a facile method for co-immobilizing multiple enzymes in metal coordinated hydrogel nanofibers. Specifically, four types of protein enzymes, including glucose oxidase, Candida rugosa lipase, α-amylase, and horseradish peroxidase, were respectively encapsulated in a gel nanofiber made of Zn2+ and adenosine monophosphate (AMP) with a simple mixing step. Most enzymes achieved quantitative loading and retained full activity. At the same time, the entrapped enzymes were more stable against temperature variation (by 7.5 °C), protease attack, extreme pH (by 2-fold), and organic solvents. After storing for 15 days, the entrapped enzyme still retained 70% activity while the free enzyme nearly completely lost its activity. Compared to nanoparticles formed with AMP and lanthanide ions, the nanofiber gels allowed much higher enzyme activity. Finally, a highly sensitive and selective biosensor for glucose was prepared using the gel nanofiber to co-immobilize glucose oxidase and horseradish peroxidase for an enzyme cascade system. A detection limit of 0.3 μM glucose with excellent selectivity was achieved. This work indicates that metal coordinated materials using nucleotides are highly useful for interfacing with biomolecules. Electronic supplementary information (ESI) available: Additional methods, IR and XRD spectroscopy, enzyme loading capacity, enzyme kinetic parameters, and enzyme stability data. See DOI: 10.1039/c5nr08734a

Functional design of electrolytic biosensor

NASA Astrophysics Data System (ADS)

Gamage Preethichandra, D. M.; Mala Ekanayake, E. M. I.; Onoda, M.

2017-11-01

A novel amperometric biosensbased on conjugated polypyrrole (PPy) deposited on a Pt modified ITO (indium tin oxide) conductive glass substrate and their performances are described. We have presented a method of developing a highly sensitive and low-cost nano-biosensor for blood glucose measurements. The fabrication method proposed decreases the cost of production significantly as the amount of noble metals used is minimized. A nano-corrugated PPy substrate was developed through pulsed electrochemical deposition. The sensitivity achieved was 325 mA/(Mcm2) and the linear range of the developed sensor was 50-60 mmol/l. Then the application of the electrophoresis helps the glucose oxidase (GOx) on the PPy substrate. The main reason behind this high enzyme loading is the high electric field applied across the sensor surface (working electrode) and the counter electrode where that pushes the nano-scale enzyme particles floating in the phosphate buffer solution towards the substrate. The novel technique used has provided an extremely high sensitivities and very high linear ranges for enzyme (GOx) and therefore can be concluded that this is a very good technique to load enzyme onto the conducting polymer substrates.

Maximizing the efficiency of multienzyme process by stoichiometry optimization.

PubMed

Dvorak, Pavel; Kurumbang, Nagendra P; Bendl, Jaroslav; Brezovsky, Jan; Prokop, Zbynek; Damborsky, Jiri

2014-09-05

Multienzyme processes represent an important area of biocatalysis. Their efficiency can be enhanced by optimization of the stoichiometry of the biocatalysts. Here we present a workflow for maximizing the efficiency of a three-enzyme system catalyzing a five-step chemical conversion. Kinetic models of pathways with wild-type or engineered enzymes were built, and the enzyme stoichiometry of each pathway was optimized. Mathematical modeling and one-pot multienzyme experiments provided detailed insights into pathway dynamics, enabled the selection of a suitable engineered enzyme, and afforded high efficiency while minimizing biocatalyst loadings. Optimizing the stoichiometry in a pathway with an engineered enzyme reduced the total biocatalyst load by an impressive 56 %. Our new workflow represents a broadly applicable strategy for optimizing multienzyme processes. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Sugar loss and enzyme inhibition due to oligosaccharides accumulation during high solids-loading enzymatic hydrolysis

USDA-ARS?s Scientific Manuscript database

Oligosaccharide accumulation occurs during high solid loading enzymatic hydrolysis of corn stover (CS) irrespective of using different pretreated corn stover (dilute acid: DA, ionic liquids: IL, ammonia fiber expansion: AFEX and extractive ammonia: EA). The methodology for large-scale separation of ...

Pretreating wheat straw by phosphoric acid plus hydrogen peroxide for enzymatic saccharification and ethanol production at high solid loading.

PubMed

Qiu, Jingwen; Ma, Lunjie; Shen, Fei; Yang, Gang; Zhang, Yanzong; Deng, Shihuai; Zhang, Jing; Zeng, Yongmei; Hu, Yaodong

2017-08-01

Wheat straw was pretreated by phosphoric acid plus hydrogen peroxide (PHP) for enzymatic hydrolysis and ethanol fermentation at high solid loadings. Results indicated solid loading could reach 20% with 77.4% cellulose-glucose conversion and glucose concentration of 164.9g/L in hydrolysate, it even was promoted to 25% with only 3.4% decrease on cellulose-glucose conversion as the pretreated-wheat straw was dewatered by air-drying. 72.9% cellulose-glucose conversion still was achieved as the minimized enzyme input of 20mg protein/g cellulose was employed for hydrolysis at 20% solid loading. In the corresponding conditions, 100g wheat straw can yield 11.2g ethanol with concentration of 71.2g/L by simultaneous saccharification and fermentation. Thus, PHP-pretreatment benefitted the glucose or ethanol yield at high solid loadings with lower enzyme input. Additionally, decreases on the maximal cellulase adsorption and the direct-orange/direct-blue indicated drying the PHP-pretreated substrates negatively affected the hydrolysis due to the shrinkage of cellulase-size-accommodable pores. Copyright © 2017 Elsevier Ltd. All rights reserved.

Co-solvent pretreatment reduces costly enzyme requirements for high sugar and ethanol yields from lignocellulosic biomass.

PubMed

Nguyen, Thanh Yen; Cai, Charles M; Kumar, Rajeev; Wyman, Charles E

2015-05-22

We introduce a new pretreatment called co-solvent-enhanced lignocellulosic fractionation (CELF) to reduce enzyme costs dramatically for high sugar yields from hemicellulose and cellulose, which is essential for the low-cost conversion of biomass to fuels. CELF employs THF miscible with aqueous dilute acid to obtain up to 95 % theoretical yield of glucose, xylose, and arabinose from corn stover even if coupled with enzymatic hydrolysis at only 2 mgenzyme  gglucan (-1) . The unusually high saccharification with such low enzyme loadings can be attributed to a very high lignin removal, which is supported by compositional analysis, fractal kinetic modeling, and SEM imaging. Subsequently, nearly pure lignin product can be precipitated by the evaporation of volatile THF for recovery and recycling. Simultaneous saccharification and fermentation of CELF-pretreated solids with low enzyme loadings and Saccharomyces cerevisiae produced twice as much ethanol as that from dilute-acid-pretreated solids if both were optimized for corn stover. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Combined substrate, enzyme and yeast feed in simultaneous saccharification and fermentation allow bioethanol production from pretreated spruce biomass at high solids loadings

PubMed Central

2014-01-01

Background Economically feasible cellulosic ethanol production requires that the process can be operated at high solid loadings, which currently imparts technical challenges including inefficient mixing leading to heat and mass transfer limitations and high concentrations of inhibitory compounds hindering microbial activity during simultaneous saccharification and fermentation (SSF) process. Consequently, there is a need to develop cost effective processes overcoming the challenges when working at high solid loadings. Results In this study we have modified the yeast cultivation procedure and designed a SSF process to address some of the challenges at high water insoluble solids (WIS) content. The slurry of non-detoxified pretreated spruce when used in a batch SSF at 19% (w/w) WIS was found to be inhibitory to Saccharomyces cerevisiae Thermosacc that produced 2 g l-1 of ethanol. In order to reduce the inhibitory effect, the non-washed solid fraction containing reduced amount of inhibitors compared to the slurry was used in the SSF. Further, the cells were cultivated in the liquid fraction of pretreated spruce in a continuous culture wherein the outflow of cell suspension was used as cell feed to the SSF reactor in order to maintain the metabolic state of the cell. Enhanced cell viability was observed with cell, enzyme and substrate feed in a SSF producing 40 g l-1 ethanol after 96 h corresponding to 53% of theoretical yield based on available hexose sugars compared to 28 g l-1 ethanol in SSF with enzyme and substrate feed but no cell feed resulting in 37% of theoretical yield at a high solids loading of 20% (w/w) WIS content. The fed-batch SSF also significantly eased the mixing, which is usually challenging in batch SSF at high solids loading. Conclusions A simple modification of the cell cultivation procedure together with a combination of yeast, enzyme and substrate feed in a fed-batch SSF process, made it possible to operate at high solids loadings in a conventional bioreactor. The proposed process strategy significantly increased the yeast cell viability and overall ethanol yield. It was also possible to obtain 4% (w/v) ethanol concentration, which is a minimum requirement for an economical distillation process. PMID:24713027

Combined substrate, enzyme and yeast feed in simultaneous saccharification and fermentation allow bioethanol production from pretreated spruce biomass at high solids loadings.

PubMed

Koppram, Rakesh; Olsson, Lisbeth

2014-04-08

Economically feasible cellulosic ethanol production requires that the process can be operated at high solid loadings, which currently imparts technical challenges including inefficient mixing leading to heat and mass transfer limitations and high concentrations of inhibitory compounds hindering microbial activity during simultaneous saccharification and fermentation (SSF) process. Consequently, there is a need to develop cost effective processes overcoming the challenges when working at high solid loadings. In this study we have modified the yeast cultivation procedure and designed a SSF process to address some of the challenges at high water insoluble solids (WIS) content. The slurry of non-detoxified pretreated spruce when used in a batch SSF at 19% (w/w) WIS was found to be inhibitory to Saccharomyces cerevisiae Thermosacc that produced 2 g l-1 of ethanol. In order to reduce the inhibitory effect, the non-washed solid fraction containing reduced amount of inhibitors compared to the slurry was used in the SSF. Further, the cells were cultivated in the liquid fraction of pretreated spruce in a continuous culture wherein the outflow of cell suspension was used as cell feed to the SSF reactor in order to maintain the metabolic state of the cell. Enhanced cell viability was observed with cell, enzyme and substrate feed in a SSF producing 40 g l-1 ethanol after 96 h corresponding to 53% of theoretical yield based on available hexose sugars compared to 28 g l-1 ethanol in SSF with enzyme and substrate feed but no cell feed resulting in 37% of theoretical yield at a high solids loading of 20% (w/w) WIS content. The fed-batch SSF also significantly eased the mixing, which is usually challenging in batch SSF at high solids loading. A simple modification of the cell cultivation procedure together with a combination of yeast, enzyme and substrate feed in a fed-batch SSF process, made it possible to operate at high solids loadings in a conventional bioreactor. The proposed process strategy significantly increased the yeast cell viability and overall ethanol yield. It was also possible to obtain 4% (w/v) ethanol concentration, which is a minimum requirement for an economical distillation process.

Of enzyme use in cost-effective high solid simultaneous saccharification and fermentation processes.

PubMed

Sóti, Valentin; Lenaerts, Silvia; Cornet, Iris

2018-03-20

Enzyme cost is considered to be one of the most significant factors defining the final product price in lignocellulose hydrolysis and fermentation. Enzyme immobilization and recycling can be a tool to decrease costs. However, high solid loading is a key factor towards high product titers, and recovery of immobilized enzymes from this thick liquid is often overlooked. This paper aims to evaluate the economic feasibility of immobilized enzymes in simultaneous saccharification and fermentation (SSF) of lignocellulose biomass in general, as well as the recuperation of magnetic immobilized enzymes (m-CLEAs) during high solid loading in simultaneous saccharification, detoxification and fermentation processes (SSDF) of lignocellulose biomass. Enzyme prices were obtained from general cost estimations by Klein-Marcuschamer et al. [Klein-Marcuschamer et al. (2012) Biotechnol. Bioeng. 109, 1083-1087]. During enzyme cost analysis, the influence of inoculum recirculation as well as a shortened fermentation time was explored. Both resulted in 15% decrease of final enzyme product price. Enzyme recuperation was investigated experimentally and 99.5 m/m% of m-CLEAs was recovered from liquid medium in one step, while 88 m/m% could still be recycled from a thick liquid with high solid concentrations (SSF fermentation broth). A mathematical model was constructed to calculate the cost of immobilized and free enzyme utilization and showed that, with current process efficiencies and commercial enzyme prices, the cost reduction obtained by enzyme immobilization can reach around 60% compared to free enzyme utilization, while lower enzyme prices will result in a lower percentage of immobilization related savings, but overall enzyme costs will decrease significantly. These results are applied in a case study, estimating the viability of shifting from sugar to lignocellulose substrate for a 100 t lactic acid fermentation batch. It was concluded that it will only be economically feasible if the enzymes are produced at the most optimistic variable cost and either the activity of the immobilized catalyst or the recovery efficiency is further increased. Copyright © 2018 Elsevier B.V. All rights reserved.

Response surface methodology optimization of partitioning of xylanase form Aspergillus Niger by metal affinity polymer-salt aqueous two-phase systems.

PubMed

Fakhari, Mohamad Ali; Rahimpour, Farshad; Taran, Mojtaba

2017-09-15

Aqueous two phase affinity partitioning system using metal ligands was applied for partitioning and purification of xylanase produced by Aspergillus Niger. To minimization the number of experiments for the design parameters and develop predictive models for optimization of the purification process, response surface methodology (RSM) with a face-centered central composite design (CCF) has been used. Polyethylene glycol (PEG) 6000 was activated using epichlorohydrin, covalently linked to iminodiacetic acid (IDA), and the specific metal ligand Cu was attached to the polyethylene glycol-iminodiacetic acid (PEG-IDA). The influence of some experimental variables such as PEG (10-18%w/w), sodium sulfate (8-12%), PEG-IDA-Cu 2+ concentration (0-50% w/w of total PEG), pH of system (4-8) and crude enzyme loading (6-18%w/w) on xylanase and total protein partitioning coefficient, enzyme yield and enzyme specific activity were systematically evaluated. Two optimal point with high enzyme partitioning factor 10.97 and yield 79.95 (including 10% PEG, 12% Na 2 SO 4 , 50% ligand, pH 8 and 6% crude enzyme loading) and high specific activity in top phase 42.21 (including 14.73% PEG, 8.02% Na 2 SO 4 , 28.43% ligand, pH 7.7 and 6.08% crude enzyme loading) were attained. The adequacy of the RSM models was verified by a good agreement between experimental and predicted results. Copyright © 2017 Elsevier B.V. All rights reserved.

Simple Fabrication of a Highly Sensitive and Fast Glucose Biosensor using Enzyme Immobilized in Mesocellular Carbon Foam

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Dohoon; Lee, Jinwoo; Kim, Jungbae

2005-12-05

We fabricated a highly sensitive and fast glucose biosensor by simply immobilizing glucose oxidase in mesocellular carbon foam. Due to its unique structure, the MSU-F-C enabled high enzyme loading without serious mass transfer limitation, resulting in high catalytic efficiency. As a result, the glucose biosensor fabricated with MSU-F-C/GOx showed a high sensitivity and fast response. Given these results and the inherent electrical conductivity, we anticipate that MSU-F-C will make a useful matrix for enzyme immobilization in various biocatalytic and electrobiocatalytic applications.

Protein microspheres for controlled drug delivery and related analysis of biopolymers

NASA Astrophysics Data System (ADS)

Kirk, James Forrest

Rheumatoid arthritis (RA) is a systemic disorder which manifests itself most notably in the synovial joints. In recent years, methotrexate (MTX), a foliate antagonist, has been used with some success for treatment of RA. MTX has a maximum cumulative dose beyond which it becomes dangerous to administer due primarily to liver toxicity. This unfortunate side effect has prompted research into means of delivering MTX to the synovial joint in hopes of making more efficient use of the drug. Both MTX and its sodium salt (Na-MTX) were loaded into microspheres (MS) composed of bovine serum albumin (BSA) stabilized by cross linking with dialdehydes or ferric ion. MS were prepared with two levels of drug loading at two different levels of cross linking. MTX loading densities as high as 46.8% w/w were achieved in the aldehyde cross linking system and as high as 46.3% w/w were achieved with ferric ion cross linking. Using Na-MTX, the values were 37.2% w/w and 31.7% w/w respectively. Both MTX and Na-MTX were elutable from the MS into phosphate buffered saline at 37sp°C. MTX elution from aldehyde cross linked microspheres was load dependent with ca. 60% eluted by 9 hours at low loading and ca. 60% eluted by 24 hours at high loading. In the ferric ion cross link system, the elution was independent of loading with 50% elution occurring between 20 and 48 hours. Na-MTX elution was independent of drug loading or cross link system with 50% elution occurring in less than two hours in all cases. Other investigations included the loading of mitoxantrone (NOV) and of enzyme. NOV was loaded onto BSA microspheres to a concentration of 19.3% w/w and was used successfully in the treatment of murine ovarian tumors. Acid phosphatase was successfully loaded onto and into BSA microspheres. This enzyme retained its initial activity up to four months on post-loaded spheres. The enzyme also remained active inside the microsphere as demonstrated by a substrate cleavage assay.

Stable metal-organic frameworks containing single-molecule traps for enzyme encapsulation.

PubMed

Feng, Dawei; Liu, Tian-Fu; Su, Jie; Bosch, Mathieu; Wei, Zhangwen; Wan, Wei; Yuan, Daqiang; Chen, Ying-Pin; Wang, Xuan; Wang, Kecheng; Lian, Xizhen; Gu, Zhi-Yuan; Park, Jihye; Zou, Xiaodong; Zhou, Hong-Cai

2015-01-19

Enzymatic catalytic processes possess great potential in chemical manufacturing, including pharmaceuticals, fuel production and food processing. However, the engineering of enzymes is severely hampered due to their low operational stability and difficulty of reuse. Here, we develop a series of stable metal-organic frameworks with rationally designed ultra-large mesoporous cages as single-molecule traps (SMTs) for enzyme encapsulation. With a high concentration of mesoporous cages as SMTs, PCN-333(Al) encapsulates three enzymes with record-high loadings and recyclability. Immobilized enzymes that most likely undergo single-enzyme encapsulation (SEE) show smaller Km than free enzymes while maintaining comparable catalytic efficiency. Under harsh conditions, the enzyme in SEE exhibits better performance than free enzyme, showing the effectiveness of SEE in preventing enzyme aggregation or denaturation. With extraordinarily large pore size and excellent chemical stability, PCN-333 may be of interest not only for enzyme encapsulation, but also for entrapment of other nanoscaled functional moieties.

Stable metal-organic frameworks containing single-molecule traps for enzyme encapsulation

NASA Astrophysics Data System (ADS)

Feng, Dawei; Liu, Tian-Fu; Su, Jie; Bosch, Mathieu; Wei, Zhangwen; Wan, Wei; Yuan, Daqiang; Chen, Ying-Pin; Wang, Xuan; Wang, Kecheng; Lian, Xizhen; Gu, Zhi-Yuan; Park, Jihye; Zou, Xiaodong; Zhou, Hong-Cai

2015-01-01

Enzymatic catalytic processes possess great potential in chemical manufacturing, including pharmaceuticals, fuel production and food processing. However, the engineering of enzymes is severely hampered due to their low operational stability and difficulty of reuse. Here, we develop a series of stable metal-organic frameworks with rationally designed ultra-large mesoporous cages as single-molecule traps (SMTs) for enzyme encapsulation. With a high concentration of mesoporous cages as SMTs, PCN-333(Al) encapsulates three enzymes with record-high loadings and recyclability. Immobilized enzymes that most likely undergo single-enzyme encapsulation (SEE) show smaller Km than free enzymes while maintaining comparable catalytic efficiency. Under harsh conditions, the enzyme in SEE exhibits better performance than free enzyme, showing the effectiveness of SEE in preventing enzyme aggregation or denaturation. With extraordinarily large pore size and excellent chemical stability, PCN-333 may be of interest not only for enzyme encapsulation, but also for entrapment of other nanoscaled functional moieties.

Enzymatic saccharification of brown seaweed for production of fermentable sugars.

PubMed

Sharma, Sandeep; Horn, Svein Jarle

2016-08-01

This study shows that high drying temperatures negatively affect the enzymatic saccharification yield of the brown seaweed Saccharina latissima. The optimal drying temperature of the seaweed in terms of enzymatic sugar release was found to be 30°C. The enzymatic saccharification process was optimized by investigating factors such as kinetics of sugar release, enzyme dose, solid loading and different blend ratios of cellulases and an alginate lyase. It was found that the seaweed biomass could be efficiently hydrolysed to fermentable sugars using a commercial cellulase cocktail. The inclusion of a mono-component alginate lyase was shown to improve the performance of the enzyme blend, in particular at high solid loadings. At 25% dry matter loading a combined glucose and mannitol concentration of 74g/L was achieved. Copyright © 2016 Elsevier Ltd. All rights reserved.

Understanding the fundamental mechanism behind accumulation of oligosaccharides during high solids loading enzymatic hydrolysis

USDA-ARS?s Scientific Manuscript database

During enzymatic hydrolysis of biomass, polysaccharides are cleaved by glycosyl hydrolases to soluble oligosaccharides and further hydrolyzed by ß-glucosidase, ß-xylosidase and other enzymes to monomeric sugars. However, commercial enzyme mixtures do not hydrolyze all of these oligosaccharides and v...

Fabrication of enzyme-based coatings on intact multi-walled carbon nanotubes as highly effective electrodes in biofuel cells

NASA Astrophysics Data System (ADS)

Kim, Byoung Chan; Lee, Inseon; Kwon, Seok-Joon; Wee, Youngho; Kwon, Ki Young; Jeon, Chulmin; An, Hyo Jin; Jung, Hee-Tae; Ha, Su; Dordick, Jonathan S.; Kim, Jungbae

2017-01-01

CNTs need to be dispersed in aqueous solution for their successful use, and most methods to disperse CNTs rely on tedious and time-consuming acid-based oxidation. Here, we report the simple dispersion of intact multi-walled carbon nanotubes (CNTs) by adding them directly into an aqueous solution of glucose oxidase (GOx), resulting in simultaneous CNT dispersion and facile enzyme immobilization through sequential enzyme adsorption, precipitation, and crosslinking (EAPC). The EAPC achieved high enzyme loading and stability because of crosslinked enzyme coatings on intact CNTs, while obviating the chemical pretreatment that can seriously damage the electron conductivity of CNTs. EAPC-driven GOx activity was 4.5- and 11-times higher than those of covalently-attached GOx (CA) on acid-treated CNTs and simply-adsorbed GOx (ADS) on intact CNTs, respectively. EAPC showed no decrease of GOx activity for 270 days. EAPC was employed to prepare the enzyme anodes for biofuel cells, and the EAPC anode produced 7.5-times higher power output than the CA anode. Even with a higher amount of bound non-conductive enzymes, the EAPC anode showed 1.7-fold higher electron transfer rate than the CA anode. The EAPC on intact CNTs can improve enzyme loading and stability with key routes of improved electron transfer in various biosensing and bioelectronics devices.

Streptavidin-mirror DNA tetrahedron hybrid as a platform for intracellular and tumor delivery of enzymes.

PubMed

Kim, Kyoung-Ran; Hwang, Dohyeon; Kim, Juhyeon; Lee, Chang-Yong; Lee, Wonseok; Yoon, Dae Sung; Shin, Dongyun; Min, Sun-Joon; Kwon, Ick Chan; Chung, Hak Suk; Ahn, Dae-Ro

2018-06-28

Despite the extremely high substrate specificity and catalytically amplified activity of enzymes, the lack of efficient cellular internalization limits their application as therapeutics. To overcome this limitation and to harness enzymes as practical biologics for targeting intracellular functions, we developed the streptavidin-mirror DNA tetrahedron hybrid as a platform for intracellular delivery of various enzymes. The hybrid consists of streptavidin, which provides a stoichiometrically controlled loading site for the enzyme cargo and an L-DNA (mirror DNA) tetrahedron, which provides the intracellular delivery potential. Due to the cell-penetrating ability of the mirror DNA tetrahedron of this hybrid, enzymes loaded on streptavidin can be efficiently delivered into the cells, intracellularly expressing their activity. In addition, we demonstrate tumor delivery of enzymes in an animal model by utilizing the potential of the hybrid to accumulate in tumors. Strikingly, the hybrid is able to transfer the apoptotic enzyme specifically into tumor cells, leading to strong suppression of tumor growth without causing significant damage to other tissues. These results suggest that the hybrid may allow anti-proliferative enzymes and proteins to be utilized as anticancer drugs. Copyright © 2018 Elsevier B.V. All rights reserved.

Techno-Economic Analysis of the Deacetylation and Disk Refining Process. Characterizing the Effect of Refining Energy and Enzyme Usage on Minimum Sugar Selling Price and Minimum Ethanol Selling Price

DOE PAGES

Chen, Xiaowen; Shekiro, Joseph; Pschorn, Thomas; ...

2015-10-29

A novel, highly efficient deacetylation and disk refining (DDR) process to liberate fermentable sugars from biomass was recently developed at the National Renewable Energy Laboratory (NREL). The DDR process consists of a mild, dilute alkaline deacetylation step followed by low-energy-consumption disk refining. The DDR corn stover substrates achieved high process sugar conversion yields, at low to modest enzyme loadings, and also produced high sugar concentration syrups at high initial insoluble solid loadings. The sugar syrups derived from corn stover are highly fermentable due to low concentrations of fermentation inhibitors. The objective of this work is to evaluate the economic feasibilitymore » of the DDR process through a techno-economic analysis (TEA). A large array of experiments designed using a response surface methodology was carried out to investigate the two major cost-driven operational parameters of the novel DDR process: refining energy and enzyme loadings. The boundary conditions for refining energy (128–468 kWh/ODMT), cellulase (Novozyme’s CTec3) loading (11.6–28.4 mg total protein/g of cellulose), and hemicellulase (Novozyme’s HTec3) loading (0–5 mg total protein/g of cellulose) were chosen to cover the most commercially practical operating conditions. The sugar and ethanol yields were modeled with good adequacy, showing a positive linear correlation between those yields and refining energy and enzyme loadings. The ethanol yields ranged from 77 to 89 gallons/ODMT of corn stover. The minimum sugar selling price (MSSP) ranged from $0.191 to $0.212 per lb of 50 % concentrated monomeric sugars, while the minimum ethanol selling price (MESP) ranged from $2.24 to $2.54 per gallon of ethanol. The DDR process concept is evaluated for economic feasibility through TEA. The MSSP and MESP of the DDR process falls within a range similar to that found with the deacetylation/dilute acid pretreatment process modeled in NREL’s 2011 design report. The DDR process is a much simpler process that requires less capital and maintenance costs when compared to conventional chemical pretreatments with pressure vessels. As a result, we feel the DDR process should be considered as an option for future biorefineries with great potential to be more cost-effective.« less

Techno-Economic Analysis of the Deacetylation and Disk Refining Process. Characterizing the Effect of Refining Energy and Enzyme Usage on Minimum Sugar Selling Price and Minimum Ethanol Selling Price

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Xiaowen; Shekiro, Joseph; Pschorn, Thomas

A novel, highly efficient deacetylation and disk refining (DDR) process to liberate fermentable sugars from biomass was recently developed at the National Renewable Energy Laboratory (NREL). The DDR process consists of a mild, dilute alkaline deacetylation step followed by low-energy-consumption disk refining. The DDR corn stover substrates achieved high process sugar conversion yields, at low to modest enzyme loadings, and also produced high sugar concentration syrups at high initial insoluble solid loadings. The sugar syrups derived from corn stover are highly fermentable due to low concentrations of fermentation inhibitors. The objective of this work is to evaluate the economic feasibilitymore » of the DDR process through a techno-economic analysis (TEA). A large array of experiments designed using a response surface methodology was carried out to investigate the two major cost-driven operational parameters of the novel DDR process: refining energy and enzyme loadings. The boundary conditions for refining energy (128–468 kWh/ODMT), cellulase (Novozyme’s CTec3) loading (11.6–28.4 mg total protein/g of cellulose), and hemicellulase (Novozyme’s HTec3) loading (0–5 mg total protein/g of cellulose) were chosen to cover the most commercially practical operating conditions. The sugar and ethanol yields were modeled with good adequacy, showing a positive linear correlation between those yields and refining energy and enzyme loadings. The ethanol yields ranged from 77 to 89 gallons/ODMT of corn stover. The minimum sugar selling price (MSSP) ranged from $0.191 to $0.212 per lb of 50 % concentrated monomeric sugars, while the minimum ethanol selling price (MESP) ranged from $2.24 to $2.54 per gallon of ethanol. The DDR process concept is evaluated for economic feasibility through TEA. The MSSP and MESP of the DDR process falls within a range similar to that found with the deacetylation/dilute acid pretreatment process modeled in NREL’s 2011 design report. The DDR process is a much simpler process that requires less capital and maintenance costs when compared to conventional chemical pretreatments with pressure vessels. As a result, we feel the DDR process should be considered as an option for future biorefineries with great potential to be more cost-effective.« less

Techno-economic analysis of the deacetylation and disk refining process: characterizing the effect of refining energy and enzyme usage on minimum sugar selling price and minimum ethanol selling price.

PubMed

Chen, Xiaowen; Shekiro, Joseph; Pschorn, Thomas; Sabourin, Marc; Tucker, Melvin P; Tao, Ling

2015-01-01

A novel, highly efficient deacetylation and disk refining (DDR) process to liberate fermentable sugars from biomass was recently developed at the National Renewable Energy Laboratory (NREL). The DDR process consists of a mild, dilute alkaline deacetylation step followed by low-energy-consumption disk refining. The DDR corn stover substrates achieved high process sugar conversion yields, at low to modest enzyme loadings, and also produced high sugar concentration syrups at high initial insoluble solid loadings. The sugar syrups derived from corn stover are highly fermentable due to low concentrations of fermentation inhibitors. The objective of this work is to evaluate the economic feasibility of the DDR process through a techno-economic analysis (TEA). A large array of experiments designed using a response surface methodology was carried out to investigate the two major cost-driven operational parameters of the novel DDR process: refining energy and enzyme loadings. The boundary conditions for refining energy (128-468 kWh/ODMT), cellulase (Novozyme's CTec3) loading (11.6-28.4 mg total protein/g of cellulose), and hemicellulase (Novozyme's HTec3) loading (0-5 mg total protein/g of cellulose) were chosen to cover the most commercially practical operating conditions. The sugar and ethanol yields were modeled with good adequacy, showing a positive linear correlation between those yields and refining energy and enzyme loadings. The ethanol yields ranged from 77 to 89 gallons/ODMT of corn stover. The minimum sugar selling price (MSSP) ranged from $0.191 to $0.212 per lb of 50 % concentrated monomeric sugars, while the minimum ethanol selling price (MESP) ranged from $2.24 to $2.54 per gallon of ethanol. The DDR process concept is evaluated for economic feasibility through TEA. The MSSP and MESP of the DDR process falls within a range similar to that found with the deacetylation/dilute acid pretreatment process modeled in NREL's 2011 design report. The DDR process is a much simpler process that requires less capital and maintenance costs when compared to conventional chemical pretreatments with pressure vessels. As a result, we feel the DDR process should be considered as an option for future biorefineries with great potential to be more cost-effective.

Understanding sugar yield loss and enzyme inhibition due to oligosaccharides accumulation during high solids-loading enzymatic hydrolisis.

USDA-ARS?s Scientific Manuscript database

During enzymatic hydrolysis of biomass, polysaccharides are cleaved by glycosyl hydrolases to soluble oligosaccharides and further hydrolyzed by ß-glucosidase, ß-xylosidase and other enzymes to monomeric sugars. However, not all oligosaccharides can be fully hydrolyzed and they may accumulate to 18-...

Enhanced enzymatic hydrolysis of lignocellulose by optimizing enzyme complexes.

PubMed

Zhang, Mingjia; Su, Rongxin; Qi, Wei; He, Zhimin

2010-03-01

To enhance the conversion of the cellulose and hemicellulose, the corncob pretreated by aqueous ammonia soaking was hydrolyzed by enzyme complexes. The saturation limit for cellulase (Spezyme CP) was determined as 15 mg protein/g glucan (50 filter paper unit (FPU)/g glucan). The accessory enzymes (beta-glucosidase, xylanase, and pectinase) were supplemented to hydrolyze cellobiose (cellulase-inhibiting product), hemicellulose, and pectin (the component covering the fiber surfaces), respectively. It was found that beta-glucosidase (Novozyme 188) loading of 1.45 mg protein/g glucan [30 cellobiase units (CBU)/g glucan] was enough to eliminate the cellobiose inhibitor, and 2.9 mg protein/g glucan (60 CBU/g glucan) was the saturation limit. The supplementation of xylanase and pectinase can increase the conversion of cellulose and hemicellulose significantly. The yields of glucose and xylose enhanced with the increasing enzyme loading, but the increasing trend became low at high loading. Compared with xylanase, pectinase was more effective to promote the hydrolysis of cellulose and hemicellulose. The supplementation of pectinase with 0.12 mg protein/g glucan could increase the yields of glucose and xylose by 7.5% and 29.3%, respectively.

Green coconut mesocarp pretreated by an alkaline process as raw material for bioethanol production.

PubMed

Soares, Jimmy; Demeke, Mekonnen M; Foulquié-Moreno, Maria R; Van de Velde, Miet; Verplaetse, Alex; Fernandes, Antonio Alberto Ribeiro; Thevelein, Johan M; Fernandes, Patricia Machado Bueno

2016-09-01

Cocos nucifera L., coconut, is a palm of high importance in the food industry, but a considerable part of the biomass is inedible. In this study, the pretreatment and saccharification parameters NaOH solution, pretreatment duration and enzyme load were evaluated for the production of hydrolysates from green coconut mesocarp using 18% (w/v) total solids (TS). Hydrolysates were not detoxified in order to preserve sugars solubilized during the pretreatment. Reduction of enzyme load from 15 to 7.5 filter paper cellulase unit (FPU)/g of biomass has little effect on the final ethanol titer. With optimized pretreatment and saccharification, hydrolysates with more than 7% (w/v) sugars were produced in 48h. Fermentation of the hydrolysate using industrial Saccharomyces cerevisiae strains produced 3.73% (v/v) ethanol. Our results showed a simple pretreatment condition with a high-solid load of biomass followed by saccharification and fermentation of undetoxified coconut mesocarp hydrolysates to produce ethanol with high titer. Copyright © 2016 Elsevier Ltd. All rights reserved.

An evaluation of serum soluble CD30 levels and serum CD26 (DPPIV) enzyme activity as markers of type 2 and type 1 cytokines in HIV patients receiving highly active antiretroviral therapy

PubMed Central

Keane, N M; Price, P; Lee, S; Stone, S F; French, M A

2001-01-01

This study evaluates serum CD26 (dipeptidyl peptidase IV, DPPIV) enzyme activity and serum levels of soluble CD30 as markers of T1 and T2 cytokine environments in HIV patients who achieved immune reconstitution after highly active antiretroviral therapy (HAART). Patients who had experienced inflammatory disease associated with pre-existent opportunistic infections after HAART (immune restoration diseases, IRD) were considered separately. Serum sCD30 levels and CD26 (DPPIV) enzyme activity were compared with IFN-γ production by PBMC cultured with cytomegalovirus (CMV) antigen in controls and patient groups. High sCD30 levels were associated with low IFN-γ production after antigenic stimulation in control subjects and, to a lesser extent, in immune reconstituted HIV patients. There was no association between serum CD26 (DPPIV) enzyme activity and IFN-γ production or sCD30 levels. Serum sCD30 levels and CD26 (DPPIV) enzyme activity were significantly increased in immune reconstituted patients with high HIV viral loads. Patients who had experienced CMV retinitis as an IRD had significantly higher sCD30 levels than all other patient groups. Hence, high sCD30 levels may be a marker of a T2 cytokine environment in HIV patients with immune reconstitution and are associated with higher HIV viral loads and a history of CMV associated IRD. PMID:11678906

Optimization of high solids fed-batch saccharification of sugarcane bagasse based on system viscosity changes.

PubMed

Liu, Yunyun; Xu, Jingliang; Zhang, Yu; Yuan, Zhenhong; Xie, Jun

2015-10-10

Viscosity trends in alkali-pretreated sugarcane bagasse (SCB) slurries undergoing high solids fed-batch enzymatic hydrolysis were measured for a range of solids loading from 15% to 36%. Solids liquefaction times were related to system viscosity changes. The viscosity decreased quickly for low solids loading, and increased with increasing solids content. Fed-batch hydrolysis was initiated with 15% solids loading, and an additional 8%, 7% and 6% were successively added after the system viscosity decreased to stable values to achieve a final solids content of 36%. Two enzyme-adding modes with 8.5FPU/g solid were investigated. The batch mode with all enzyme being added at the beginning of the reaction produced the highest yields, with approximately 231.7g/L total sugars and 134.9g/L glucose being obtained after 96h with nearly 60% of the final glucan conversion rate. This finding indicates that under the right conditions, the fed-batch strategy might be a plausible way to produce high sugars under high solids. Copyright © 2015 Elsevier B.V. All rights reserved.

Self-assembly of amphiphilic janus particles into monolayer capsules for enhanced enzyme catalysis in organic media.

PubMed

Cao, Wei; Huang, Renliang; Qi, Wei; Su, Rongxin; He, Zhimin

2015-01-14

Encapsulation of enzymes during the creation of an emulsion is a simple and efficient route for enhancing enzyme catalysis in organic media. Herein, we report a capsule with a shell comprising a monolayer of silica Janus particles (JPs) (referred to as a monolayer capsule) and a Pickering emulsion for the encapsulation of enzyme molecules for catalysis purposes in organic media using amphiphilic silica JPs as building blocks. We demonstrate that the JP capsules had a monolayer shell consisting of closely packed silica JPs (270 nm). The capsules were on average 5-50 μm in diameter. The stability of the JP capsules (Pickering emulsion) was investigated with the use of homogeneous silica nanoparticles as a control. The results show that the emulsion stabilized via amphiphilic silica JPs presented no obvious changes in physical appearance after 15 days, indicating the high stability of the emulsions and JP capsules. Furthermore, the lipase from Candida sp. was chosen as a model enzyme for encapsulation within the JP capsules during their formation. The catalytic performance of lipase was evaluated according to the esterification of 1-hexanol with hexanoic acid. It was found that the specific activity of the encapsulated enzymes (28.7 U mL(-1)) was more than 5.6 times higher than that of free enzymes in a biphasic system (5.1 U mL(-1)). The enzyme activity was further increased by varying the volume ratio of water to oil and the JPs loadings. The enzyme-loaded capsule also exhibited high stability during the reaction process and good recyclability. In particular, the jellification of agarose in the JP capsules further enhanced their operating stability. We believe that the monolayer structure of the JP capsules, together with their high stability, rendered the capsules to be ideal enzyme carriers and microreactors for enzyme catalysis in organic media because they created a large interfacial area and had low mass transfer resistance through the monolayer shell.

Polyelectrolyte Complex Optimization for Macrophage Delivery of Redox Enzyme Nanoparticles

PubMed Central

Zhao, Yuling; Haney, Matthew J.; Klyachko, Natalia L.; Li, Shu; Booth, Stephanie L.; Higginbotham, Sheila M.; Jones, Jocelyn; Zimmerman, Matthew C.; Mosley, R. Lee; Kabanov, Alexander V.; Gendelman, Howard E.; Batrakova, Elena V.

2011-01-01

Background We posit that cell-mediated drug delivery can improve transport of therapeutic enzymes to the brain and decrease inflammation and neurodegeneration induced during Parkinson’s disease. Our prior work demonstrated that macrophages loaded with nanoformulated catalase (“nanozyme”) protect the nigrostriatum in a murine model of Parkinson’s disease. Packaging of catalase into block ionomer complex with a synthetic polyelectrolyte block copolymers protects the enzyme degradation in macrophages. Methods We examined relationships between the composition and structure of block ionomer complexes, their physicochemical characteristics, and loadings, release rates, and catalase activity in bone marrow-derived macrophages. Results Formation of block-ionomer complexes resulted in improved aggregation stability. Block ionomer complexes with ε-polylisine, and poly-L-glutamic acid -poly(ethylene glycol) demonstrated the least cytotoxicity and high loading and release rates, however, did not efficiently protect catalase inside macrophages. Conclusion nanozymes with polyethyleneimine- and poly(L-lysine)10-poly(ethylene glycol) provided the best protection of enzymatic activity for cell-mediated drug delivery. PMID:21182416

A three-dimensional nitrogen-doped graphene structure: a highly efficient carrier of enzymes for biosensors

NASA Astrophysics Data System (ADS)

Guo, Jingxing; Zhang, Tao; Hu, Chengguo; Fu, Lei

2015-01-01

In recent years, graphene-based enzyme biosensors have received considerable attention due to their excellent performance. Enormous efforts have been made to utilize graphene oxide and its derivatives as carriers of enzymes for biosensing. However, the performance of these sensors is limited by the drawbacks of graphene oxide such as slow electron transfer rate, low catalytic area and poor conductivity. Here, we report a new graphene-based enzyme carrier, i.e. a highly conductive 3D nitrogen-doped graphene structure (3D-NG) grown by chemical vapour deposition, for highly effective enzyme-based biosensors. Owing to the high conductivity, large porosity and tunable nitrogen-doping ratio, this kind of graphene framework shows outstanding electrical properties and a large surface area for enzyme loading and biocatalytic reactions. Using glucose oxidase (GOx) as a model enzyme and chitosan (CS) as an efficient molecular binder of the enzyme, our 3D-NG based biosensors show extremely high sensitivity for the sensing of glucose (226.24 μA mM-1 m-2), which is almost an order of magnitude higher than those reported in most of the previous studies. The stable adsorption and outstanding direct electrochemical behaviour of the enzyme on the nanocomposite indicate the promising application of this 3D enzyme carrier in high-performance electrochemical biosensors or biofuel cells.In recent years, graphene-based enzyme biosensors have received considerable attention due to their excellent performance. Enormous efforts have been made to utilize graphene oxide and its derivatives as carriers of enzymes for biosensing. However, the performance of these sensors is limited by the drawbacks of graphene oxide such as slow electron transfer rate, low catalytic area and poor conductivity. Here, we report a new graphene-based enzyme carrier, i.e. a highly conductive 3D nitrogen-doped graphene structure (3D-NG) grown by chemical vapour deposition, for highly effective enzyme-based biosensors. Owing to the high conductivity, large porosity and tunable nitrogen-doping ratio, this kind of graphene framework shows outstanding electrical properties and a large surface area for enzyme loading and biocatalytic reactions. Using glucose oxidase (GOx) as a model enzyme and chitosan (CS) as an efficient molecular binder of the enzyme, our 3D-NG based biosensors show extremely high sensitivity for the sensing of glucose (226.24 μA mM-1 m-2), which is almost an order of magnitude higher than those reported in most of the previous studies. The stable adsorption and outstanding direct electrochemical behaviour of the enzyme on the nanocomposite indicate the promising application of this 3D enzyme carrier in high-performance electrochemical biosensors or biofuel cells. Electronic supplementary information (ESI) available: Procedures for CVD growth of 3D-NG, XRD and TEM measurements, a comparison with other graphene-based biosensors, a detailed study on the universality of 3D-NG as an enzyme carrier and more CV data on selectivity and stability. See DOI: 10.1039/c4nr05325g

Toxicity reduction and MMP-2 stimulation of papain and bromelain loaded in elastic niosomes.

PubMed

Manosroi, Aranya; Chankhampan, Charinya; Manosroi, Worapaka; Manosroi, Jiradej

2012-10-01

The elastic niosomes (Tween 61/cholesterol/sodium cholate at 1:1:0.1 molar ratio) loaded with the protease enzymes (papain and bromelain) gave the vesicular sizes of 109.5 to 143.9 nm with the negative zeta potential of -14.7 to -30.1 mv. The elastic niosomes loaded with the standard papain (PS), extracted papain (PE), standard bromelain (BS) and extracted bromelain (BE) showed deformability index (DI values) of 1.35, 1.81, 1.22 and 1.61 times higher than their corresponding non-elastic niosomes, respectively. The elastic niosomes did not only improve the entrapment efficiency of the enzymes over the non-elastic niosomes of about 1.35 times, but also reduced the toxicity on skin human fibroblasts by SRB assay of the PS, PE, BS and BE at 1.68, 2.10, 1.56 and 1.52 times, respectively. The relative MMP-2 stimulation of PS, PE, BS and BE loaded in elastic niosomes were 1.26 +/- 0.14, 1.34 +/- 0.15, 1.09 +/- 0.09 and 1.20 +/- 0.04 for the pro MMP-2 and 1.26 +/- 0.12, 1.41 +/- 0.23, 1.01 +/- 0.08 and 1.03 +/- 0.12 for the active MMP-2, respectively in comparing to the control which were similar activity to their free enzymes. The PE loaded in elastic niosomes gave superior characteristics (low cytotoxicity and high MMP-2 stimulation) to other enzymes. The elastic niosomes can enhance the chemical stability of PE, which exhibited higher remaining contents than the free PE of 1.36 times when kept at 27 +/- 2 degrees C after 8 weeks. Therefore, the extracted papain loaded in elastic niosomes appeared to have potential to be developed as a topical product for scar treatment.

Enzymatic Hydrolysis of Pretreated Fibre Pressed Oil Palm Frond by using Sacchariseb C6

NASA Astrophysics Data System (ADS)

Hashim, F. S.; Yussof, H. W.; Zahari, M. A. K. M.; Rahman, R. A.; Illias, R. M.

2017-06-01

Enzymatic hydrolysis becomes a prominent technology for conversion of cellulosic biomass to its glucose monomers that requires an action of cellulolytic enzymes in a sequential and synergistic manner. In this study, the effect of agitation speed, glucan loading, enzyme loading, temperature and reaction time on the production of glucose from fibre pressed oil palm frond (FPOPF) during enzymatic hydrolysis was screened by a half factorial design 25-1 using Response Surface Methodology (RSM). The FPOPF sample was first delignified by alkaline pretreatment at 4.42 (w/v) sodium hydroxide for an hour prior to enzymatic hydrolysis using commercial cellulase enzyme, Sacchariseb C6. The effect of enzymatic hydrolysis on the structural of FPOPF has been evaluated by Scanning Electron Microscopy (SEM) analysis. Characterization of raw FPOPF comprised of 4.5 extractives, 40.7 glucan, 26.1 xylan, 26.2 lignin and 1.8 ash, whereas for pretreated FPOPF gave 0.3 extractives, 61.4 glucan, 20.4 xylan, 13.3 lignin and 1.3 ash. From this study, it was found that the best enzymatic hydrolysis condition yielded 33.01 ± 0.73 g/L of glucose when performed at 200 rpm of agitation speed, 60 FPU/mL of enzyme loading, 4 (w/w) of glucan loading, temperature at 55 □ and 72 hours of reaction time. The model obtained was significant with p-value <0.0001 as verified by the analysis of variance (ANOVA). The coefficient of determination (R2) from ANOVA study was 0.9959. Overall, it can be concluded that addition of Sacchariseb C6 during enzymatic hydrolysis from pretreated FPOPF produce high amount of glucose that enhances it potential for industrial application. This glucose can be further used to produce high-value products.

Probing collagen-enzyme mechanochemistry in native tissue with dynamic, enzyme-induced creep

PubMed Central

Zareian, Ramin; Church, Kelli P.; Saeidi, Nima; Flynn, Brendan P.; Beale, John W.; Ruberti, Jeffrey W.

2012-01-01

Mechanical strain or stretch of collagen has been shown to be protective of fibrils against both thermal and enzymatic degradation. The details of this mechanochemical relationship could change our understanding of load-bearing tissue formation, growth, maintenance and disease in vertebrate animals. However, extracting a quantitative relationship between strain and the rate of enzymatic degradation is extremely difficult in bulk tissue due to confounding diffusion effects. In this investigation, we develop a dynamic, enzyme-induced creep assay and diffusion/reaction rate scaling arguments to extract a lower bound on the relationship between strain and the cutting rate of bacterial collagenase (BC) at low strains. The assay method permits continuous, forced probing of enzyme-induced strain which is very sensitive to degradation rate differences between specimens at low initial strain. The results, obtained on uniaxially-loaded strips of bovine corneal tissue (0.1, 0.25 or 0.5 N), demonstrate that small differences in strain alter the enzymatic cutting rate of the BC substantially. It was estimated that a change in tissue elongation of only 1.5% (at ~5% strain) reduces the maximum cutting-rate of the enzyme by more than half. Estimation of the average load per monomer in the tissue strips indicates that this protective “cutoff” occurs when the collagen monomers are transitioning from an entropic to an energetic mechanical regime. The continuous tracking of the enzymatic cleavage rate as a function of strain during the initial creep response indicates that the decrease in the cleavage rate of the BC is non-linear (initially-steep between 4.5 and 6.5% then flattens out from 6.5–9.5%). The high sensitivity to strain at low strain implies that even lightly-loaded collagenous tissue may exhibit significant strain-protection. The dynamic, enzyme-induced creep assay described herein has the potential to permit the rapid characterization of collagen/enzyme mechanochemistry in many different tissue types. PMID:20429513

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Hua; Li, Shuai; Si, Yanmei

Fe3O4 nanoparticles as nanocatalysts may present peroxidase-like catalysis activities and high electrocatalysis if loaded on conductive carbon nanotube (CNT) supports; however, their catalysis performances in an aqueous system might still be challenged by the poor aqueous dispersion of hydrophobic carbon supports and/or low stability of loaded iron catalysts. In this work, amphiphilic graphene oxide nanosheets were employed as “surfactant” to disperse CNTs to create stable graphene oxide-dispersed CNT (GCNT) supports in water for covalently loading cubic Fe3O4 nanoparticles with improved distribution and binding efficiency. Compared with original Fe3O4 nanos and CNT-loaded Fe3O4 nanocomplex, the prepared GCNT–Fe3O4 nanocomposite could achieve highermore » aqueous stability and, especially, much stronger peroxidase-like catalysis and electrocatalysis to H2O2, presumably resulting from the synergetic effects of two conductive carbon supports and cubic Fe3O4 nanocatalysts effectively loaded. Colorimetric and direct electrochemical detections of H2O2 and glucose using the GCNT–Fe3O4 nanocomposite were conducted with high detection sensitivities, demonstrating the feasibility of practical sensing applications. Such a magnetically recyclable “enzyme mimic” may circumvent some disadvantages of natural protein enzymes and common inorganic catalysts, featuring the multi-functions of high peroxidase-like catalysis, strong electrocatalysis, magnetic separation/recyclability, environmental stability, and direct H2O2 electrochemistry.« less

Pie waste - A component of food waste and a renewable substrate for producing ethanol.

PubMed

Magyar, Margaret; da Costa Sousa, Leonardo; Jayanthi, Singaram; Balan, Venkatesh

2017-04-01

Sugar-rich food waste is a sustainable feedstock that can be converted into ethanol without an expensive thermochemical pretreatment that is commonly used in first and second generation processes. In this manuscript we have outlined the pie waste conversion to ethanol through a two-step process, namely, enzyme hydrolysis using commercial enzyme products mixtures and microbial fermentation using yeast. Optimized enzyme cocktail was found to be 45% alpha amylase, 45% gamma amylase, and 10% pectinase at 2.5mg enzyme protein/g glucan produced a hydrolysate with high glucose concentration. All three solid loadings (20%, 30%, and 40%) produced sugar-rich hydrolysates and ethanol with little to no enzyme or yeast inhibition. Enzymatic hydrolysis and fermentation process mass balance was carried out using pie waste on a 1000g dry weight basis that produced 329g ethanol at 20% solids loading. This process clearly demonstrate how food waste could be efficiently converted to ethanol that could be used for making biodiesel by reacting with waste cooking oil. Copyright © 2017 Elsevier Ltd. All rights reserved.

Saccharification efficiencies of multi-enzyme complexes produced by aerobic fungi.

PubMed

Badhan, Ajay; Huang, Jiangli; Wang, Yuxi; Abbott, D Wade; Di Falco, Marcos; Tsang, Adrian; McAllister, Tim

2018-05-24

In the present study, we have characterized high molecular weight multi-enzyme complexes in two commercial enzymes produced by Trichoderma reesei (Spezyme CP) and Penicillium funiculosum (Accellerase XC). We successfully identified 146-1000 kDa complexes using Blue native polyacrylamide gel electrophoresis (BN-PAGE) to fractionate the protein profile in both preparations. Identified complexes dissociated into lower molecular weight constituents when loaded on SDS PAGE. Unfolding of the secondary structure of multi-enzyme complexes with trimethylamine (pH >10) suggested that they were not a result of unspecific protein aggregation. Cellulase (CMCase) profiles of extracts of BN-PAGE fractionated protein bands confirmed cellulase activity within the multi-enzyme complexes. A microassay was used to identify protein bands that promoted high levels of glucose release from barley straw. Those with high saccharification yield were subjected to LC-MS analysis to identify the principal enzymatic activities responsible. The results suggest that secretion of proteins by aerobic fungi leads to the formation of high molecular weight multi-enzyme complexes that display activity against carboxymethyl cellulose and barley straw. Copyright © 2018. Published by Elsevier B.V.

Enzymatic conversion of pretreated biomass into fermentable sugars for biorefinery operation

NASA Astrophysics Data System (ADS)

Gao, Dahai

2011-12-01

Depleting petroleum reserves and potential climate change caused by fossil fuel consumption have attracted significant attention towards the use of alternative renewable resources for production of fuels and chemicals. Lignocellulosic biomass provides a plentiful resource for the sustainable production of biofuels and biochemicals and could serve as an important contributor to the world energy portfolio in the near future. Successful biological conversion of lignocellulosic biomass requires an efficient and economical pretreatment method, high glucose/xylose yields during enzymatic hydrolysis and fermentation of both hexose and pentose to ethanol. High enzyme loading is a major economic bottleneck for the commercial processing of pretreated lignocellulosic biomass to produce fermentable sugars. Optimizing the enzyme cocktail for specific types of pretreated biomass allows for a significant reduction in enzyme loading without sacrificing hydrolysis yield. Core glycosyl hydrolases were isolated and purified from various sources to help rationally optimize an enzyme cocktail to digest ammonia fiber expansion (AFEX) treated corn stover. The four core cellulases were endoglucanase I (EG I), cellobiohydrolase I (CBH I), cellobiohydrolase II (CBH II) and beta-Glucosidase (betaG). The two core hemicellulases were an endoxylanase (EX) and a beta-xylosidase (betaX). A diverse set of accessory hemicellulases from bacterial sources was found necessary to enhance the synergistic action of cellulases hydrolysing AFEX pretreated corn stover. High glucose (around 80%) and xylose (around 70%) yields were achieved with a moderate enzyme loading (˜20 mg protein/g glucan) using an in-house developed enzyme cocktail and this cocktail was compared to commercial enzyme. Studying the binding properties of cellulases to lignocellulosic substrates is critical to achieving a fundamental understanding of plant cell wall saccharification. Lignin auto-fluorescence and degradation products formed during pretreatment impede accurate quantification of individual glycosyl hydrolases (GH) binding to pretreated cell walls. A high-throughput Fast Protein Liquid Chromatography (HT-FPLC) based method has been developed to quantify CBH I, CBH II and EG I present in hydrolyzates of untreated, AFEX, and dilute-acid pretreated corn stover. This method can accurately quantify individual enzymes present in complex binary and ternary protein mixtures without interference from plant cell wall derived components. The binding characteristics of CBH I, CBH II and EG I during 48 hours hydrolysis were studied on different cellulose allomorphs: microcrystalline cellulose Avicel (cellulose Ibeta), liquid ammonia treated cellulose (cellulose III), sodium hydroxide treated cellulose (cellulose II) and phosphoric acid swollen amorphous cellulose (AC). The digestibility ranking is AC>cellulose III>cellulose II>cellulose I. However, AC has the highest initial enzyme binding capacity while cellulose III had the lowest. CBH II is less stable during hydrolysis. Time course binding studies were also performed for pretreated biomass. Ammonia Fiber Expansion (AFEX) treated corn stover (CS), dilute acid (ACID) treated CS and ionic liquid (IL) pretreated CS were compared. The results indicate that presence of lignin is responsible for significant unproductive cellulase binding. These results are critical for improving our understanding of enzyme synergism, productive/unproductive enzyme binding and the role of pretreatment on enzyme accessibility to lignocellulosic plant cell walls. The results also assist in engineering novel low unproductive binding enzyme systems and developing economic enzyme recycle options.

Conversion of lignocellulosic agave residues into liquid biofuels using an AFEX™-based biorefinery.

PubMed

Flores-Gómez, Carlos A; Escamilla Silva, Eleazar M; Zhong, Cheng; Dale, Bruce E; da Costa Sousa, Leonardo; Balan, Venkatesh

2018-01-01

Agave-based alcoholic beverage companies generate thousands of tons of solid residues per year in Mexico. These agave residues might be used for biofuel production due to their abundance and favorable sustainability characteristics. In this work, agave leaf and bagasse residues from species Agave tequilana and Agave salmiana were subjected to pretreatment using the ammonia fiber expansion (AFEX) process. The pretreatment conditions were optimized using a response surface design methodology. We also identified commercial enzyme mixtures that maximize sugar yields for AFEX-pretreated agave bagasse and leaf matter, at ~ 6% glucan (w/w) loading enzymatic hydrolysis. Finally, the pretreated agave hydrolysates (at a total solids loading of ~ 20%) were used for ethanol fermentation using the glucose- and xylose-consuming strain Saccharomyces cerevisiae 424A (LNH-ST), to determine ethanol yields at industrially relevant conditions. Low-severity AFEX pretreatment conditions are required (100-120 °C) to enable efficient enzymatic deconstruction of the agave cell wall. These studies showed that AFEX-pretreated A. tequilana bagasse, A. tequilana leaf fiber, and A. salmiana bagasse gave ~ 85% sugar conversion during enzyme hydrolysis and over 90% metabolic yields of ethanol during fermentation without any washing step or nutrient supplementation. On the other hand, although lignocellulosic A. salmiana leaf gave high sugar conversions, the hydrolysate could not be fermented at high solids loadings, apparently due to the presence of natural inhibitory compounds. These results show that AFEX-pretreated agave residues can be effectively hydrolyzed at high solids loading using an optimized commercial enzyme cocktail (at 25 mg protein/g glucan) producing > 85% sugar conversions and over 40 g/L bioethanol titers. These results show that AFEX technology has considerable potential to convert lignocellulosic agave residues to bio-based fuels and chemicals in a biorefinery.

Recycling cellulases for cellulosic ethanol production at industrial relevant conditions: potential and temperature dependency at high solid processes.

PubMed

Lindedam, Jane; Haven, Mai Østergaard; Chylenski, Piotr; Jørgensen, Henning; Felby, Claus

2013-11-01

Different versions of two commercial cellulases were tested for their recyclability of enzymatic activity at high dry matter processes (12% or 25% DM). Recyclability was assessed by measuring remaining enzyme activity in fermentation broth and the ability of enzymes to hydrolyse fresh, pretreated wheat straw. Industrial conditions were used to study the impact of hydrolysis temperature (40 or 50°C) and residence time on recyclability. Enzyme recycling at 12% DM indicated that hydrolysis at 50°C, though ideal for ethanol yield, should be kept short or carried out at lower temperature to preserve enzymatic activity. Best results for enzyme recycling at 25% DM was 59% and 41% of original enzyme load for a Celluclast:Novozyme188 mixture and a modern cellulase preparation, respectively. However, issues with stability of enzymes and their strong adsorption to residual solids still pose a challenge for applicable methods in enzyme recycling. Copyright © 2013 Elsevier Ltd. All rights reserved.

Alkaline peroxide pretreatment of corn stover: effects of biomass, peroxide, and enzyme loading and composition on yields of glucose and xylose

PubMed Central

2011-01-01

Background Pretreatment is a critical step in the conversion of lignocellulose to fermentable sugars. Although many pretreatment processes are currently under investigation, none of them are entirely satisfactory in regard to effectiveness, cost, or environmental impact. The use of hydrogen peroxide at pH 11.5 (alkaline hydrogen peroxide (AHP)) was shown by Gould and coworkers to be an effective pretreatment of grass stovers and other plant materials in the context of animal nutrition and ethanol production. Our earlier experiments indicated that AHP performed well when compared against two other alkaline pretreatments. Here, we explored several key parameters to test the potential of AHP for further improvement relevant to lignocellulosic ethanol production. Results The effects of biomass loading, hydrogen peroxide loading, residence time, and pH control were tested in combination with subsequent digestion with a commercial enzyme preparation, optimized mixtures of four commercial enzymes, or optimized synthetic mixtures of pure enzymes. AHP pretreatment was performed at room temperature (23°C) and atmospheric pressure, and after AHP pretreatment the biomass was neutralized with HCl but not washed before enzyme digestion. Standard enzyme digestion conditions were 0.2% glucan loading, 15 mg protein/g glucan, and 48 h digestion at 50°C. Higher pretreatment biomass loadings (10% to 20%) gave higher monomeric glucose (Glc) and xylose (Xyl) yields than the 2% loading used in earlier studies. An H2O2 loading of 0.25 g/g biomass was almost as effective as 0.5 g/g, but 0.125 g/g was significantly less effective. Optimized mixtures of four commercial enzymes substantially increased post-AHP-pretreatment enzymatic hydrolysis yields at all H2O2 concentrations compared to any single commercial enzyme. At a pretreatment biomass loading of 10% and an H2O2 loading of 0.5 g/g biomass, an optimized commercial mixture at total protein loadings of 8 or 15 mg/g glucan gave monomeric Glc yields of 83% or 95%, respectively. Yields of Glc and Xyl after pretreatment at a low hydrogen peroxide loading (0.125 g H2O2/g biomass) could be improved by extending the pretreatment residence time to 48 h and readjusting the pH to 11.5 every 6 h during the pretreatment. A Glc yield of 77% was obtained using a pretreatment of 15% biomass loading, 0.125 g H2O2/g biomass, and 48 h with pH adjustment, followed by digestion with an optimized commercial enzyme mixture at an enzyme loading of 15 mg protein/g glucan. Conclusions Alkaline peroxide is an effective pretreatment for corn stover. Particular advantages are the use of reagents with low environmental impact and avoidance of special reaction chambers. Reasonable yields of monomeric Glc can be obtained at an H2O2 concentration one-quarter of that used in previous AHP research. Additional improvements in the AHP process, such as peroxide stabilization, peroxide recycling, and improved pH control, could lead to further improvements in AHP pretreatment. PMID:21658263

Enzyme-free ethanol sensor based on electrospun nickel nanoparticle-loaded carbon fiber paste electrode.

PubMed

Liu, Yang; Zhang, Lei; Guo, Qiaohui; Hou, Haoqing; You, Tianyan

2010-03-24

We have developed a novel nickel nanoparticle-loaded carbon fiber paste (NiCFP) electrode for enzyme-free determination of ethanol. An electrospinning technique was used to prepare the NiCF composite with large amounts of spherical nanoparticles firmly embedded in carbon fibers (CF). In application to electroanalysis of ethanol, the NiCFP electrode exhibited high amperometric response and good operational stability. The calibration curve was linear up to 87.5 mM with a detection limit of 0.25 mM, which is superior to that obtained with other transition metal based electrodes. For detection of ethanol present in liquor samples, the values obtained with the NiCFP electrode were in agreement with the ones declared on the label. The attractive analytical performance and simple preparation method make this novel material promising for the development of effective enzyme-free sensors. Copyright 2010 Elsevier B.V. All rights reserved.

Synthetic enzyme mixtures for biomass deconstruction: production and optimization of a core set.

PubMed

Banerjee, Goutami; Car, Suzana; Scott-Craig, John S; Borrusch, Melissa S; Aslam, Nighat; Walton, Jonathan D

2010-08-01

The high cost of enzymes is a major bottleneck preventing the development of an economically viable lignocellulosic ethanol industry. Commercial enzyme cocktails for the conversion of plant biomass to fermentable sugars are complex mixtures containing more than 80 proteins of suboptimal activities and relative proportions. As a step toward the development of a more efficient enzyme cocktail for biomass conversion, we have developed a platform, called GENPLAT, that uses robotic liquid handling and statistically valid experimental design to analyze synthetic enzyme mixtures. Commercial enzymes (Accellerase 1000 +/- Multifect Xylanase, and Spezyme CP +/- Novozyme 188) were used to test the system and serve as comparative benchmarks. Using ammonia-fiber expansion (AFEX) pretreated corn stover ground to 0.5 mm and a glucan loading of 0.2%, an enzyme loading of 15 mg protein/g glucan, and 48 h digestion at 50 degrees C, commercial enzymes released 53% and 41% of the available glucose and xylose, respectively. Mixtures of three, five, and six pure enzymes of Trichoderma species, expressed in Pichia pastoris, were systematically optimized. Statistical models were developed for the optimization of glucose alone, xylose alone, and the average of glucose + xylose for two digestion durations, 24 and 48 h. The resulting models were statistically significant (P < 0.0001) and indicated an optimum composition for glucose release (values for optimized xylose release are in parentheses) of 29% (5%) cellobiohydrolase 1, 5% (14%) cellobiohydrolase 2, 25% (25%) endo-beta1,4-glucanase 1, 14% (5%) beta-glucosidase, 22% (34%) endo-beta1,4-xylanase 3, and 5% (17%) beta-xylosidase in 48 h at a protein loading of 15 mg/g glucan. Comparison of two AFEX-treated corn stover preparations ground to different particle sizes indicated that particle size (100 vs. 500 microm) makes a large difference in total digestibility. The assay platform and the optimized "core" set together provide a starting point for the rapid testing and optimization of alternate core enzymes from other microbial and recombinant sources as well as for the testing of "accessory" proteins for development of superior enzyme mixtures for biomass conversion. (c) 2010 Wiley Periodicals, Inc.

Quantitative evolutionary design

PubMed Central

Diamond, Jared

2002-01-01

The field of quantitative evolutionary design uses evolutionary reasoning (in terms of natural selection and ultimate causation) to understand the magnitudes of biological reserve capacities, i.e. excesses of capacities over natural loads. Ratios of capacities to loads, defined as safety factors, fall in the range 1.2-10 for most engineered and biological components, even though engineered safety factors are specified intentionally by humans while biological safety factors arise through natural selection. Familiar examples of engineered safety factors include those of buildings, bridges and elevators (lifts), while biological examples include factors of bones and other structural elements, of enzymes and transporters, and of organ metabolic performances. Safety factors serve to minimize the overlap zone (resulting in performance failure) between the low tail of capacity distributions and the high tail of load distributions. Safety factors increase with coefficients of variation of load and capacity, with capacity deterioration with time, and with cost of failure, and decrease with costs of initial construction, maintenance, operation, and opportunity. Adaptive regulation of many biological systems involves capacity increases with increasing load; several quantitative examples suggest sublinear increases, such that safety factors decrease towards 1.0. Unsolved questions include safety factors of series systems, parallel or branched pathways, elements with multiple functions, enzyme reaction chains, and equilibrium enzymes. The modest sizes of safety factors imply the existence of costs that penalize excess capacities. Those costs are likely to involve wasted energy or space for large or expensive components, but opportunity costs of wasted space at the molecular level for minor components. PMID:12122135

An Optical Biosensing Platform using Reprecipitated Polyaniline Microparticles

NASA Astrophysics Data System (ADS)

Nemzer, Louis; Epstein, Arthur

2009-03-01

A great deal of effort remains focused on the goal of developing a continuous in vivo glucose monitoring system for patients with diabetes mellitus. We report a proof-of-concept study on a reagentless optical biosensing platform that circumvents the problems usually associated with direct glucose detection by utilizing the UV-VIS absorption properties of polyaniline, a biocompatible polymer. When the enzyme glucose oxidase is entrapped within reprecipitated polyaniline microparticles, a glucose molecule readily donates two protons and two electrons to the polyaniline, reversibly altering the polymer's oxidation state. The resultant change can be monitored by measuring the absorption at wavelengths that fall within the ``optical window'' for skin. The micro-structured morphology also insures a high surface-area to volume ratio. Data from in vitro prototype devices indicate that in the low enzyme-loading regime, the response can be fit to the Michaelis-Menten model for enzyme kinetics, but at higher enzyme loading, diffusion effects dominate. As a biosensing platform, the system also has the potential to be adapted to detect other biologically relevant analytes, including cholesterol and ethanol.

Co-immobilization of multiple enzymes by metal coordinated nucleotide hydrogel nanofibers: improved stability and an enzyme cascade for glucose detection.

PubMed

Liang, Hao; Jiang, Shuhui; Yuan, Qipeng; Li, Guofeng; Wang, Feng; Zhang, Zijie; Liu, Juewen

2016-03-21

Preserving enzyme activity and promoting synergistic activity via co-localization of multiple enzymes are key topics in bionanotechnology, materials science, and analytical chemistry. This study reports a facile method for co-immobilizing multiple enzymes in metal coordinated hydrogel nanofibers. Specifically, four types of protein enzymes, including glucose oxidase, Candida rugosa lipase, α-amylase, and horseradish peroxidase, were respectively encapsulated in a gel nanofiber made of Zn(2+) and adenosine monophosphate (AMP) with a simple mixing step. Most enzymes achieved quantitative loading and retained full activity. At the same time, the entrapped enzymes were more stable against temperature variation (by 7.5 °C), protease attack, extreme pH (by 2-fold), and organic solvents. After storing for 15 days, the entrapped enzyme still retained 70% activity while the free enzyme nearly completely lost its activity. Compared to nanoparticles formed with AMP and lanthanide ions, the nanofiber gels allowed much higher enzyme activity. Finally, a highly sensitive and selective biosensor for glucose was prepared using the gel nanofiber to co-immobilize glucose oxidase and horseradish peroxidase for an enzyme cascade system. A detection limit of 0.3 μM glucose with excellent selectivity was achieved. This work indicates that metal coordinated materials using nucleotides are highly useful for interfacing with biomolecules.

IMPACTS OF NITROGEN LOADING ON SALT MARSH INTEGRITY IN NEW ENGLAND, USA

EPA Science Inventory

Salt marsh habitat integrity is linked with the ability to provide good water quality and high biodiversity. We measured high denitrification enzyme activity (DEA) in ten coastal salt marshes in Narragansett Bay, Rhode Island (seasonal means ranging from 7,476 - 53,494 kg N-1ha-...

Strong cellulase inhibition by Mannan polysaccharides in cellulose conversion to sugars.

PubMed

Kumar, Rajeev; Wyman, Charles E

2014-07-01

Cellulase enzymes contribute a major fraction of the total cost for biological conversion of lignocellulosic biomass to fuels and chemicals. Although a several fold reduction in cellulase production costs and enhancement of cellulase activity and stability have been reported in recent years, sugar yields are still lower at low enzyme doses than desired commercially. We recently reported that hemicellulose xylan and its oligomers strongly inhibit cellulase and that supplementation of cellulase with xylanase and β-xylosidase would significantly reduce such inhibition. In this study, mannan polysaccharides and their enzymatically prepared hydrolyzates were discovered to be strongly inhibitory to fungal cellulase in cellulose conversion (>50% drop in % relative conversion), even at a small concentration of 0.1 g/L, and inhibition was much greater than experienced by other known inhibitors such as cellobiose, xylooligomers, and furfural. Furthermore, cellulase inhibition dramatically increased with heteromannan loading and mannan substitution with galactose side units. In general, enzymatically prepared hydrolyzates were less inhibitory than their respective mannan polysaccharides except highly substituted ones. Supplementation of cellulase with commercial accessory enzymes such as xylanase, pectinase, and β-glucosidase was effective in greatly relieving inhibition but only for less substituted heteromannans. However, cellulase supplementation with purified heteromannan specific enzymes relieved inhibition by these more substituted heteromannans as well, suggesting that commercial preparations need to have higher amounts of such activities to realize high sugar yields at the low enzyme protein loadings needed for low cost fuels production. © 2014 Wiley Periodicals, Inc.

Synthesis of polymeric microcapsule arrays and their use for enzyme immobilization

NASA Astrophysics Data System (ADS)

Parthasarathy, Ranjani V.; Martin, Charles R.

1994-05-01

CURRENT methods for immobilizing enzymes for use in bioreactors and biosensors1-20 include adsorption on or covalent attachment to a support2-4, micro-encapsulation5,6, and entrapment within a membrane/film7,8,11-20 or gel9. The ideal immobilization method should employ mild chemical conditions, allow for large quantities of enzyme to be immobilized, provide a large surface area for enzyme-substrate contact within a small total volume, minimize barriers to mass transport of substrate and product, and provide a chemically and mechanically robust system. Here we describe a method for enzyme immobilization that satisfies all of these criteria. We have developed a template-based synthetic method that yields hollow polymeric microcapsules of uniform diameter and length. These microcapsules are arranged in a high-density array in which the individual capsules protrude from a surface like the bristles of a brush. We have developed procedures for filling these microcapsules with high concentrations of enzymes. The enzyme-loaded microcapsule arrays function as enzymatic bioreactors in both aqueous solution and organic solvents.

Cellulase stability, adsorption/desorption profiles and recycling during successive cycles of hydrolysis and fermentation of wheat straw.

PubMed

Rodrigues, Ana Cristina; Felby, Claus; Gama, Miguel

2014-03-01

The potential of enzymes recycling after hydrolysis and fermentation of wheat straw under a variety of conditions was investigated, monitoring the activity of the enzymes in the solid and liquid fractions, using low molecular weight substrates. A significant amount of active enzymes could be recovered by recycling the liquid phase. In the early stage of the process, enzyme adsorb to the substrate, then gradually returning to the solution as the saccharification proceeds. At 50°C, normally regarded as an acceptable operational temperature for saccharification, the enzymes (Celluclast) significantly undergo thermal deactivation. The hydrolysis yield and enzyme recycling efficiency in consecutive recycling rounds can be increased by using high enzyme loadings and moderate temperatures. Indeed, the amount of enzymes in the liquid phase increased with its thermostability and hydrolytic efficiency. This study contributes towards developing effective enzymes recycling strategies and helping to reduce the enzyme costs on bioethanol production. Copyright © 2014 Elsevier Ltd. All rights reserved.

Load-induced modulation of signal transduction networks.

PubMed

Jiang, Peng; Ventura, Alejandra C; Sontag, Eduardo D; Merajver, Sofia D; Ninfa, Alexander J; Del Vecchio, Domitilla

2011-10-11

Biological signal transduction networks are commonly viewed as circuits that pass along information--in the process amplifying signals, enhancing sensitivity, or performing other signal-processing tasks--to transcriptional and other components. Here, we report on a "reverse-causality" phenomenon, which we call load-induced modulation. Through a combination of analytical and experimental tools, we discovered that signaling was modulated, in a surprising way, by downstream targets that receive the signal and, in doing so, apply what in physics is called a load. Specifically, we found that non-intuitive changes in response dynamics occurred for a covalent modification cycle when load was present. Loading altered the response time of a system, depending on whether the activity of one of the enzymes was maximal and the other was operating at its minimal rate or whether both enzymes were operating at submaximal rates. These two conditions, which we call "limit regime" and "intermediate regime," were associated with increased or decreased response times, respectively. The bandwidth, the range of frequency in which the system can process information, decreased in the presence of load, suggesting that downstream targets participate in establishing a balance between noise-filtering capabilities and a circuit's ability to process high-frequency stimulation. Nodes in a signaling network are not independent relay devices, but rather are modulated by their downstream targets.

High-throughput microplate technique for enzymatic hydrolysis of lignocellulosic biomass.

PubMed

Chundawat, Shishir P S; Balan, Venkatesh; Dale, Bruce E

2008-04-15

Several factors will influence the viability of a biochemical platform for manufacturing lignocellulosic based fuels and chemicals, for example, genetically engineering energy crops, reducing pre-treatment severity, and minimizing enzyme loading. Past research on biomass conversion has focused largely on acid based pre-treatment technologies that fractionate lignin and hemicellulose from cellulose. However, for alkaline based (e.g., AFEX) and other lower severity pre-treatments it becomes critical to co-hydrolyze cellulose and hemicellulose using an optimized enzyme cocktail. Lignocellulosics are appropriate substrates to assess hydrolytic activity of enzyme mixtures compared to conventional unrealistic substrates (e.g., filter paper, chromogenic, and fluorigenic compounds) for studying synergistic hydrolysis. However, there are few, if any, high-throughput lignocellulosic digestibility analytical platforms for optimizing biomass conversion. The 96-well Biomass Conversion Research Lab (BCRL) microplate method is a high-throughput assay to study digestibility of lignocellulosic biomass as a function of biomass composition, pre-treatment severity, and enzyme composition. The most suitable method for delivering milled biomass to the microplate was through multi-pipetting slurry suspensions. A rapid bio-enzymatic, spectrophotometric assay was used to determine fermentable sugars. The entire procedure was automated using a robotic pipetting workstation. Several parameters that affect hydrolysis in the microplate were studied and optimized (i.e., particle size reduction, slurry solids concentration, glucan loading, mass transfer issues, and time period for hydrolysis). The microplate method was optimized for crystalline cellulose (Avicel) and ammonia fiber expansion (AFEX) pre-treated corn stover. Copyright 2008 Wiley Periodicals, Inc.

Thermoascus aurantiacus is a promising source of enzymes for biomass deconstruction under thermophilic conditions.

PubMed

McClendon, Shara D; Batth, Tanveer; Petzold, Christopher J; Adams, Paul D; Simmons, Blake A; Singer, Steven W

2012-07-28

Thermophilic fungi have attracted increased interest for their ability to secrete enzymes that deconstruct biomass at high temperatures. However, development of thermophilic fungi as enzyme producers for biomass deconstruction has not been thoroughly investigated. Comparing the enzymatic activities of thermophilic fungal strains that grow on targeted biomass feedstocks has the potential to identify promising candidates for strain development. Thielavia terrestris and Thermoascus aurantiacus were chosen for characterization based on literature precedents. Thermoascus aurantiacus and Thielavia terrestris were cultivated on various biomass substrates and culture supernatants assayed for glycoside hydrolase activities. Supernatants from both cultures possessed comparable glycoside hydrolase activities when incubated with artificial biomass substrates. In contrast, saccharifications of ionic liquid pretreated switchgrass (Panicum virgatum) revealed that T. aurantiacus enzymes released more glucose than T. terrestris enzymes over a range of protein mass loadings and temperatures. Temperature-dependent saccharifications demonstrated that the T. aurantiacus proteins retained higher levels of activity compared to a commercial enzyme mixture sold by Novozymes, Cellic CTec2, at elevated temperatures. Enzymes secreted by T. aurantiacus released glucose at similar protein loadings to CTec2 on dilute acid, ammonia fiber expansion, or ionic liquid pretreated switchgrass. Proteomic analysis of the T. aurantiacus culture supernatant revealed dominant glycoside hydrolases from families 5, 7, 10, and 61, proteins that are key enzymes in commercial cocktails. T. aurantiacus produces a complement of secreted proteins capable of higher levels of saccharification of pretreated switchgrass than T. terrestris enzymes. The T. aurantiacus enzymatic cocktail performs at the same level as commercially available enzymatic cocktail for biomass deconstruction, without strain development or genetic modifications. Therefore, T. aurantiacus provides an excellent platform to develop a thermophilic fungal system for enzyme production for the conversion of biomass to biofuels.

Thermoascus aurantiacus is a promising source of enzymes for biomass deconstruction under thermophilic conditions

PubMed Central

2012-01-01

Background Thermophilic fungi have attracted increased interest for their ability to secrete enzymes that deconstruct biomass at high temperatures. However, development of thermophilic fungi as enzyme producers for biomass deconstruction has not been thoroughly investigated. Comparing the enzymatic activities of thermophilic fungal strains that grow on targeted biomass feedstocks has the potential to identify promising candidates for strain development. Thielavia terrestris and Thermoascus aurantiacus were chosen for characterization based on literature precedents. Results Thermoascus aurantiacus and Thielavia terrestris were cultivated on various biomass substrates and culture supernatants assayed for glycoside hydrolase activities. Supernatants from both cultures possessed comparable glycoside hydrolase activities when incubated with artificial biomass substrates. In contrast, saccharifications of ionic liquid pretreated switchgrass (Panicum virgatum) revealed that T. aurantiacus enzymes released more glucose than T. terrestris enzymes over a range of protein mass loadings and temperatures. Temperature-dependent saccharifications demonstrated that the T. aurantiacus proteins retained higher levels of activity compared to a commercial enzyme mixture sold by Novozymes, Cellic CTec2, at elevated temperatures. Enzymes secreted by T. aurantiacus released glucose at similar protein loadings to CTec2 on dilute acid, ammonia fiber expansion, or ionic liquid pretreated switchgrass. Proteomic analysis of the T. aurantiacus culture supernatant revealed dominant glycoside hydrolases from families 5, 7, 10, and 61, proteins that are key enzymes in commercial cocktails. Conclusions T. aurantiacus produces a complement of secreted proteins capable of higher levels of saccharification of pretreated switchgrass than T. terrestris enzymes. The T. aurantiacus enzymatic cocktail performs at the same level as commercially available enzymatic cocktail for biomass deconstruction, without strain development or genetic modifications. Therefore, T. aurantiacus provides an excellent platform to develop a thermophilic fungal system for enzyme production for the conversion of biomass to biofuels. PMID:22839529

Batch fermentation options for high titer bioethanol production from a SPORL pretreated Douglas-Fir forest residue without detoxification

Treesearch

Mingyan Yang; Hairui Ji; Junyong Zhu

2016-01-01

This study evaluated batch fermentation modes, namely, separate hydrolysis and fermentation (SHF), quasi-simultaneous saccharification and fermentation (Q-SSF), and simultaneous saccharification and fermentation (SSF), and fermentation conditions, i.e., enzyme and yeast loadings, nutrient supplementation and sterilization, on high titer bioethanol production from SPORL...

Xylanase and feruloyl esterase from actinomycetes cultures could enhance sugarcane bagasse hydrolysis in the production of fermentable sugars.

PubMed

Rahmani, Nanik; Kahar, Prihardi; Lisdiyanti, Puspita; Hermiati, Euis; Lee, Jaemin; Yopi; Prasetya, Bambang; Ogino, Chiaki; Kondo, Akihiko

2018-02-23

The addition of enzymes that are capable of degrading hemicellulose has a potential to reduce the need for commercial enzymes during biomass hydrolysis in the production of fermentable sugars. In this study, a high xylanase producing actinomycete strain (Kitasatospora sp. ID06-480) and the first ethyl ferulate producing actinomycete strain (Nonomuraea sp. ID06-094) were selected from 797 rare actinomycetes, respectively, which were isolated in Indonesia. The addition (30%, v/v) of a crude enzyme supernatant from the selected strains in sugarcane bagasse hydrolysis with low-level loading (1 FPU/g-biomass) of Cellic® CTec2 enhanced both the released amount of glucose and reducing sugars. When the reaction with Ctec2 was combined with crude enzymes containing either xylanase or feruloyl esterase, high conversion yield of glucose from cellulose at 60.5% could be achieved after 72 h-saccharification.

Preserving catalytic activity and enhancing biochemical stability of the therapeutic enzyme asparaginase by biocompatible multilayered polyelectrolyte microcapsules.

PubMed

Karamitros, Christos S; Yashchenok, Alexey M; Möhwald, Helmuth; Skirtach, Andre G; Konrad, Manfred

2013-12-09

The present study focuses on the formation of microcapsules containing catalytically active L-asparaginase (L-ASNase), a protein drug of high value in antileukemic therapy. We make use of the layer-by-layer (LbL) technique to coat protein-loaded calcium carbonate (CaCO3) particles with two or three poly dextran/poly-L-arginine-based bilayers. To achieve high loading efficiency, the CaCO3 template was generated by coprecipitation with the enzyme. After assembly of the polymer shell, the CaCO3 core material was dissolved under mild conditions by dialysis against 20 mM EDTA. Biochemical stability of the encapsulated L-asparaginase was analyzed by treating the capsules with the proteases trypsin and thrombin, which are known to degrade and inactivate the enzyme during leukemia treatment, allowing us to test for resistance against proteolysis by physiologically relevant proteases through measurement of residual l-asparaginase activities. In addition, the thermal stability, the stability at the physiological temperature, and the long-term storage stability of the encapsulated enzyme were investigated. We show that encapsulation of l-asparaginase remarkably improves both proteolytic resistance and thermal inactivation at 37 °C, which could considerably prolong the enzyme's in vivo half-life during application in acute lymphoblastic leukemia (ALL). Importantly, the use of low EDTA concentrations for the dissolution of CaCO3 by dialysis could be a general approach in cases where the activity of sensitive biomacromolecules is inhibited, or even irreversibly damaged, when standard protocols for fabrication of such LbL microcapsules are used. Encapsulated and free enzyme showed similar efficacies in driving leukemic cells to apoptosis.

Rapid optimization of enzyme mixtures for deconstruction of diverse pretreatment/biomass feedstock combinations.

PubMed

Banerjee, Goutami; Car, Suzana; Scott-Craig, John S; Borrusch, Melissa S; Walton, Jonathan D

2010-10-12

Enzymes for plant cell wall deconstruction are a major cost in the production of ethanol from lignocellulosic biomass. The goal of this research was to develop optimized synthetic mixtures of enzymes for multiple pretreatment/substrate combinations using our high-throughput biomass digestion platform, GENPLAT, which combines robotic liquid handling, statistical experimental design and automated Glc and Xyl assays. Proportions of six core fungal enzymes (CBH1, CBH2, EG1, β-glucosidase, a GH10 endo-β1,4-xylanase, and β-xylosidase) were optimized at a fixed enzyme loading of 15 mg/g glucan for release of Glc and Xyl from all combinations of five biomass feedstocks (corn stover, switchgrass, Miscanthus, dried distillers' grains plus solubles [DDGS] and poplar) subjected to three alkaline pretreatments (AFEX, dilute base [0.25% NaOH] and alkaline peroxide [AP]). A 16-component mixture comprising the core set plus 10 accessory enzymes was optimized for three pretreatment/substrate combinations. Results were compared to the performance of two commercial enzymes (Accellerase 1000 and Spezyme CP) at the same protein loadings. When analyzed with GENPLAT, corn stover gave the highest yields of Glc with commercial enzymes and with the core set with all pretreatments, whereas corn stover, switchgrass and Miscanthus gave comparable Xyl yields. With commercial enzymes and with the core set, yields of Glc and Xyl were highest for grass stovers pretreated by AP compared to AFEX or dilute base. Corn stover, switchgrass and DDGS pretreated with AFEX and digested with the core set required a higher proportion of endo-β1,4-xylanase (EX3) and a lower proportion of endo-β1,4-glucanase (EG1) compared to the same materials pretreated with dilute base or AP. An optimized enzyme mixture containing 16 components (by addition of α-glucuronidase, a GH11 endoxylanase [EX2], Cel5A, Cel61A, Cip1, Cip2, β-mannanase, amyloglucosidase, α-arabinosidase, and Cel12A to the core set) was determined for AFEX-pretreated corn stover, DDGS, and AP-pretreated corn stover. The optimized mixture for AP-corn stover contained more exo-β1,4-glucanase (i.e., the sum of CBH1 + CBH2) and less endo-β1,4-glucanase (EG1 + Cel5A) than the optimal mixture for AFEX-corn stover. Amyloglucosidase and β-mannanase were the two most important enzymes for release of Glc from DDGS but were not required (i.e., 0% optimum) for corn stover subjected to AP or AFEX. As a function of enzyme loading over the range 0 to 30 mg/g glucan, Glc release from AP-corn stover reached a plateau of 60-70% Glc yield at a lower enzyme loading (5-10 mg/g glucan) than AFEX-corn stover. Accellerase 1000 was superior to Spezyme CP, the core set or the 16-component mixture for Glc yield at 12 h, but the 16-component set was as effective as the commercial enzyme mixtures at 48 h. The results in this paper demonstrate that GENPLAT can be used to rapidly produce enzyme cocktails for specific pretreatment/biomass combinations. Pretreatment conditions and feedstock source both influence the Glc and Xyl yields as well as optimal enzyme proportions. It is predicted that it will be possible to improve synthetic enzyme mixtures further by the addition of additional accessory enzymes.

GENPLAT: an Automated Platform for Biomass Enzyme Discovery and Cocktail Optimization

PubMed Central

Walton, Jonathan; Banerjee, Goutami; Car, Suzana

2011-01-01

The high cost of enzymes for biomass deconstruction is a major impediment to the economic conversion of lignocellulosic feedstocks to liquid transportation fuels such as ethanol. We have developed an integrated high throughput platform, called GENPLAT, for the discovery and development of novel enzymes and enzyme cocktails for the release of sugars from diverse pretreatment/biomass combinations. GENPLAT comprises four elements: individual pure enzymes, statistical design of experiments, robotic pipeting of biomass slurries and enzymes, and automated colorimeteric determination of released Glc and Xyl. Individual enzymes are produced by expression in Pichia pastoris or Trichoderma reesei, or by chromatographic purification from commercial cocktails or from extracts of novel microorganisms. Simplex lattice (fractional factorial) mixture models are designed using commercial Design of Experiment statistical software. Enzyme mixtures of high complexity are constructed using robotic pipeting into a 96-well format. The measurement of released Glc and Xyl is automated using enzyme-linked colorimetric assays. Optimized enzyme mixtures containing as many as 16 components have been tested on a variety of feedstock and pretreatment combinations. GENPLAT is adaptable to mixtures of pure enzymes, mixtures of commercial products (e.g., Accellerase 1000 and Novozyme 188), extracts of novel microbes, or combinations thereof. To make and test mixtures of ˜10 pure enzymes requires less than 100 μg of each protein and fewer than 100 total reactions, when operated at a final total loading of 15 mg protein/g glucan. We use enzymes from several sources. Enzymes can be purified from natural sources such as fungal cultures (e.g., Aspergillus niger, Cochliobolus carbonum, and Galerina marginata), or they can be made by expression of the encoding genes (obtained from the increasing number of microbial genome sequences) in hosts such as E. coli, Pichia pastoris, or a filamentous fungus such as T. reesei. Proteins can also be purified from commercial enzyme cocktails (e.g., Multifect Xylanase, Novozyme 188). An increasing number of pure enzymes, including glycosyl hydrolases, cell wall-active esterases, proteases, and lyases, are available from commercial sources, e.g., Megazyme, Inc. (www.megazyme.com), NZYTech (www.nzytech.com), and PROZOMIX (www.prozomix.com). Design-Expert software (Stat-Ease, Inc.) is used to create simplex-lattice designs and to analyze responses (in this case, Glc and Xyl release). Mixtures contain 4-20 components, which can vary in proportion between 0 and 100%. Assay points typically include the extreme vertices with a sufficient number of intervening points to generate a valid model. In the terminology of experimental design, most of our studies are "mixture" experiments, meaning that the sum of all components adds to a total fixed protein loading (expressed as mg/g glucan). The number of mixtures in the simplex-lattice depends on both the number of components in the mixture and the degree of polynomial (quadratic or cubic). For example, a 6-component experiment will entail 63 separate reactions with an augmented special cubic model, which can detect three-way interactions, whereas only 23 individual reactions are necessary with an augmented quadratic model. For mixtures containing more than eight components, a quadratic experimental design is more practical, and in our experience such models are usually statistically valid. All enzyme loadings are expressed as a percentage of the final total loading (which for our experiments is typically 15 mg protein/g glucan). For "core" enzymes, the lower percentage limit is set to 5%. This limit was derived from our experience in which yields of Glc and/or Xyl were very low if any core enzyme was present at 0%. Poor models result from too many samples showing very low Glc or Xyl yields. Setting a lower limit in turn determines an upper limit. That is, for a six-component experiment, if the lower limit for each single component is set to 5%, then the upper limit of each single component will be 75%. The lower limits of all other enzymes considered as "accessory" are set to 0%. "Core" and "accessory" are somewhat arbitrary designations and will differ depending on the substrate, but in our studies the core enzymes for release of Glc from corn stover comprise the following enzymes from T. reesei: CBH1 (also known as Cel7A), CBH2 (Cel6A), EG1(Cel7B), BG (β-glucosidase), EX3 (endo-β1,4-xylanase, GH10), and BX (β-xylosidase). PMID:22042431

GENPLAT: an automated platform for biomass enzyme discovery and cocktail optimization.

PubMed

Walton, Jonathan; Banerjee, Goutami; Car, Suzana

2011-10-24

The high cost of enzymes for biomass deconstruction is a major impediment to the economic conversion of lignocellulosic feedstocks to liquid transportation fuels such as ethanol. We have developed an integrated high throughput platform, called GENPLAT, for the discovery and development of novel enzymes and enzyme cocktails for the release of sugars from diverse pretreatment/biomass combinations. GENPLAT comprises four elements: individual pure enzymes, statistical design of experiments, robotic pipeting of biomass slurries and enzymes, and automated colorimeteric determination of released Glc and Xyl. Individual enzymes are produced by expression in Pichia pastoris or Trichoderma reesei, or by chromatographic purification from commercial cocktails or from extracts of novel microorganisms. Simplex lattice (fractional factorial) mixture models are designed using commercial Design of Experiment statistical software. Enzyme mixtures of high complexity are constructed using robotic pipeting into a 96-well format. The measurement of released Glc and Xyl is automated using enzyme-linked colorimetric assays. Optimized enzyme mixtures containing as many as 16 components have been tested on a variety of feedstock and pretreatment combinations. GENPLAT is adaptable to mixtures of pure enzymes, mixtures of commercial products (e.g., Accellerase 1000 and Novozyme 188), extracts of novel microbes, or combinations thereof. To make and test mixtures of ˜10 pure enzymes requires less than 100 μg of each protein and fewer than 100 total reactions, when operated at a final total loading of 15 mg protein/g glucan. We use enzymes from several sources. Enzymes can be purified from natural sources such as fungal cultures (e.g., Aspergillus niger, Cochliobolus carbonum, and Galerina marginata), or they can be made by expression of the encoding genes (obtained from the increasing number of microbial genome sequences) in hosts such as E. coli, Pichia pastoris, or a filamentous fungus such as T. reesei. Proteins can also be purified from commercial enzyme cocktails (e.g., Multifect Xylanase, Novozyme 188). An increasing number of pure enzymes, including glycosyl hydrolases, cell wall-active esterases, proteases, and lyases, are available from commercial sources, e.g., Megazyme, Inc. (www.megazyme.com), NZYTech (www.nzytech.com), and PROZOMIX (www.prozomix.com). Design-Expert software (Stat-Ease, Inc.) is used to create simplex-lattice designs and to analyze responses (in this case, Glc and Xyl release). Mixtures contain 4-20 components, which can vary in proportion between 0 and 100%. Assay points typically include the extreme vertices with a sufficient number of intervening points to generate a valid model. In the terminology of experimental design, most of our studies are "mixture" experiments, meaning that the sum of all components adds to a total fixed protein loading (expressed as mg/g glucan). The number of mixtures in the simplex-lattice depends on both the number of components in the mixture and the degree of polynomial (quadratic or cubic). For example, a 6-component experiment will entail 63 separate reactions with an augmented special cubic model, which can detect three-way interactions, whereas only 23 individual reactions are necessary with an augmented quadratic model. For mixtures containing more than eight components, a quadratic experimental design is more practical, and in our experience such models are usually statistically valid. All enzyme loadings are expressed as a percentage of the final total loading (which for our experiments is typically 15 mg protein/g glucan). For "core" enzymes, the lower percentage limit is set to 5%. This limit was derived from our experience in which yields of Glc and/or Xyl were very low if any core enzyme was present at 0%. Poor models result from too many samples showing very low Glc or Xyl yields. Setting a lower limit in turn determines an upper limit. That is, for a six-component experiment, if the lower limit for each single component is set to 5%, then the upper limit of each single component will be 75%. The lower limits of all other enzymes considered as "accessory" are set to 0%. "Core" and "accessory" are somewhat arbitrary designations and will differ depending on the substrate, but in our studies the core enzymes for release of Glc from corn stover comprise the following enzymes from T. reesei: CBH1 (also known as Cel7A), CBH2 (Cel6A), EG1(Cel7B), BG (β-glucosidase), EX3 (endo-β1,4-xylanase, GH10), and BX (β-xylosidase).

Effect of Degeneration on Fluid-Solid Interaction within Intervertebral Disk Under Cyclic Loading - A Meta-Model Analysis of Finite Element Simulations.

PubMed

Nikkhoo, Mohammad; Khalaf, Kinda; Kuo, Ya-Wen; Hsu, Yu-Chun; Haghpanahi, Mohammad; Parnianpour, Mohamad; Wang, Jaw-Lin

2015-01-01

The risk of low back pain resulted from cyclic loadings is greater than that resulted from prolonged static postures. Disk degeneration results in degradation of disk solid structures and decrease of water contents, which is caused by activation of matrix digestive enzymes. The mechanical responses resulted from internal solid-fluid interactions of degenerative disks to cyclic loadings are not well studied yet. The fluid-solid interactions in disks can be evaluated by mathematical models, especially the poroelastic finite element (FE) models. We developed a robust disk poroelastic FE model to analyze the effect of degeneration on solid-fluid interactions within disk subjected to cyclic loadings at different loading frequencies. A backward analysis combined with in vitro experiments was used to find the elastic modulus and hydraulic permeability of intact and enzyme-induced degenerated porcine disks. The results showed that the averaged peak-to-peak disk deformations during the in vitro cyclic tests were well fitted with limited FE simulations and a quadratic response surface regression for both disk groups. The results showed that higher loading frequency increased the intradiscal pressure, decreased the total fluid loss, and slightly increased the maximum axial stress within solid matrix. Enzyme-induced degeneration decreased the intradiscal pressure and total fluid loss, and barely changed the maximum axial stress within solid matrix. The increase of intradiscal pressure and total fluid loss with loading frequency was less sensitive after the frequency elevated to 0.1 Hz for the enzyme-induced degenerated disk. Based on this study, it is found that enzyme-induced degeneration decreases energy attenuation capability of disk, but less change the strength of disk.

Biliary copper excretion by hepatocyte lysosomes in the rat. Major excretory pathway in experimental copper overload

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gross, J.B. Jr.; Myers, B.M.; Kost, L.J.

1989-01-01

We investigated the hypothesis that lysosomes are the main source of biliary copper in conditions of hepatic copper overload. We used a rat model of oral copper loading and studied the relationship between the biliary output of copper and lysosomal hydrolases. Male Sprague-Dawley rats were given tap water with or without 0.125% copper acetate for up to 36 wk. Copper loading produced a 23-fold increase in the hepatic copper concentration and a 30-65% increase in hepatic lysosomal enzyme activity. Acid phosphatase histochemistry showed that copper-loaded livers contained an increased number of hepatocyte lysosomes; increased copper concentration of these organelles wasmore » confirmed directly by both x ray microanalysis and tissue fractionation. The copper-loaded rats showed a 16-fold increase in biliary copper output and a 50-300% increase in biliary lysosomal enzyme output. In the basal state, excretory profiles over time were similar for biliary outputs of lysosomal enzymes and copper in the copper-loaded animals but not in controls. After pharmacologic stimulation of lysosomal exocytosis, biliary outputs of copper and lysosomal hydrolases in the copper-loaded animals remained coupled: injection of colchicine or vinblastine produced an acute rise in the biliary output of both lysosomal enzymes and copper to 150-250% of baseline rates. After these same drugs, control animals showed only the expected increase in lysosomal enzyme output without a corresponding increase in copper output. We conclude that the hepatocyte responds to an increased copper load by sequestering excess copper in an increased number of lysosomes that then empty their contents directly into bile. The results provide direct evidence that exocytosis of lysosomal contents into biliary canaliculi is the major mechanism for biliary copper excretion in hepatic copper overload.« less

Effect of Degeneration on Fluid–Solid Interaction within Intervertebral Disk Under Cyclic Loading – A Meta-Model Analysis of Finite Element Simulations

PubMed Central

Nikkhoo, Mohammad; Khalaf, Kinda; Kuo, Ya-Wen; Hsu, Yu-Chun; Haghpanahi, Mohammad; Parnianpour, Mohamad; Wang, Jaw-Lin

2015-01-01

The risk of low back pain resulted from cyclic loadings is greater than that resulted from prolonged static postures. Disk degeneration results in degradation of disk solid structures and decrease of water contents, which is caused by activation of matrix digestive enzymes. The mechanical responses resulted from internal solid–fluid interactions of degenerative disks to cyclic loadings are not well studied yet. The fluid–solid interactions in disks can be evaluated by mathematical models, especially the poroelastic finite element (FE) models. We developed a robust disk poroelastic FE model to analyze the effect of degeneration on solid–fluid interactions within disk subjected to cyclic loadings at different loading frequencies. A backward analysis combined with in vitro experiments was used to find the elastic modulus and hydraulic permeability of intact and enzyme-induced degenerated porcine disks. The results showed that the averaged peak-to-peak disk deformations during the in vitro cyclic tests were well fitted with limited FE simulations and a quadratic response surface regression for both disk groups. The results showed that higher loading frequency increased the intradiscal pressure, decreased the total fluid loss, and slightly increased the maximum axial stress within solid matrix. Enzyme-induced degeneration decreased the intradiscal pressure and total fluid loss, and barely changed the maximum axial stress within solid matrix. The increase of intradiscal pressure and total fluid loss with loading frequency was less sensitive after the frequency elevated to 0.1 Hz for the enzyme-induced degenerated disk. Based on this study, it is found that enzyme-induced degeneration decreases energy attenuation capability of disk, but less change the strength of disk. PMID:25674562

Enzyme recycle and fed-batch addition for high-productivity soybean flour processing to produce enriched soy protein and concentrated hydrolysate of fermentable sugars.

PubMed

Loman, Abdullah Al; Islam, S M Mahfuzul; Li, Qian; Ju, Lu-Kwang

2017-10-01

Despite having high protein and carbohydrate, soybean flour utilization is limited to partial replacement of animal feed to date. Enzymatic process can be exploited to increase its value by enriching protein content and separating carbohydrate for utilization as fermentation feedstock. Enzyme hydrolysis with fed-batch and recycle designs were evaluated here for achieving this goal with high productivities. Fed-batch process improved carbohydrate conversion, particularly at high substrate loadings of 250-375g/L. In recycle process, hydrolysate retained a significant portion of the limiting enzyme α-galactosidase to accelerate carbohydrate monomerization rate. At single-pass retention time of 6h and recycle rate of 62.5%, reducing sugar concentration reached up to 120g/L using 4ml/g enzyme. When compared with batch and fed-batch processes, the recycle process increased the volumetric productivity of reducing sugar by 36% (vs. fed-batch) to 57% (vs. batch) and that of protein product by 280% (vs. fed-batch) to 300% (vs. batch). Copyright © 2017 Elsevier Ltd. All rights reserved.

Early evolution of efficient enzymes and genome organization

PubMed Central

2012-01-01

Background Cellular life with complex metabolism probably evolved during the reign of RNA, when it served as both information carrier and enzyme. Jensen proposed that enzymes of primordial cells possessed broad specificities: they were generalist. When and under what conditions could primordial metabolism run by generalist enzymes evolve to contemporary-type metabolism run by specific enzymes? Results Here we show by numerical simulation of an enzyme-catalyzed reaction chain that specialist enzymes spread after the invention of the chromosome because protocells harbouring unlinked genes maintain largely non-specific enzymes to reduce their assortment load. When genes are linked on chromosomes, high enzyme specificity evolves because it increases biomass production, also by reducing taxation by side reactions. Conclusion The constitution of the genetic system has a profound influence on the limits of metabolic efficiency. The major evolutionary transition to chromosomes is thus proven to be a prerequisite for a complex metabolism. Furthermore, the appearance of specific enzymes opens the door for the evolution of their regulation. Reviewers This article was reviewed by Sándor Pongor, Gáspár Jékely, and Rob Knight. PMID:23114029

Deactivation of Cellulase at the Air-Liquid Interface Is the Main Cause of Incomplete Cellulose Conversion at Low Enzyme Loadings.

PubMed

Bhagia, Samarthya; Dhir, Rachna; Kumar, Rajeev; Wyman, Charles E

2018-01-22

Amphiphilic additives such as bovine serum albumin (BSA) and Tween have been used to improve cellulose hydrolysis by cellulases. However, there has been a lack of clarity to explain their mechanism of action in enzymatic hydrolysis of pure or low-lignin cellulosic substrates. In this work, a commercial Trichoderma reesei enzyme preparation and the amphiphilic additives BSA and Tween 20 were applied for hydrolysis of pure Avicel cellulose. The results showed that these additives only had large effects on cellulose conversion at low enzyme to substrate ratios when the reaction flasks were shaken. Furthermore, changes in the air-liquid interfacial area profoundly affected cellulose conversion, but surfactants reduced or prevented cellulase deactivation at the air-liquid interface. Not shaking the flasks or adding low amounts of surfactant resulted in near theoretical cellulose conversion at low enzyme loadings given enough reaction time. At low enzyme loadings, hydrolysis of cellulose in lignocellulosic biomass with low lignin content suffered from enhanced enzyme deactivation at the air-liquid interface.

Determinants on an efficient cellulase recycling process for the production of bioethanol from recycled paper sludge under high solid loadings.

PubMed

Gomes, Daniel; Gama, Miguel; Domingues, Lucília

2018-01-01

In spite of the continuous efforts and investments in the last decades, lignocellulosic ethanol is still not economically competitive with fossil fuels. Optimization is still required in different parts of the process. Namely, the cost effective usage of enzymes has been pursued by different strategies, one of them being recycling. Cellulase recycling was analyzed on recycled paper sludge (RPS) conversion into bioethanol under intensified conditions. Different cocktails were studied regarding thermostability, hydrolysis efficiency, distribution in the multiphasic system and recovery from solid. Celluclast showed inferior stability at higher temperatures (45-55 °C), nevertheless its performance at moderate temperatures (40 °C) was slightly superior to other cocktails (ACCELLERASE ® 1500 and Cellic ® CTec2). Celluclast distribution in the solid-liquid medium was also more favorable, enabling to recover 88% of final activity at the end of the process. A central composite design studied the influence of solid concentration and enzyme dosage on RPS conversion by Celluclast. Solids concentration showed a significant positive effect on glucose production, no major limitations being found from utilizing high amounts of solids under the studied conditions. Increasing enzyme loading from 20 to 30 FPU/g cellulose had no significant effect on sugars production, suggesting that 22% solids and 20 FPU/g cellulose are the best operational conditions towards an intensified process. Applying these, a system of multiple rounds of hydrolysis with enzyme recycling was implemented, allowing to maintain the steady levels of enzyme activity with only 50% of enzyme on each recycling stage. Additionally, interesting levels of solid conversion (70-81%) were also achieved, leading to considerable improvements on glucose and ethanol production comparatively with the reports available so far (3.4- and 3.8-fold, respectively). Enzyme recycling viability depends on enzyme distribution between the solid and liquid phases at the end of hydrolysis, as well as enzymes thermostability. Both are critical features to be observed for a judicious choice of enzyme cocktail. This work demonstrates that enzyme recycling in intensified biomass degradation can be achieved through simple means. The process is possibly much more effective at larger scale, hence novel enzyme formulations favoring this possibility should be developed for industrial usage.

Nanobiotechnology as a novel paradigm for enzyme immobilisation and stabilisation with potential applications in biodiesel production.

PubMed

Verma, Madan Lal; Barrow, Colin J; Puri, Munish

2013-01-01

Nanobiotechnology is emerging as a new frontier of biotechnology. The potential applications of nanobiotechnology in bioenergy and biosensors have encouraged researchers in recent years to investigate new novel nanoscaffolds to build robust nanobiocatalytic systems. Enzymes, mainly hydrolytic class of enzyme, have been extensively immobilised on nanoscaffold support for long-term stabilisation by enhancing thermal, operational and storage catalytic potential. In the present report, novel nanoscaffold variants employed in the recent past for enzyme immobilisation, namely nanoparticles, nanofibres, nanotubes, nanopores, nanosheets and nanocomposites, are discussed in the context of lipase-mediated nanobiocatalysis. These nanocarriers have an inherently large surface area that leads to high enzyme loading and consequently high volumetric enzyme activity. Due to their high tensile strengths, nanoscale materials are often robust and resistant to breakage through mechanical shear in the running reactor making them suitable for multiple reuses. The optimisation of various nanosupports process parameters, such as the enzyme type and selection of suitable immobilisation method may help lead to the development of an efficient enzyme reactor. This might in turn offer a potential platform for exploring other enzymes for the development of stable nanobiocatalytic systems, which could help to address global environmental issues by facilitating the production of green energy. The successful validation of the feasibility of nanobiocatalysis for biodiesel production represents the beginning of a new field of research. The economic hurdles inherent in viably scaling nanobiocatalysts from a lab-scale to industrial biodiesel production are also discussed.

Green synthesis of isopropyl myristate in novel single phase medium Part I: Batch optimization studies.

PubMed

Vadgama, Rajeshkumar N; Odaneth, Annamma A; Lali, Arvind M

2015-12-01

Isopropyl myristate finds many applications in food, cosmetic and pharmaceutical industries as an emollient, thickening agent, or lubricant. Using a homogeneous reaction phase, non-specific lipase derived from Candida antartica, marketed as Novozym 435, was determined to be most suitable for the enzymatic synthesis of isopropyl myristate. The high molar ratio of alcohol to acid creates novel single phase medium which overcomes mass transfer effects and facilitates downstream processing. The effect of various reaction parameters was optimized to obtain a high yield of isopropyl myristate. Effect of temperature, agitation speed, organic solvent, biocatalyst loading and batch operational stability of the enzyme was systematically studied. The conversion of 87.65% was obtained when the molar ratio of isopropyl alcohol to myristic acid (15:1) was used with 4% (w/w) catalyst loading and agitation speed of 150 rpm at 60 °C. The enzyme has also shown good batch operational stability under optimized conditions.

Simultaneously and separately immobilizing incompatible dual-enzymes on polymer substrate via visible light induced graft polymerization

NASA Astrophysics Data System (ADS)

Zhu, Xing; He, Bin; Zhao, Changwen; Ma, Yuhong; Yang, Wantai

2018-04-01

Developing facile and mild strategy to construct multi-enzymes immobilization system has attracted considerable attentions in recent years. Here a simple immobilization strategy called visible light induced graft polymerization that can simultaneously and separately encapsulate two kinds of enzymes on one polymer film was proposed. Two incompatible enzymes, trypsin and transglutaminase (TGase) were selected as model dual-enzymes system and simultaneously immobilized on two sides of low-density polyethylene (LDPE) film. After immobilization, it was found that more than 90% of the enzymes can be embedded into dual-enzymes loaded film without leakage. And the activities of both separately immobilized enzymes were higher than the activities of mixed co-immobilized enzymes or the sequential immobilized ones. This dual-enzymes loaded film (DEL film) showed excellent recyclability and can retain >87% activities of both enzymes after 4 cycles of utilization. As an example, this DEL film was used to conjugate a prodrug of cytarabine with a target peptide. The successful preparation of expected product demonstrated that the separately immobilized two enzymes can worked well together to catalyze a two-step reaction.

Immobilization of Chloroperoxidase on Aminopropyl-Glass

PubMed Central

Kadima, Tenshuk A.; Pickard, Michael A.

1990-01-01

Chloroperoxidase (CPO) purified from Caldariomyces fumago CMI 89362 was covalently bound to aminopropyl-glass by using a modification of an established method. Acid-washed glass was derivatized by using aminopropyltriethoxysilane, and the enzyme was ionically bound at low ionic strength. Further treatment with glutaraldehyde covalently linked the enzyme to the glass beads in an active form. No elution of bound activity from glass beads could be detected with a variety of washings. The loading of enzyme protein to the glass beads was highest, 100 mg of CPO per g of glass, at high reaction ratios of CPO to glass, but the specific activity of the immobilized enzyme was highest, 36% of theoretical, at low enzyme-to-carrier ratios. No differences in the properties of the soluble and immobilized enzymes could be detected by a number of criteria: their pH-activity and pH-stability profiles were similar, as were their thermal stabilities. After five uses, the immobilized enzyme retained full activity between pH 6.0 and 6.7. PMID:16348352

Simultaneous glucose production from cellulose and fouling reduction using a magnetic responsive membrane reactor with superparamagnetic nanoparticles carrying cellulolytic enzymes.

PubMed

Gebreyohannes, Abaynesh Yihdego; Dharmjeet, Madhav; Swusten, Tom; Mertens, Matthias; Verspreet, Joran; Verbiest, Thierry; Courtin, Christophe M; Vankelecom, Ivo F J

2018-05-02

This work aimed at investigating simultaneous hydrolysis of cellulose and in-situ foulant degradation in a cellulose fed superparamagnetic biocatalytic membrane reactor (BMR SP ). In this reactor, a dynamic layer of superparamagnetic bionanocomposites with immobilized cellulolytic enzymes were reversibly immobilized on superparamagnetic polymeric membrane using an external magnetic field. The formation of a dynamic layer of bionanocomposites on the membrane helped to prevent direct membrane-foulant interaction. Due to in-situ biocatalysis, there was limited filtration resistance. Simultaneous separation of the product helped to avoid enzyme product inhibition, achieve constant reaction rate over time and 50% higher enzyme efficiency than batch reactor. Stable enzyme immobilization and the ability to keep enzyme in the system for long period helped to achieve continuous productivity at very low enzyme but high solid loading, while also reducing the extent of membrane fouling. Hence, the BMR SP paves a path for sustainable production of bioethanol from the cheaply available lignocellulose. Copyright © 2018 Elsevier Ltd. All rights reserved.

Chitosan-based biocatalytic nanoparticles for pollutant removal from wastewater.

PubMed

Alarcón-Payán, Dulce A; Koyani, Rina D; Vazquez-Duhalt, Rafael

2017-05-01

Chitosan, a renewable biopolymer has the prospective applications in different fields due to its gelation capacity. Nanoconfiguration of chitosan through ionotropic gelation to encapsulate enzymatic activity offers numerous potential applications. In the present study, the preparation and characterization of chitosan nanoparticles loaded with versatile peroxidase are reported. Their performance in bioremediation process and the resistance enhancement against natural microbial biodegradation were studied. The average diameter of enzymatic nanoparticles was 120nm and showed a high enzyme loading capacity. The kinetic parameters of nanoparticles exhibited a slightly lower catalytic activity (k cat ), similar affinity constant (Km) for hydrogen peroxide and higher Km value for the phenolic compound when compared with the free enzyme. The enzymatic nanoparticles showed higher thermostability and the same pH activity profile than those from free enzyme. Ten phenolic compounds, including pesticides, halogenated compounds, endocrine disruptors and antibacterials were transformed by the enzymatic nanoparticles. The transformation rate was lower than those obtained with free enzyme suggesting mass transfer limitations. But very importantly, the enzymatic nanoparticles showed a significant increase of the operational stability in real conditions of wastewater treatment process. Moreover, chemical modification of nanoparticles with different aldehydes still enhanced the operational stability of nanoparticulated enzymes. This enhancement of stability in real conditions and the potential use of biocatalytic nanoparticles in bioremediation processes are discussed. Copyright © 2017 Elsevier Inc. All rights reserved.

Influence of steam pretreatment severity on post-treatments used to enhance the enzymatic hydrolysis of pretreated softwoods at low enzyme loadings.

PubMed

Kumar, Linoj; Chandra, Richard; Saddler, Jack

2011-10-01

It is recognized that some form of post-treatment will usually be required if reasonable hydrolysis yields (>60%) of steam pretreated softwood are to be achieved when using low enzyme loadings (5 FPU/g cellulose). In the work reported here we modified/removed lignin from steam pretreated softwood while investigating the influence that the severity of pretreatment might have on the effectiveness of subsequent post-treatments. Although treatment at a lower severity could provide better overall hemicellulose recovery, post-treatment was not as effective on the cellulosic component. Pretreatment at medium severity resulted in the best compromise, providing reasonable recovery of the water soluble hemicellulose sugars and the use of post-treatment conditions that significantly increased the enzymatic hydrolysis of the water insoluble cellulosic component. Post-treatment with alkaline hydrogen peroxide or neutral sulfonation resulted in 62% cellulose hydrolysis at an enzyme loading of 5 FPU/g cellulose, which was four times greater than was obtained when the cellulosic fraction was not post-treated. When the enzyme loading was increased to 15 FPU/g cellulose, the post-treated cellulosic fraction was almost completely hydrolyzed to glucose. Despite the higher lignin content (44%) of the sulfonated substrate, similar hydrolysis yields to those achieved after alkaline peroxide post-treatment (14% lignin content) indicated that, in addition to lignin removal, lignin modification also plays an important role in influencing the effectiveness of hydrolysis when low enzyme loadings are used. Copyright © 2011 Wiley Periodicals, Inc.

Straightforward and effective protein encapsulation in polypeptide-based artificial cells.

PubMed

Zhi, Zheng-Liang; Haynie, Donald T

2006-01-01

A simple and straightforward approach to encapsulating an enzyme and preserving its function in polypeptide-based artificial cells is demonstrated. A model enzyme, glucose oxidase (GOx), was encapsulated by repeated stepwise adsorption of poly(L-lysine) and poly(L-glutamic acid) onto GOx-coated CaCO3 templates. These polypeptides are known from previous research to exhibit nanometer-scale organization in multilayer films. Templates were dissolved by ethylenediaminetetraacetic acid (EDTA) at neutral pH. Addition of polyethylene glycol (PEG) to the polypeptide assembly solutions greatly increased enzyme retention on the templates, resulting in high-capacity, high-activity loading of the enzyme into artificial cells. Assay of enzyme activity showed that over 80 mg-mL(-1) GOx was retained in artificial cells after polypeptide multilayer film formation and template dissolution in the presence of PEG, but only one-fifth as much was retained in the absence of PEG. Encapsulation is a means of improving the availability of therapeutic macromolecules in biomedicine. This work therefore represents a means of developing polypeptide-based artificial cells for use as therapeutic biomacromolecule delivery vehicles.

Cellulose conversion of corn pericarp without pretreatment.

PubMed

Kim, Daehwan; Orrego, David; Ximenes, Eduardo A; Ladisch, Michael R

2017-12-01

We report enzyme hydrolysis of cellulose in unpretreated pericarp at a cellulase loading of 0.25FPU/g pericarp solids using a phenol tolerant Aspergillus niger pectinase preparation. The overall protein added was 5mg/g and gave 98% cellulose conversion in 72h. However, for double the amount of enzyme from Trichoderma reesei, which is significantly less tolerant to phenols, conversion was only 16%. The key to achieving high conversion without pretreatment is combining phenol inhibition-resistant enzymes (such as from A. niger) with unground pericarp from which release of phenols is minimal. Size reduction of the pericarp, which is typically carried out in a corn-to-ethanol process, where corn is first ground to a fine powder, causes release of highly inhibitory phenols that interfere with cellulase enzyme activity. This work demonstrates hydrolysis without pretreatment of large particulate pericarp is a viable pathway for directly producing cellulose ethanol in corn ethanol plants. Copyright © 2017 Elsevier Ltd. All rights reserved.

Secretome analysis of Trichoderma reesei and Aspergillus niger cultivated by submerged and sequential fermentation processes: Enzyme production for sugarcane bagasse hydrolysis.

PubMed

Florencio, Camila; Cunha, Fernanda M; Badino, Alberto C; Farinas, Cristiane S; Ximenes, Eduardo; Ladisch, Michael R

2016-08-01

Cellulases and hemicellulases from Trichoderma reesei and Aspergillus niger have been shown to be powerful enzymes for biomass conversion to sugars, but the production costs are still relatively high for commercial application. The choice of an effective microbial cultivation process employed for enzyme production is important, since it may affect titers and the profile of protein secretion. We used proteomic analysis to characterize the secretome of T. reesei and A. niger cultivated in submerged and sequential fermentation processes. The information gained was key to understand differences in hydrolysis of steam exploded sugarcane bagasse for enzyme cocktails obtained from two different cultivation processes. The sequential process for cultivating A. niger gave xylanase and β-glucosidase activities 3- and 8-fold higher, respectively, than corresponding activities from the submerged process. A greater protein diversity of critical cellulolytic and hemicellulolytic enzymes were also observed through secretome analyses. These results helped to explain the 3-fold higher yield for hydrolysis of non-washed pretreated bagasse when combined T. reesei and A. niger enzyme extracts from sequential fermentation were used in place of enzymes obtained from submerged fermentation. An enzyme loading of 0.7 FPU cellulase activity/g glucan was surprisingly effective when compared to the 5-15 times more enzyme loadings commonly reported for other cellulose hydrolysis studies. Analyses showed that more than 80% consisted of proteins other than cellulases whose role is important to the hydrolysis of a lignocellulose substrate. Our work combined proteomic analyses and enzymology studies to show that sequential and submerged cultivation methods differently influence both titers and secretion profile of key enzymes required for the hydrolysis of sugarcane bagasse. The higher diversity of feruloyl esterases, xylanases and other auxiliary hemicellulolytic enzymes observed in the enzyme mixtures from the sequential fermentation could be one major reason for the more efficient enzyme hydrolysis that results when using the combined secretomes from A. niger and T. reesei. Copyright © 2016 Elsevier Inc. All rights reserved.

Effects of combined pressure and temperature on enzymes related to quality of fruits and vegetables: from kinetic information to process engineering aspects.

PubMed

Ludikhuyze, L; Van Loey, A; Indrawati; Smout, C; Hendrickx, M

2003-01-01

Throughout the last decade, high pressure technology has been shown to offer great potential to the food processing and preservation industry in delivering safe and high quality products. Implementation of this new technology will be largely facilitated when a scientific basis to assess quantitatively the impact of high pressure processes on food safety and quality becomes available. Besides, quantitative data on the effects of pressure and temperature on safety and quality aspects of foods are indispensable for design and evaluation of optimal high pressure processes, i.e., processes resulting in maximal quality retention within the constraints of the required reduction of microbial load and enzyme activity. Indeed it has to be stressed that new technologies should deliver, apart from the promised quality improvement, an equivalent or preferably enhanced level of safety. The present paper will give an overview from a quantitative point of view of the combined effects of pressure and temperature on enzymes related to quality of fruits and vegetables. Complete kinetic characterization of the inactivation of the individual enzymes will be discussed, as well as the use of integrated kinetic information in process engineering.

High titer ethanol production from simultaneous enzymatic saccharification and fermentation of aspen at high solids : a comparison between SPORL and dilute acid pretreatments

Treesearch

J.Y. Zhu; R. Gleisner; C.T. Scott; X.L. Luo; S. Tian

2011-01-01

Native aspen (Populus tremuloides) was pretreated using sulfuric acid and sodium bisulfite (SPORL) and dilute sulfuric acid alone (DA). Simultaneous enzymatic saccharification and fermentation (SSF) was conducted at 18% solids using commercial enzymes with cellulase loadings ranging from 6 to 15 FPU/g glucan and Saccharomyces cerevisiae Y5. Compared with DA...

Saccharification of ozonated sugarcane bagasse using enzymes from Myceliophthora thermophila JCP 1-4 for sugars release and ethanol production.

PubMed

de Cassia Pereira, Josiani; Travaini, Rodolfo; Paganini Marques, Natalia; Bolado-Rodríguez, Silvia; Bocchini Martins, Daniela Alonso

2016-03-01

The saccharification of ozonated sugarcane bagasse (SCB) by enzymes from Myceliophthora thermophila JCP 1-4 was studied. Fungal enzymes provided slightly higher sugar release than commercial enzymes, working at 50°C. Sugar release increased with temperature increase. Kinetic studies showed remarkable glucose release (4.99 g/L, 3%w/w dry matter) at 60°C, 8 h of hydrolysis, using an enzyme load of 10 FPU (filter paper unit). FPase and β-glucosidase activities increased during saccharification (284% and 270%, respectively). No further significant improvement on glucose release was observed increasing the enzyme load above 7.5 FPU per g of cellulose. Higher dry matter contents increased sugars release, but not yields. The fermentation of hydrolysates by Saccharomyces cerevisiae provided glucose-to-ethanol conversions around to 63%. Copyright © 2015 Elsevier Ltd. All rights reserved.

Cellulose-Hemicellulose Interactions at Elevated Temperatures Increase Cellulose Recalcitrance to Biological Conversion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mittal, Ashutosh; Himmel, Michael E; Kumar, Rajeev

It has been previously shown that cellulose-lignin droplets' strong interactions, resulting from lignin coalescence and redisposition on cellulose surface during thermochemical pretreatments, increase cellulose recalcitrance to biological conversion, especially at commercially viable low enzyme loadings. However, information on the impact of cellulose-hemicellulose interactions on cellulose recalcitrance following relevant pretreatment conditions are scarce. Here, to investigate the effects of plausible hemicellulose precipitation and re-association with cellulose on cellulose conversion, different pretreatments were applied to pure Avicel(R) PH101 cellulose alone and Avicel mixed with model hemicellulose compounds followed by enzymatic hydrolysis of resulting solids at both low and high enzyme loadings. Solidsmore » produced by pretreatment of Avicel mixed with hemicelluloses (AMH) were found to contain about 2 to 14.6% of exogenous, precipitated hemicelluloses and showed a remarkably much lower digestibility (up to 60%) than their respective controls. However, the exogenous hemicellulosic residues that associated with Avicel following high temperature pretreatments resulted in greater losses in cellulose conversion than those formed at low temperatures, suggesting that temperature plays a strong role in the strength of cellulose-hemicellulose association. Molecular dynamics simulations of hemicellulosic xylan and cellulose were found to further support this temperature effect as the xylan-cellulose interactions were found to substantially increase at elevated temperatures. Furthermore, exogenous, precipitated hemicelluloses in pretreated AMH solids resulted in a larger drop in cellulose conversion than the delignified lignocellulosic biomass containing comparably much higher natural hemicellulose amounts. Increased cellulase loadings or supplementation of cellulase with xylanases enhanced cellulose conversion for most pretreated AMH solids; however, this approach was less effective for solids containing mannan polysaccharides, suggesting stronger association of cellulose with (hetero) mannans or lack of enzymes in the mixture required to hydrolyze such polysaccharides.« less

Production of β-xylosidase from Trichoderma asperellum KIF125 and its application in efficient hydrolysis of pretreated rice straw with fungal cellulase.

PubMed

Inoue, Hiroyuki; Kitao, Chiaki; Yano, Shinichi; Sawayama, Shigeki

2016-11-01

On-site cellulase and hemicellulase production is a promising way to reduce enzyme cost in the commercialization of the lignocellulose-to-ethanol process. A hemicellulase-producing fungal strain suitable for on-site enzyme production was selected from cultures prepared using wet disc-milling rice straw (WDM-RS) and identified as Trichoderma asperellum KIF125. KIF125 hemicellulase showed uniquely high abundance of β-xylosidase in the xylanolytic enzyme system compared to other fungal hemicellulase preparations. Supplementation of Talaromyces cellulolyticus cellulase with KIF125 hemicellulase was more effective than that with the hemicellulases from other fungal sources in reducing the total enzyme loading for the improvement of xylose yield in the hydrolysis of ball-milling RS, due to its high β-xylosidase dominance. β-Xylosidase in KIF125 hemicellulase was purified and classified as a glycosyl hydrolase family 3 enzyme with relatively high specificity for xylobiose. The production of KIF125 β-xylosidase in the fermentor was estimated as 118 U/g-WDM-RS (2350 U/L culture) at 48 h. These results demonstrate that KIF125 is promising as a practical hemicellulase source to combine with on-site cellulase production using T. cellulolyticus.

Utilization of solid catfish manure waste as carbon and nutrient source for lactic acid production.

PubMed

Shi, Suan; Li, Jing; Blersch, David M

2018-06-01

The aim of this work was to study the solid waste (manure) produced by catfish as a potential feedstock for the production of lactic acid (LA) via fermentation. The solid waste contains high levels of both carbohydrates and nutrients that are sufficient for LA bacteria. Simultaneous saccharification and co-fermentation (SSCF) was applied using enzyme and Lactobacillus pentosus, and different loadings of enzyme and solid waste were tested. Results showed LA concentrations of 35.7 g/L were obtained at 15% solids content of catfish waste. Because of the high nutrient content in the fish waste, it could also be used as supplementary substrate for nitrogen and carbon sources with other lignocellulosic materials. A combined feedstock of catfish waste and paper mill sludge was tested, increasing the final LA concentration to 43.1 g/L at 12% solids loading. The catfish waste was shown to be a potential feedstock to provide both carbon and nutrients for LA production, suggesting its use as a sole substrate or in combination with other lignocellulosic materials.

Ultrasound assisted intensification of biodiesel production using enzymatic interesterification.

PubMed

Subhedar, Preeti B; Gogate, Parag R

2016-03-01

Ultrasound assisted intensification of synthesis of biodiesel from waste cooking oil using methyl acetate and immobilized lipase obtained from Thermomyces lanuginosus (Lipozyme TLIM) as a catalyst has been investigated in the present work. The reaction has also been investigated using the conventional approach based on stirring so as to establish the beneficial effects obtained due to the use of ultrasound. Effect of operating conditions such as reactant molar ratio (oil and methyl acetate), temperature and enzyme loading on the yield of biodiesel has been investigated. Optimum conditions for the conventional approach (without ultrasound) were established as reactant molar ratio of 1:12 (oil:methyl acetate), enzyme loading of 6% (w/v), temperature of 40 °C and reaction time of 24 h and under these conditions, 90.1% biodiesel yield was obtained. The optimum conditions for the ultrasound assisted approach were oil to methyl acetate molar ratio of 1:9, enzyme loading of 3% (w/v), and reaction time of 3 h and the biodiesel yield obtained under these conditions was 96.1%. Use of ultrasound resulted in significant reduction in the reaction time with higher yields and lower requirement of the enzyme loading. The obtained results have clearly established that ultrasound assisted interesterification was a fast and efficient approach for biodiesel production giving significant benefits, which can help in reducing the costs of production. Reusability studies for the enzyme were also performed but it was observed that reuse of the catalyst under the optimum experimental condition resulted in reduced enzyme activity and biodiesel yield. Copyright © 2015 Elsevier B.V. All rights reserved.

Stellate macroporous silica nanospheres in bio-macromolecules encapsulation and delivery

NASA Astrophysics Data System (ADS)

Chi, Hao-Hsin

This project focused on using mesoporous silica as a solid support to encapsulate enzymes for operating a highly economic, and recyclable biomass processing system. The main objective is to turn non-food biomass sources into food products. Enzymes are macromolecules with the structural backbone of proteins or ribonucleic acid sequences (RNAs) which work as catalysts in living organisms. Enzymes have the advantage of being the least contaminating catalyst due to normal catalyst might generate toxic by-product, and preferable to organic and inorganic catalysts, especially when used for product related to human used, which require biocompatibility of final product. However, there are several disadvantages in enzyme utilization. Their fabrication is time-consuming and requires elaborated molecular biology processes. Most of the enzymes need well-defined reaction conditions to be functional and operate at high yield. Unfortunately, although they are reusable as normal catalysts, it proves difficult to extract or reuse the enzymes from a reaction. Also, enzyme molecules are easily degradable and demand proper storage. To overcome some of the disadvantages, especially regarding stability to degradation, recovery, and reusability, immobilization of enzyme on solid support has become a thriving methodology. In recent years, mesoporous silica nanomaterials(MSN) have been at the forefront of enzyme immobilization given their extensive surface area, which provides capability to increase enzyme loading and for their demonstrate ability to protect enzyme from degradation, thus enabling high recyclability. Mesoporous silica is biocompatible and has already been used for several applications included. Catalysis, drug delivery, and Bio-imaging. Previously published research utilized mesoporous silica to deliver drugs, DNAs, RNAs or encapsulate single enzyme. The objective of this research is completed to develop a new porous silica platform that is unique in its porosity structure and develop it into a dual-enzyme platform with the scope of demonstrating a multi-reaction bio nanocatalyst. In regard to the further applications, the stellate MSN can be used as drug delivery or become a package of the biomacromolecule delivery system kit.

DECREASE Final Technical Report: Development of a Commercial Ready Enzyme Application System for Ethanol

DOE Office of Scientific and Technical Information (OSTI.GOV)

Teter, Sarah A

Conversion of biomass to sugars plays a central in reducing our dependence on petroleum, as it allows production of a wide range of biobased fuels and chemicals, through fermentation of those sugars. The DECREASE project delivers an effective enzyme cocktail for this conversion, enabling reduced costs for producing advanced biofuels such as cellulosic ethanol. Benefits to the public contributed by growth of the advanced biofuels industry include job creation, economic growth, and energy security. The DECREASE primary project objective was to develop a two-fold improved enzyme cocktail, relative to an advanced cocktail (CZP00005) that had been developed previously (from 2000-more » 2007). While the final milestone was delivery of all enzyme components as an experimental mixture, a secondary objective was to deploy an improved cocktail within 3 years following the close of the project. In February 2012, Novozymes launched Cellic CTec3, a multi-enzyme cocktail derived in part from components developed under DECREASE. The externally validated performance of CTec3 and an additional component under project benchmarking conditions indicated a 1.8-fold dose reduction in enzyme dose required for 90% conversion (based on all available glucose and xylose sources) of NREL dilute acid pretreated PCS, relative to the starting advanced enzyme cocktail. While the ability to achieve 90% conversion is impressive, targeting such high levels of biomass digestion is likely not the most cost effective strategy. Novozymes techno economic modeling showed that for NREL's dilute acid pretreated corn stover (PCS), 80% target conversion enables a lower total production cost for cellulosic ethanol than for 90% conversion, and this was also found to be the case when cost assumptions were based on the NREL 2002 Design Report. A 1.8X dose-reduction was observed for 80% conversion in the small scale (50 g) DECREASE benchmark assay for CTec3 and an additional component. An upscaled experiment (in 0.5 kg kettle reactors) was performed to compare the starting enzyme mixture CZP00005 with CTec3 alone; these results indicated a 1.9X dose- reduction for 80% conversion. The CTec3 composition does not include the best available enzyme components from the DECREASE effort. While these components are not yet available in a commercial product, experimental mixtures were assayed in a smaller scale assay using DECREASE PCS, at high solids loadings (21.5% TS). The results indicated that the newer mixtures required 2.9X-less enzyme for 90% conversion, and 3.2X-less enzyme for 80% conversion, relative to the starting enzyme cocktail. In conclusion, CTec3 delivers a 1.8-1.9X dose reduction on NREL PCS at high solids loadings, and the next generation enzyme from Novozymes will continue to show dramatically improved biochemical performance. CTec3 allows reduced costs today, and the experimental cocktails point to continued biotechnological improvements that will further drive down costs for biorefineries of tomorrow.« less

Atopy: a risk factor of refractory mycoplasma pneumoniae pneumonia?

PubMed

Bao, Yi-Xiao; Li, Jing; Tian, Ye; Liu, Quang-Hua; Bao, Jun

2017-11-01

To investigate the relationship of pathogen DNA copies with clinic and laboratory features among children with Mycoplasma pneumoniae (MP) pneumonia. A total of 95 enrolled children with MP pneumonia were assigned into the high-MP-load group (>10 6 /mL) and the low-MP-load group (≤10 6 /mL) according to MP-DNA copies in bronchoalveolar lavage fluid (BALF). Clinical characteristics and any allergy history were collected. Aeroallergens and food allergens were detected with a skin test. Serum IgE and eosinophil cationic protein (ECP) were assessed using enzyme immunoassay. BALF levels of IL-4, IFN-γ, IL-8 and TNF-α were assessed by ELISA. Compared with the low-MP-load group, 72.7% in the high-MP-load group developed refractory MP pneumonia who failed to respond to at least 1-week treatment with macrolides (72.7% vs 41.9%, P = 0.005). More children in the high-load group than those in the low-load group presented with extrapulmonary manifestations, lung consolidation, pleural effusion and atopic conditions including any allergy history, positive findings of aeroallergen test and increased serum IgE and ECP (P < 0.05). A significant higher BALF IL-4 level was seen in the high-load group versus the low-load group (23.00 ± 11.24 vs 14.68 ± 7.12; pg/mL; P < 0.01). There were no significant differences in BALF levels of IFN-γ, IL-8 and TNF-α between the two groups (P > 0.05). Atopy may be a risk factor for the presence and severity of refractory MP pneumonia due to the high pathogen load in airway. © 2016 John Wiley & Sons Ltd.

Assessment of cytomegalovirus and cell-mediated immunity for predicting outcomes in non-HIV-infected patients with Pneumocystis jirovecii pneumonia.

PubMed

Kim, Taeeun; Park, Se Yoon; Lee, Hyun-Jung; Kim, Sun-Mi; Sung, Heungsup; Chong, Yong Pil; Lee, Sang-Oh; Choi, Sang-Ho; Kim, Yang Soo; Woo, Jun Hee; Kim, Sung-Han

2017-07-01

The clinical importance of pulmonary cytomegalovirus (CMV) co-infection in patients with Pneumocystis jirovecii pneumonia (PCP) is uncertain. We therefore determined the association of CMV infection with outcomes in non-HIV-infected patients with PCP by assessing CMV viral load and CMV-specific T-cell response.We prospectively enrolled all non-HIV-infected patients with confirmed PCP, over a 2-year period. Real-time polymerase chain reaction from bronchoalveolar lavage was performed to measure CMV viral load, and CMV enzyme-linked immunospot assays of peripheral blood were used to measure CMV-specific T-cell responses. The primary outcome was 30-day mortality.A total of 76 patients were finally analyzed. The mortality in patients with high BAL CMV viral load (>2.52 log copies/mL, 6/32 [18%]) showed a nonsignificant trend to be higher than in those with low CMV viral load (2/44 [5%], P = .13). However, the mortality in patients with low CMV-specific T-cell responses (<5 spots/2.0 × 10 PBMC, 6/29 [21%]) was significantly higher than in patients with high CMV-specific T-cell response (2/47 [4%], P = .048). Moreover, the 2 strata with high CMV viral load and low CMV-specific T-cell responses (4/14 [29%]) and low CMV viral load and low CMV-specific T-cell responses (2/15 [13%]) had poorer outcomes than the 2 strata with high CMV viral load and high CMV-specific T-cell responses (2/18 [11%]) and low CMV viral load and high CMV-specific T-cell responses (0/29 [0%]).These data suggest that the CMV replication and impaired CMV-specific T-cell responses adversely affect the outcomes in non-HIV-infected patients with PCP.

Is the alkaline tide a signal to activate metabolic or ionoregulatory enzymes in the dogfish shark (Squalus acanthias)?

PubMed

Wood, Chris M; Kajimura, Makiko; Mommsen, Thomas P; Walsh, Patrick J

2008-01-01

Experimental metabolic alkalosis is known to stimulate whole-animal urea production and active ion secretion by the rectal gland in the dogfish shark. Furthermore, recent evidence indicates that a marked alkaline tide (systemic metabolic alkalosis) follows feeding in this species and that the activities of the enzymes of the ornithine-urea cycle (OUC) for urea synthesis in skeletal muscle and liver and of energy metabolism and ion transport in the rectal gland are increased at this time. We therefore evaluated whether alkalosis and/or NaCl/volume loading (which also occurs with feeding) could serve as a signal for activation of these enzymes independent of nutrient loading. Fasted dogfish were infused for 20 h with either 500 mmol L(-1) NaHCO3 (alkalosis + volume expansion) or 500 mmol L(-1) NaCl (volume expansion alone), both isosmotic to dogfish plasma, at a rate of 3 mL kg(-1) h(-1). NaHCO3 infusion progressively raised arterial pH to 8.28 (control = 7.85) and plasma [HCO3-] to 20.8 mmol L(-1) (control = 4.5 mmol L(-1)) at 20 h, with unchanged arterial P(CO2), whereas NaCl/volume loading had no effect on blood acid-base status. Rectal gland Na+,K+-ATPase activity was increased 50% by NaCl loading and more than 100% by NaHCO3 loading, indicating stimulatory effects of both volume expansion and alkalosis. Rectal gland lactate dehydrogenase activity was elevated 25% by both treatments, indicating volume expansion effects only, whereas neither treatment increased the activities of the aerobic enzymes citrate synthase, NADP-isocitrate dehydrogenase, or the ketone body-utilizing enzyme beta-hydroxybutyrate dehydrogenase in the rectal gland or liver. The activity of ornithine-citrulline transcarbamoylase in skeletal muscle was doubled by NaHCO3 infusion, but neither treatment altered the activities of other OUC-related enzymes (glutamine synthetase, carbamoylphosphate synthetase III). We conclude that both the alkaline tide and salt loading/volume expansion act as signals to activate some but not all of the elevated metabolic pathways and ionoregulatory mechanisms needed during processing of a meal.

Kinetics and Optimization of Lipophilic Kojic Acid Derivative Synthesis in Polar Aprotic Solvent Using Lipozyme RMIM and Its Rheological Study.

PubMed

Ishak, Nurazwa; Lajis, Ahmad Firdaus B; Mohamad, Rosfarizan; Ariff, Arbakariya B; Mohamed, Mohd Shamzi; Halim, Murni; Wasoh, Helmi

2018-02-24

The synthesis of kojic acid derivative (KAD) from kojic and palmitic acid (C16:0) in the presence of immobilized lipase from Rhizomucor miehei (commercially known as Lipozyme RMIM), was studied using a shake flask system. Kojic acid is a polyfunctional heterocycles that acts as a source of nucleophile in this reaction allowing the formation of a lipophilic KAD. In this study, the source of biocatalyst, Lipozyme RMIM, was derived from the lipase of Rhizomucor miehei immobilized on weak anion exchange macro-porous Duolite ES 562 by the adsorption technique. The effects of solvents, enzyme loading, reaction temperature, and substrate molar ratio on the reaction rate were investigated. In one-factor-at-a-time (OFAT) experiments, a high reaction rate (30.6 × 10 -3 M·min -1 ) of KAD synthesis was recorded using acetone, enzyme loading of 1.25% ( w / v ), reaction time of 12 h, temperature of 50 °C and substrate molar ratio of 5:1. Thereafter, a yield of KAD synthesis was optimized via the response surface methodology (RSM) whereby the optimized molar ratio (fatty acid: kojic acid), enzyme loading, reaction temperature and reaction time were 6.74, 1.97% ( w / v ), 45.9 °C, and 20 h respectively, giving a high yield of KAD (64.47%). This condition was reevaluated in a 0.5 L stirred tank reactor (STR) where the agitation effects of two impellers; Rushton turbine (RT) and pitch-blade turbine (PBT), were investigated. In the STR, a very high yield of KAD synthesis (84.12%) was achieved using RT at 250 rpm, which was higher than the shake flask, thus indicating better mixing quality in STR. In a rheological study, a pseudoplastic behavior of KAD mixture was proposed for potential application in lotion formulation.

Immobilization of β-galactosidase from Kluyveromyces lactis onto polymeric membrane surfaces: effect of surface characteristics.

PubMed

Güleç, Hacı Ali

2013-04-01

The aim of this study was to investigate the effects of surface characteristics of plain and plasma modified cellulose acetate (CA) membranes on the immobilization yield of β-galactosidases from Kluyveromyces lactis (KLG) and its galacto-oligosaccharide (GOS) yield, respectively. Low pressure plasma treatments involving oxygen plasma activation, plasma polymerization (PlsP) of ethylenediamine (EDA) and PlsP of 2-mercaptoethanol were used to modify plain CA membrane surfaces. KLG enzyme was immobilized onto plain and oxygen plasma treated membrane surfaces by simple adsorption. Oxygen plasma activation increased the hydrophylicity of CA membrane surfaces and it improved the immobilization yield of the enzyme by 42%. KLG enzyme was also immobilized onto CA membrane surfaces through amino groups created by PlsP of EDA via covalent binding. Plasma action at 60W plasma power and 15 min. exposure time improved the amount of membrane bounded enzyme by 3.5-fold. The enrichment of the amount of amino groups via polyethyleneimine (PEI) addition enhanced this increase from 3.5-fold to 4.5-fold. Although high enzyme loading was achived (65-83%), both of the methods dramatically decreased the enzyme activity (11-12%) and GOS yield due to probably negative effects of active amino groups. KLG enzyme was more effectively immobilized onto thiolated CA membrane surface created by PlsP of 2-mercaptoethanol with high immobilization yield (70%) and especially high enzyme activity (46%). Immobilized enzymes on the CA membranes treated by PlsP were successively reutilized for 5-8 cycles at 25°C and enzymatic derivatives retained approximately 75-80% of their initial activites at the end of the reactions. Copyright © 2012 Elsevier B.V. All rights reserved.

Enzyme-aided extraction of lycopene from high-pigment tomato cultivars by supercritical carbon dioxide.

PubMed

Lenucci, Marcello Salvatore; De Caroli, Monica; Marrese, Pier Paolo; Iurlaro, Andrea; Rescio, Leonardo; Böhm, Volker; Dalessandro, Giuseppe; Piro, Gabriella

2015-03-01

This work reports a novel enzyme-assisted process for lycopene concentration into a freeze-dried tomato matrix and describes the results of laboratory scale lycopene supercritical CO2 (SC-CO2) extractions carried out with untreated (control) and enzyme-digested matrices. The combined use of food-grade commercial plant cell-wall glycosidases (Celluclast/Novozyme plus Viscozyme) allows to increase lycopene (∼153%) and lipid (∼137%) concentration in the matrix and rises substrate load onto the extraction vessel (∼46%) compared to the control. The addition of an oleaginous co-matrix (hazelnut seeds) to the tomato matrix (1:1 by weight) increases CO2 diffusion through the highly dense enzyme-treated matrix bed and provides lipids that are co-extracted increasing lycopene yield. Under the same operative conditions (50 MPa, 86 °C, 4 mL min(-1) SC-CO2 flow) extraction yield from control and Celluclast/Novozyme+Viscozyme-treated tomato matrix/co-matrix mixtures was similar, exceeding 75% after 4.5h of extraction. However, the total extracted lycopene was ∼3 times higher in enzyme-treated matrix than control. Copyright © 2014 Elsevier Ltd. All rights reserved.

DICER-ARGONAUTE2 Complex in Continuous Fluorogenic Assays of RNA Interference Enzymes

PubMed Central

Bernard, Mark A.; Wang, Leyu; Tachado, Souvenir D.

2015-01-01

Mechanistic studies of RNA processing in the RNA-Induced Silencing Complex (RISC) have been hindered by lack of methods for continuous monitoring of enzymatic activity. “Quencherless” fluorogenic substrates of RNAi enzymes enable continuous monitoring of enzymatic reactions for detailed kinetics studies. Recombinant RISC enzymes cleave the fluorogenic substrates targeting human thymidylate synthase (TYMS) and hypoxia-inducible factor 1-α subunit (HIF1A). Using fluorogenic dsRNA DICER substrates and fluorogenic siRNA, DICER+ARGONAUTE2 mixtures exhibit synergistic enzymatic activity relative to either enzyme alone, and addition of TRBP does not enhance the apparent activity. Titration of AGO2 and DICER in enzyme assays suggests that AGO2 and DICER form a functional high-affinity complex in equimolar ratio. DICER and DICER+AGO2 exhibit Michaelis-Menten kinetics with DICER substrates. However, AGO2 cannot process the fluorogenic siRNA without DICER enzyme, suggesting that AGO2 cannot self-load siRNA into its active site. The DICER+AGO2 combination processes the fluorogenic siRNA substrate (K m=74 nM) with substrate inhibition kinetics (K i=105 nM), demonstrating experimentally that siRNA binds two different sites that affect Dicing and AGO2-loading reactions in RISC. This result suggests that siRNA (product of DICER) bound in the active site of DICER may undergo direct transfer (as AGO2 substrate) to the active site of AGO2 in the DICER+AGO2 complex. Competitive substrate assays indicate that DICER+AGO2 cleavage of fluorogenic siRNA is specific, since unlabeled siRNA and DICER substrates serve as competing substrates that cause a concentration-dependent decrease in fluorescent rates. Competitive substrate assays of a series of DICER substrates in vitro were correlated with cell-based assays of HIF1A mRNA knockdown (log-log slope=0.29), suggesting that improved DICER substrate designs with 10-fold greater processing by the DICER+AGO2 complex can provide a strong (~2800-fold) improvement in potency for mRNA knockdown. This study lays the foundation of a systematic biochemical approach to optimize nucleic acid-based therapeutics for Dicing and ARGONAUTE2-loading for improving efficacy. PMID:25793518

Techno-economic potential of bioethanol from bamboo in China

PubMed Central

2013-01-01

Background Bamboo is potentially an interesting feedstock for advanced bioethanol production in China due to its natural abundance, rapid growth, perennial nature and low management requirements. Liquid hot water (LHW) pretreatment was selected as a promising technology to enhance sugar release from bamboo lignocellulose whilst keeping economic and environmental costs to a minimum. The present research was conducted to assess: 1) by how much LHW pretreatment can enhance sugar yields in bamboo, and 2) whether this process has the potential to be economically feasible for biofuel use at the commercial scale. Pretreatments were performed at temperatures of 170-190°C for 10–30 minutes, followed by enzymatic saccharification with a commercial enzyme cocktail at various loadings. These data were then used as inputs to a techno-economic model using AspenPlus™ to determine the production cost of bioethanol from bamboo in China. Results At the selected LHW pretreatment of 190°C for 10 minutes, 69% of the initial sugars were released under a standardised enzyme loading; this varied between 59-76% when 10–140 FPU/g glucan of commercial enzyme Cellic CTec2 was applied. Although the lowest enzyme loading yielded the least amount of bioethanol, the techno-economic evaluation revealed it to be the most economically viable scenario with a production cost of $0.484 per litre (with tax exemption and a $0.16/litre subsidy). The supply-chain analysis demonstrated that bioethanol could be economically competitive with petrol at the pump at enzyme loadings up to 60 FPU/g glucan. However, in a prospective scenario with reduced government support, this enzyme loading threshold would be reduced to 30 FPU/g glucan. Conclusions Bioethanol from bamboo is shown to be both technically and economically feasible, as well as competitive with petrol in China. Alternative approaches to reduce bioethanol production costs are still needed however, to ensure its competitiveness in a possible future scenario where neither tax exemptions nor subsidies are granted to producers. These measures may include improving sugar release with more effective pretreatments and reduced enzyme usage, accessing low cost bamboo feedstock or selecting feedstocks with higher/more accessible cellulose. PMID:24286490

Synthesis of PEGylated polyglutamic acid peptide dendrimer and its application in dissolving thrombus.

PubMed

Zhang, Shao-Fei; Gao, Chunmei; Lü, Shaoyu; He, Jiujun; Liu, Mingzhu; Wu, Can; Liu, Yijing; Zhang, Xinyu; Liu, Zhen

2017-11-01

Nattokinase (NK) has been used as a new generation thrombolytic drug, due to its high safety, low cost and low side effects. However, it is sensitive to external environment and may lose the enzyme activity easily. Peptide dendrimer possesses functional groups on its surface, adjustable sizes, biodegradability, biocompatibility, and low toxicity, which could be used as ideal carrier for drug protection and delivery. Demonstrated for the first time in this paper, a PEGylated dendrimer (G n -PEG-G n ) composed of polyglutamic acid is designed and synthesized as delivery platform of NK for thrombus treatment. A panel of PEGylated dendrimers with three different generations of 2, 3, 4 was prepared to investigate the effect of dendrimer architecture on the properties and therapeutic efficacy of the resultant NK-loaded delivery systems in terms of the morphology, dimension and enzyme activity. The results demonstrated that the NK-loaded G 3 -PEG-G 3 (G 3 -PEG-G 3 /NK ratio of 6/1), of all the formulations, displayed the optimal enzyme activity for dissolving thrombus in vitro, thus offering great potential for the treatment of thrombus. Copyright © 2017 Elsevier B.V. All rights reserved.

Portable Enzyme-Paper Biosensors Based on Redox-Active CeO2 Nanoparticles.

PubMed

Karimi, A; Othman, A; Andreescu, S

2016-01-01

Portable, nanoparticle (NP)-enhanced enzyme sensors have emerged as powerful devices for qualitative and quantitative analysis of a variety of analytes for biomedicine, environmental applications, and pharmaceutical fields. This chapter describes a method for the fabrication of a portable, paper-based, inexpensive, robust enzyme biosensor for the detection of substrates of oxidase enzymes. The method utilizes redox-active NPs of cerium oxide (CeO2) as a sensing platform which produces color in response to H2O2 generated by the action of oxidase enzymes on their corresponding substrates. This avoids the use of peroxidases which are routinely used in conjunction with glucose oxidase. The CeO2 particles serve dual roles, as high surface area supports to anchor high loadings of the enzyme as well as a color generation reagent, and the particles are recycled multiple times for the reuse of the biosensor. These sensors are small, light, disposable, inexpensive, and they can be mass produced by standard, low-cost printing methods. All reagents needed for the analysis are embedded within the paper matrix, and sensors stored over extended periods of time without performance loss. This novel sensor is a general platform for the in-field detection of analytes that are substrates for oxidase enzymes in clinical, food, and environmental samples. © 2016 Elsevier Inc. All rights reserved.

Lecithin/chitosan controlled release nanopreparations of tamoxifen citrate: loading, enzyme-trigger release and cell uptake.

PubMed

Barbieri, Stefano; Sonvico, Fabio; Como, Caterina; Colombo, Gaia; Zani, Franca; Buttini, Francesca; Bettini, Ruggero; Rossi, Alessandra; Colombo, Paolo

2013-05-10

Tamoxifen citrate (TAM), an anticancer drug with amphiphilic properties, was loaded in lecithin/chitosan nanoparticles (LCN) with a view to oral administration. The influence of tamoxifen loading on the physico-chemical properties of nanoparticles was studied. Size, surface charge and morphological properties of tamoxifen-loaded nanoparticles (LCN-TAM) were assessed. The increase in the tamoxifen amount in the LCN-TAM preparation up to 60 mg/100 ml maintained the positive zeta potential value of about +45 mV. A statistically significant decrease in particle size was observed for TAM amounts between 5 and 20mg. A strong influence of loaded tamoxifen on the structure of lecithin/chitosan nanoparticles was observed, supported by the quantification of free chitosan and morphological analysis. A loading of tamoxifen in nanoparticles of around 19% was obtained. The release of the drug from the LCN-TAM colloidal dispersion was measured, showing that tamoxifen citrate was released very slowly in simulated gastro-intestinal fluids without enzymes. When enzymes able to dismantle the nanoparticle structure were added to the dissolution medium, drug release was triggered and continued in a prolonged manner. Tamoxifen-loaded nanoparticles showed cytotoxicity towards MCF-7 cells comparable to that obtained with tamoxifen citrate solution, but the rate of this toxic effect was dependent on drug release. Caco-2 cells, used as a model of the intestinal epithelium, were shown to take up the TAM loaded nanoparticles extensively. Copyright © 2013 Elsevier B.V. All rights reserved.

Fungal secretomes enhance sugar beet pulp hydrolysis.

PubMed

Kracher, Daniel; Oros, Damir; Yao, Wanying; Preims, Marita; Rezic, Iva; Haltrich, Dietmar; Rezic, Tonci; Ludwig, Roland

2014-04-01

The recalcitrance of lignocellulose makes enzymatic hydrolysis of plant biomass for the production of second generation biofuels a major challenge. This work investigates an efficient and economic approach for the enzymatic hydrolysis of sugar beet pulp (SBP), which is a difficult to degrade, hemicellulose-rich by-product of the table sugar industry. Three fungal strains were grown on different substrates and the production of various extracellular hydrolytic and oxidative enzymes involved in pectin, hemicellulose, and cellulose breakdown were monitored. In a second step, the ability of the culture supernatants to hydrolyze thermally pretreated SBP was tested in batch experiments. The supernatant of Sclerotium rolfsii, a soil-borne facultative plant pathogen, was found to have the highest hydrolytic activity on SBP and was selected for further hydrolyzation experiments. A low enzyme load of 0.2 mg g(-1) protein from the culture supernatant was sufficient to hydrolyze a large fraction of the pectin and hemicelluloses present in SBP. The addition of Trichoderma reesei cellulase (1-17.5 mg g(-1) SBP) resulted in almost complete hydrolyzation of cellulose. It was found that the combination of pectinolytic, hemicellulolytic, and cellulolytic activities works synergistically on the complex SBP composite, and a combination of these hydrolytic enzymes is required to achieve a high degree of enzymatic SBP hydrolysis with a low enzyme load. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Statistical optimization of arsenic biosorption by microbial enzyme via Ca-alginate beads.

PubMed

Banerjee, Suchetana; Banerjee, Anindita; Sarkar, Priyabrata

2018-04-16

Bioremediation of arsenic using green technology via microbial enzymes has attracted scientists due to its simplicity and cost effectiveness. Statistical optimization of arsenate bioremediation was conducted by the enzyme arsenate reductase extracted from arsenic tolerant bacterium Pseudomonas alcaligenes. Response surface methodology based on Box-Behnken design matrix was performed to determine the optimal operational conditions of a multivariable system and their interactive effects on the bioremediation process. The highest biosorptive activity of 96.2 µg gm -1 of beads was achieved under optimized conditions (pH = 7.0; As (V) concentration = 1000 ppb; time = 2 h). SEM analysis showed the morphological changes on the surface of enzyme immobilized gluteraldehyde crosslinked Ca-alginate beads. The immobilized enzyme retained its activity for 8 cycles. ANOVA with a high correlation coefficient (R 2 > 0.99) and lower "Prob > F"value (<0.0001) corroborated the second-order polynomial model for the biosorption process. This study on the adsorptive removal of As (V) by enzyme-loaded biosorbent revealed a possible way of its application in large scale treatment of As (V)-contaminated water bodies.

Moving college students to a better understanding of substrate specificity of enzymes through utilizing multimedia pre-training and an interactive enzyme model

NASA Astrophysics Data System (ADS)

Saleh, Mounir R.

Scientists' progress in understanding enzyme specificity uncovered a complex natural phenomenon. However, not all of the currently available biology textbooks seem to be up to date on this progress. Students' understanding of how enzymes work is a core requirement in biochemistry and biology tertiary education. Nevertheless, current pre-college science education does not provide students with enough biochemical background to enable them to understand complex material such as this. To bridge this gap, a multimedia pre-training presentation was prepared to fuel the learner's prior knowledge with discrete facts necessary to understand the presented concept. This treatment is also known to manage intrinsic cognitive load during the learning process. An interactive instructional enzyme model was also built to motivate students to learn about substrate specificity of enzymes. Upon testing the effect of this combined treatment on 111 college students, desirable learning outcomes were found in terms of cognitive load, motivation, and achievement. The multimedia pre-training group reported significantly less intrinsic cognitive load, higher motivation, and demonstrated higher transfer performance than the control and post-training groups. In this study, a statistical mediation model is also proposed to explain how cognitive load and motivation work in concert to foster learning from multimedia pre-training. This type of research goes beyond simple forms of "what works" to a deeper understanding of "how it works", thus enabling informed decisions for multimedia instructional design. Multimedia learning plays multiple roles in science education. Therefore, science learners would be some of the first to benefit from improving multimedia instructional design. Accordingly, complex scientific phenomena can be introduced to college students in a motivating, informative, and cognitively efficient learning environment.

Biofunctionalization of aqueous dispersed, alumina membrane-templated polymer nanorods for use in enzymatic chemiluminescence assays.

PubMed

Mark, Sonny S; Stolper, Samuel I; Baratti, Carla; Park, Jason Y; Kricka, Larry J

2008-09-01

The noncovalent immobilization of alkaline phosphatase (ALP) onto aqueous dispersed nylon 6 nanorods ( approximately 310 nm mean diameter; approximately 6 microm mean length) prepared by anodic aluminum oxide (AAO) membrane templating was studied. Using multi-stacked layer-by-layer (LBL) assembly with the cationic quaternary ammonium polymer Sapphire II , the amount of ALP enzyme loaded onto the polymer nanostructures was found to be 115+/-7 microg mg(-1) nanorod. The biofunctionalized nanorods were also characterized for their chemiluminescent activity with the dioxetane substrate, CSPD . The results indicate that the kinetic parameters, K(m) and V(max), for the catalytic activity of the nanostructure-bound ALP enzyme are different from those of soluble ('free') ALP. While the K(m) value was measured to be 156 microM for free ALP, the apparent K(m) value determined for the LBL-immobilized ALP is approximately 20% lower (122 microM). Furthermore, despite the relatively high enzyme loading capacity of the nanorods, the specific activity of the bound ALP enzyme was found to be almost nine times lower than that measured for free ALP. Finally, additional experiments revealed that the catalytic activities of both free ALP and nanorod-conjugated ALP are affected similarly by changes in pH, with optimal performance levels occurring under conditions of pH 9.5. To the best of our knowledge, this study represents the first report examining the preparation of aqueous dispersed, AAO-templated polymer nanorods for potential application as enzyme scaffolds in chemiluminescent-based assay systems.

European Congress on Biotechnology (4th) Held in Amsterdam, The Netherlands, on June 1987

DTIC Science & Technology

1988-02-19

car be used and thus higher Nmaraso Fumarate enzyme loadings can be achieved with a Glucose isomerase High- fructose corn syrup large effectiveness...is only after most of the the following reactions: glucose + oxy- glucose has been metabolized that aerobic gen + glucose oxidasa + water - gluconic...are obtained by mixing water solutions of two water -soluble * Biocatalysis polymers. Both phases have a high water * Animal cell cultures content and do

Process evaluation of enzymatic hydrolysis with filtrate recycle for the production of high concentration sugars.

PubMed

Xue, Ying; Rusli, Jannov; Chang, Hou-Min; Phillips, Richard; Jameel, Hasan

2012-02-01

Process simulation and lab trials were carried out to demonstrate and confirm the efficiency of the concept that recycling hydrolysate at low total solid enzymatic hydrolysis is one of the options to increase the sugar concentration without mixing problems. Higher sugar concentration can reduce the capital cost for fermentation and distillation because of smaller retention volume. Meanwhile, operation cost will also decrease for less operating volume and less energy required for distillation. With the computer simulation, time and efforts can be saved to achieve the steady state of recycling process, which is the scenario for industrial production. This paper, to the best of our knowledge, is the first paper discussing steady-state saccharification with recycling of the filtrate form enzymatic hydrolysis to increase sugar concentration. Recycled enzymes in the filtrate (15-30% of the original enzyme loading) resulted in 5-10% higher carbohydrate conversion compared to the case in which recycled enzymes were denatured. The recycled hydrolysate yielded 10% higher carbohydrate conversion compared to pure sugar simulated hydrolysate at the same enzyme loading, which indicated hydrolysis by-products could boost enzymatic hydrolysis. The high sugar concentration (pure sugar simulated) showed inhibition effect, since about 15% decrease in carbohydrate conversion was observed compared with the case with no sugar added. The overall effect of hydrolysate recycling at WinGEMS simulated steady-state conditions with 5% total solids was increasing the sugar concentration from 35 to 141 g/l, while the carbohydrate conversion was 2% higher for recycling at steady state (87%) compared with no recycling strategy (85%). Ten percent and 15% total solid processes were also evaluated in this study.

Encapsulation and immobilization of papain in electrospun nanofibrous membranes of PVA cross-linked with glutaraldehyde vapor.

PubMed

Moreno-Cortez, Iván E; Romero-García, Jorge; González-González, Virgilio; García-Gutierrez, Domingo I; Garza-Navarro, Marco A; Cruz-Silva, Rodolfo

2015-01-01

In this paper, papain enzyme (E.C. 3.4.22.2, 1.6 U/mg) was successfully immobilized in poly(vinyl alcohol) (PVA) nanofibers prepared by electrospinning. The morphology of the electrospun nanofibers was characterized by scanning electron microscopy (SEM) and the diameter distribution was in the range of 80 to 170 nm. The presence of the enzyme within the PVA nanofibers was confirmed by infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS) and energy dispersive X-ray spectroscopy (EDXS) analyses. The maximum catalytic activity was reached when the enzyme loading was 13%. The immobilization of papain in the nanofiber membrane was achieved by chemical crosslinking with a glutaraldehyde vapor treatment (GAvt). The catalytic activity of the immobilized papain was 88% with respect to the free enzyme. The crosslinking time by GAvt to immobilize the enzyme onto the nanofiber mat was 24h, and the enzyme retained its catalytic activity after six cycles. The crosslinked samples maintained 40% of their initial activity after being stored for 14 days. PVA electrospun nanofibers are excellent matrices for the immobilization of enzymes due to their high surface area and their nanoporous structure. Copyright © 2015. Published by Elsevier B.V.

Use of Artificial Neural Networks to Examine Parameters Affecting the Immobilization of Streptokinase in Chitosan

PubMed Central

Modaresi, Seyed Mohamad Sadegh; Faramarzi, Mohammad Ali; Soltani, Arash; Baharifar, Hadi; Amani, Amir

2014-01-01

Streptokinase is a potent fibrinolytic agent which is widely used in treatment of deep vein thrombosis (DVT), pulmonary embolism (PE) and acute myocardial infarction (MI). Major limitation of this enzyme is its short biological half-life in the blood stream. Our previous report showed that complexing streptokinase with chitosan could be a solution to overcome this limitation. The aim of this research was to establish an artificial neural networks (ANNs) model for identifying main factors influencing the loading efficiency of streptokinase, as an essential parameter determining efficacy of the enzyme. Three variables, namely, chitosan concentration, buffer pH and enzyme concentration were considered as input values and the loading efficiency was used as output. Subsequently, the experimental data were modeled and the model was validated against a set of unseen data. The developed model indicated chitosan concentration as probably the most important factor, having reverse effect on the loading efficiency. PMID:25587327

Effects of enzyme loading and β-glucosidase supplementation on enzymatic hydrolysis of switchgrass processed by leading pretreatment technologies.

PubMed

Pallapolu, Venkata Ramesh; Lee, Y Y; Garlock, Rebecca J; Balan, Venkatesh; Dale, Bruce E; Kim, Youngmi; Mosier, Nathan S; Ladisch, Michael R; Falls, Matthew; Holtzapple, Mark T; Sierra-Ramirez, Rocio; Shi, Jian; Ebrik, Mirvat A; Redmond, Tim; Yang, Bin; Wyman, Charles E; Donohoe, Bryon S; Vinzant, Todd B; Elander, Richard T; Hames, Bonnie; Thomas, Steve; Warner, Ryan E

2011-12-01

The objective of this work is to investigate the effects of cellulase loading and β-glucosidase supplementation on enzymatic hydrolysis of pretreated Dacotah switchgrass. To assess the difference among various pretreatment methods, the profiles of sugars and intermediates were determined for differently treated substrates. For all pretreatments, 72 h glucan/xylan digestibilities increased sharply with enzyme loading up to 25mg protein/g-glucan, after which the response varied depending on the pretreatment method. For a fixed level of enzyme loading, dilute sulfuric acid (DA), SO(2), and Lime pretreatments exhibited higher digestibility than the soaking in aqueous ammonia (SAA) and ammonia fiber expansion (AFEX). Supplementation of Novozyme-188 to Spezyme-CP improved the 72 h glucan digestibility only for the SAA treated samples. The effect of β-glucosidase supplementation was discernible only at the early phase of hydrolysis where accumulation of cellobiose and oligomers is significant. Addition of β-glucosidase increased the xylan digestibility of alkaline treated samples due to the β-xylosidase activity present in Novozyme-188. Copyright © 2011 Elsevier Ltd. All rights reserved.

Nanobio interfaces: charge control of enzyme/inorganic interfaces for advanced biocatalysis.

PubMed

Deshapriya, Inoka K; Kumar, Challa V

2013-11-19

Specific approaches to the rational design of nanobio interfaces for enzyme and protein binding to nanomaterials are vital for engineering advanced, functional nanobiomaterials for biocatalysis, sensing, and biomedical applications. This feature article presents an overview of our recent discoveries on structural, functional, and mechanistic details of how enzymes interact with inorganic nanomaterials and how they can be controlled in a systematic manner using α-Zr(IV)phosphate (α-ZrP) as a model system. The interactions of a number of enzymes having a wide array of surface charges, sizes, and functional groups are investigated. Interactions are carefully controlled to screen unfavorable repulsions and enhance favorable interactions for high affinity, structure retention, and activity preservation. In specific cases, catalytic activities and substrate selectivities are improved over those of the pristine enzymes, and two examples of high activity near the boiling point of water have been demonstrated. Isothermal titration calorimetric studies indicated that enzyme binding is coupled to ion sequestration or release to or from the nanobio interface, and binding is controlled in a rational manner. We learned that (1) bound enzyme stabilities are improved by lowering the entropy of the denatured state; (2) maximal loadings are obtained by matching charge footprints of the enzyme and the nanomaterial surface; (3) binding affinities are improved by ion sequestration at the nanobio interface; and (4) maximal enzyme structure retention is obtained by biophilizing the nanobio interface with protein glues. The chemical and physical manipulations of the nanobio interface are significant not only for understanding the complex behaviors of enzymes at biological interfaces but also for desiging better functional nanobiomaterials for a wide variety of practical applications.

Optimal immobilization of β-galactosidase onto κ-carrageenan gel beads using response surface methodology and its applications.

PubMed

Elnashar, Magdy M; Awad, Ghada E; Hassan, Mohamed E; Mohy Eldin, Mohamed S; Haroun, Bakry M; El-Diwany, Ahmed I

2014-01-01

β-Galactosidase (β-gal) was immobilized by covalent binding on novel κ-carrageenan gel beads activated by two-step method; the gel beads were soaked in polyethyleneimine followed by glutaraldehyde. 2(2) full-factorial central composite experiment designs were employed to optimize the conditions for the maximum enzyme loading efficiency. 11.443 U of enzyme/g gel beads was achieved by soaking 40 units of enzyme with the gel beads for eight hours. Immobilization process increased the pH from 4.5 to 5.5 and operational temperature from 50 to 55 °C compared to the free enzyme. The apparent K(m) after immobilization was 61.6 mM compared to 22.9 mM for free enzyme. Maximum velocity Vmax was 131.2 μ mol · min(-1) while it was 177.1 μ mol · min(-1) for free enzyme. The full conversion experiment showed that the immobilized enzyme form is active as that of the free enzyme as both of them reached their maximum 100% relative hydrolysis at 4 h. The reusability test proved the durability of the κ-carrageenan beads loaded with β -galactosidase for 20 cycles with retention of 60% of the immobilized enzyme activity to be more convenient for industrial uses.

Sensitive detection of respiratory syncytial virus based on a dual signal amplified plasmonic enzyme-linked immunosorbent assay.

PubMed

Zhan, Lei; Wu, Wen Bi; Yang, Lin; Huang, Cheng Zhi

2017-04-15

The timely detection of infectious pathogen is critical in clinical early diagnosis and treatment of infectious diseases. Plasmonic enzyme-linked immunosorbent assay (ELISA), by means of enzyme-mediated growth or aggregation of AuNPs, has received considerable attention because it allows a naked-eye detection of target in very low numbers. In this work, a dual-signal amplified plasmonic ELISA combined the high loading capacity of magnetic beads with the establishing stimulation effect of zinc ion has been developed to detect RSV as a model pathogen based on alkaline phosphatase-triggered dispersion of aggregated AuNPs. In ideal conditions, the proposed immunoassay can conveniently distinguish the concentration of RSV in a range of 0.1-30 pg/mL. In addition, the limit of detection of RSV of this immunoassay exceeds that of conventional ELISA by about 50 times. The high sensitivity makes this approach a good alternative to existing colorimetric immunoassays for pathogen detection. Copyright © 2017 Elsevier B.V. All rights reserved.

Effects of nutrient load on microbial activities within a seagrass-dominated ecosystem: Implications of changes in seagrass blue carbon.

PubMed

Liu, Songlin; Jiang, Zhijian; Wu, Yunchao; Zhang, Jingping; Arbi, Iman; Ye, Feng; Huang, Xiaoping; Macreadie, Peter Ian

2017-04-15

Nutrient loading is a leading cause of global seagrass decline, triggering shifts from seagrass- to macroalgal-dominance. Within seagrass meadows of Xincun Bay (South China Sea), we found that nutrient loading (due to fish farming) increased sediment microbial biomass and extracellular enzyme activity associated with carbon cycling (polyphenol oxidase, invertase and cellulase), with a corresponding decrease in percent sediment organic carbon (SOC), suggesting that nutrients primed microorganism and stimulated SOC remineralization. Surpisingly, however, the relative contribution of seagrass-derived carbon to bacteria (δ 13 C bacteria ) increased with nutrient loading, despite popular theory being that microbes switch to consuming macroalgae which are assumed to provide a more labile carbon source. Organic carbon sources of fungi were unaffected by nutrient loading. Overall, this study suggests that nutrient loading changes the relative contribution of seagrass and algal sources to SOC pools, boosting sediment microbial biomass and extracellular enzyme activity, thereby possibly changing seagrass blue carbon. Copyright © 2017 Elsevier Ltd. All rights reserved.

Novel epoxy activated hydrogels for solving lactose intolerance.

PubMed

Elnashar, Magdy M M; Hassan, Mohamed E

2014-01-01

"Lactose intolerance" is a medical problem for almost 70% of the world population. Milk and dairy products contain 5-10% w/v lactose. Hydrolysis of lactose by immobilized lactase is an industrial solution. In this work, we succeeded to increase the lactase loading capacity to more than 3-fold to 36.3 U/g gel using epoxy activated hydrogels compared to 11 U/g gel using aldehyde activated carrageenan. The hydrogel's mode of interaction was proven by FTIR, DSC, and TGA. The high activity of the epoxy group was regarded to its ability to attach to the enzyme's -SH, -NH, and -OH groups, whereas the aldehyde group could only bind to the enzyme's -NH2 group. The optimum conditions for immobilization such as epoxy chain length and enzyme concentration have been studied. Furthermore, the optimum enzyme conditions were also deliberated and showed better stability for the immobilized enzyme and the Michaelis constants, K m and V max, were doubled. Results revealed also that both free and immobilized enzymes reached their maximum rate of lactose conversion after 2 h, albeit, the aldehyde activated hydrogel could only reach 63% of the free enzyme. In brief, the epoxy activated hydrogels are more efficient in immobilizing more enzymes than the aldehyde activated hydrogel.

Using an artificial neural network to predict the optimal conditions for enzymatic hydrolysis of apple pomace.

PubMed

Gama, Repson; Van Dyk, J Susan; Burton, Mike H; Pletschke, Brett I

2017-06-01

The enzymatic degradation of lignocellulosic biomass such as apple pomace is a complex process influenced by a number of hydrolysis conditions. Predicting optimal conditions, including enzyme and substrate concentration, temperature and pH can improve conversion efficiency. In this study, the production of sugar monomers from apple pomace using commercial enzyme preparations, Celluclast 1.5L, Viscozyme L and Novozyme 188 was investigated. A limited number of experiments were carried out and then analysed using an artificial neural network (ANN) to model the enzymatic hydrolysis process. The ANN was used to simulate the enzymatic hydrolysis process for a range of input variables and the optimal conditions were successfully selected as was indicated by the R 2 value of 0.99 and a small MSE value. The inputs for the ANN were substrate loading, enzyme loading, temperature, initial pH and a combination of these parameters, while release profiles of glucose and reducing sugars were the outputs. Enzyme loadings of 0.5 and 0.2 mg/g substrate and a substrate loading of 30% were optimal for glucose and reducing sugar release from apple pomace, respectively, resulting in concentrations of 6.5 g/L glucose and 28.9 g/L reducing sugars. Apple pomace hydrolysis can be successfully carried out based on the predicted optimal conditions from the ANN.

Site-directed decapsulation of bolaamphiphilic vesicles with enzymatic cleavable surface groups.

PubMed

Popov, Mary; Grinberg, Sarina; Linder, Charles; Waner, Tal; Levi-Hevroni, Bosmat; Deckelbaum, Richard J; Heldman, Eliahu

2012-06-10

Stable nano-sized vesicles with a monolayer encapsulating membrane were prepared from novel bolaamphiphiles with choline ester head groups. The head groups were covalently bound to the alkyl chain of the bolaamphiphiles either via the nitrogen atom of the choline moiety, or via the choline ester's methyl group. Both types of bolaamphiphiles competed with acetylthiocholine for binding to acetylcholine esterase (AChE), yet, only the choline ester head groups bound to the alkyl chain via the nitrogen atom of the choline moiety were hydrolyzed by the enzyme. Likewise, only vesicles composed of bolaamphiphiles with head groups that were hydrolyzed by AChE released their encapsulated material upon exposure to the enzyme. Injection of carboxyfluorescein (CF)-loaded vesicles with cleavable choline ester head groups into mice resulted in the accumulation of CF in tissues that express high AChE activity, including the brain. By comparison, when vesicles with choline ester head groups that are not hydrolyzed by AChE were injected into mice, there was no accumulation of CF in tissues that highly express the enzyme. These results imply that bolaamphiphilic vesicles with surface groups that are substrates to enzymes which are highly expressed in target organs may potentially be used as a drug delivery system with controlled site-directed drug release. Copyright © 2011 Elsevier B.V. All rights reserved.

Evolution of enzymes in a series is driven by dissimilar functional demands.

PubMed

Salvador, Armindo; Savageau, Michael A

2006-02-14

That distinct enzyme activities in an unbranched metabolic pathway are evolutionarily tuned to a single functional requirement is a pervasive assumption. Here we test this assumption by examining the activities of two consecutively acting enzymes in human erythrocytes with an approach to quantitative evolutionary design that avoids the above-mentioned assumption. We previously found that avoidance of NADPH depletion during the pulses of oxidative load to which erythrocytes are normally exposed is the main functional requirement mediating selection for high glucose-6-phosphate dehydrogenase activity. In the present study, we find that, in contrast, the maintenance of oxidized glutathione at low concentrations is the main functional requirement mediating selection for high glutathione reductase activity. The results in this case show that, contrary to the assumption of a single functional requirement, natural selection for the normal activities of the distinct enzymes in the pathway is mediated by different requirements. On the other hand, the results agree with the more general principles that underlie our approach. Namely, that (i) the values of biochemical parameters evolve so as to fulfill the various performance requirements that are relevant to achieve high fitness, and (ii) these performance requirements can be inferred from quantitative systems theory considerations, informed by knowledge of specific aspects of the biochemistry, physiology, genetics, and ecology of the organism.

A Factorial Analysis Study on Enzymatic Hydrolysis of Fiber Pressed Oil Palm Frond for Bioethanol Production

NASA Astrophysics Data System (ADS)

Hashim, F. S.; Yussof, H. W.; Zahari, M. A. K. M.; Illias, R. M.; Rahman, R. A.

2016-03-01

Different technologies have been developed to for the conversion of lignocellulosic biomass to suitable fermentation substrates for bioethanol production. The enzymatic conversion of cellulose seems to be the most promising technology as it is highly specific and does not produce substantial amounts of unwanted byproducts. The effects of agitation speed, enzyme loading, temperature, pH and reaction time on the conversion of glucose from fiber pressed oil palm frond (FPOPF) for bioethanol production were screened by statistical analysis using response surface methodology (RSM). A half fraction two-level factorial analysis with five factors was selected for the experimental design to determine the best enzymatic conditions that produce maximum amount of glucose. FPOPF was pre-treated with alkaline prior to enzymatic hydrolysis. The enzymatic hydrolysis was performed using a commercial enzyme Cellic CTec2. From this study, the highest yield of glucose concentration was 9.736 g/L at 72 hours reaction time at 35 °C, pH 5.6, and 1.5% (w/v) of enzyme loading. The model obtained was significant with p-value <0.0001. It is suggested that this model had a maximum point which is likely to be the optimum point and possible for the optimization process.

Highly sensitive single-fibril erosion assay demonstrates mechanochemical switch in native collagen fibrils

PubMed Central

Flynn, Brendan P.; Tilburey, Graham E.

2013-01-01

It has been established that the enzyme susceptibility of collagen, the predominant load-bearing protein in vertebrates, is altered by applied tension. However, whether tensile force increases or decreases the susceptibility to enzyme is a matter of contention. It is critical to establish a definitive understanding of the direction and magnitude of the force versus catalysis rate (kC) relationship if we are to properly interpret connective tissue development, growth, remodeling, repair, and degeneration. In this investigation, we examine collagen/enzyme mechanochemistry at the smallest scale structurally relevant to connective tissue: the native collagen fibril. A single-fibril mechanochemical erosion assay with nN force resolution was developed which permits detection of the loss of a few layers of monomer from the fibril surface. Native type I fibrils (bovine) held at three levels of tension were exposed to Clostridium histolyticum collagenase A. Fibrils held at zero-load failed rapidly and consistently (20 min) while fibrils at 1.8 pN/monomer failed more slowly (35–55 min). Strikingly, fibrils at 23.9 pN/monomer did not exhibit detectable degradation. The extracted force versus kC data were combined with previous single-molecule results to produce a “master curve” which suggests that collagen degradation is governed by an extremely sensitive mechanochemical switch. PMID:22584606

A novel aqueous micellar two-phase system composed of surfactant and sorbitol for purification of pectinase enzyme from Psidium guajava and recycling phase components.

PubMed

Amid, Mehrnoush; Murshid, Fara Syazana; Manap, Mohd Yazid; Hussin, Muhaini

2015-01-01

A novel aqueous two-phase system composed of a surfactant and sorbitol was employed for the first time to purify pectinase from Psidium guajava. The influences of different parameters, including the type and concentration of the surfactant and the concentration and composition of the surfactant/sorbitol ratio, on the partitioning behavior and recovery of pectinase were investigated. Moreover, the effects of system pH and the crude load on purification fold and the yield of purified pectinase were studied. The experimental results indicated that the pectinase was partitioned into surfactant-rich top phase, and the impurities were partitioned into the sorbitol-rich bottom phase with the novel method involving an ATPS composed of 26% (w/w) Triton X-100 and 23% (w/w) sorbitol at 54.2% of the TLL crude load of 20% (w/w) at pH 6.0. The enzyme was successfully recovered by this method with a high purification factor of 15.2 and a yield of 98.3%, whereas the phase components were also recovered and recycled at rates above 96%. This study demonstrated that this novel ATPS method can be used as an efficient and economical alternative to the traditional ATPS for the purification and recovery of the valuable enzyme.

Deficiency of cellulase activity measurements for enzyme evaluation.

PubMed

Pryor, Scott W; Nahar, Nurun

2010-11-01

Switchgrass was used as a model feedstock to determine the influence of pretreatment conditions and biomass quality on enzymatic hydrolysis using different enzyme products. Dilute sulfuric acid and soaking in aqueous ammonia pretreatments were used to produce biomass with varied levels of hemicellulose and lignin sheathing. Pretreated switchgrass solids were tested with simple enzymatic hydrolysis and simultaneous saccharification and fermentation (SSF) with three commercial enzyme products: Accellerase 1000 (Genencor), Spezyme CP (Genencor)/Novozyme 188 (Novozymes), and Celluclast/Novozyme 188 (Novozymes). Enzymes were loaded on a common activity basis (FPU/g cellulose and CBU/g cellulose). Despite identical enzyme loadings, glucose yields were significantly different for both acid and alkaline pretreatments but differences diminished as hydrolysis progressed for acid-pretreated biomass. Cellobiose concentrations in Accellerase treatments indicated an initial beta-glucosidase limitation that became less significant over time. SSF experiments showed that differences in glucose and ethanol yields could not be attributed to enzyme product inhibition. Yield discrepancies of glucose or ethanol in acid pretreatment, alkaline pretreatment, and acid pretreatment/SSF were as much as 15%, 19%, and 5%. These results indicate that standardized protocols for measuring enzyme activity may not be adequate for assessing activity using pretreated biomass substrates.

Inulin hydrolysis by inulinase immobilized covalently on magnetic nanoparticles prepared with wheat gluten hydrolysates.

PubMed

Torabizadeh, Homa; Mahmoudi, Asieh

2018-03-01

Inulinase can produce a high amount of fructose syrup from inulin in a one-step enzymatic process. Inulinase from Aspergillus niger was immobilized covalently on Fe 3 O 4 magnetic nanoparticles functionalized with wheat gluten hydrolysates (WGHs). Wheat gluten was enzymatically hydrolyzed by two endopeptidases Alcalase and Neutrase and related nanoparticles were prepared by desolvation method. Magnetite nanoparticles were coated with WGHs nanoparticles and then inulinase was immobilized onto it using glutaraldehyde as crosslinking agent. Parallel studies employing differential scanning calorimetry and field emmision scanning electron microscopy were carried out to observe functional and structural variations in free inulinase during immobilization. Optimum temperature of immobilized inulinase was increased, while, pH and K m values were decreased compared to free enzyme. Overall, a 12.3 folds rise was detected in enzyme half-life value after Immobilization at 75 °C and enzyme preserved 70% of its initial activity after 12 cycles of hydrolysis with 75% of enzyme loading.

An enhanced chemoenzymatic method for loading substrates onto carrier protein domains.

PubMed

Kittilä, Tiia; Cryle, Max J

2018-06-01

Non-ribosomal peptide synthetase (NRPS) machineries produce many medically relevant peptides that cannot be easily accessed by chemical synthesis. Thus, understanding NRPS mechanism is of crucial importance to allow efficient redesign of these machineries to produce new compounds. During NRPS-mediated synthesis, substrates are covalently attached to peptidyl carrier proteins (PCPs), and studies of NRPSs are impeded by difficulties in producing PCPs loaded with substrates. Different approaches to load substrates onto PCP domains have been described, but all suffer from difficulties in either the complexity of chemical synthesis or low enzymatic efficiency. Here, we describe an enhanced chemoenzymatic loading method that combines 2 approaches into a single, highly efficient one-pot loading reaction. First, d-pantetheine and ATP are converted into dephospho-coenzyme A via the actions of 2 enzymes from coenzyme A (CoA) biosynthesis. Next, phosphoadenylates are dephosphorylated using alkaline phosphatase to allow linker attachment to PCP domain by Sfp mutant R4-4, which is inhibited by phosphoadenylates. This route does not depend on activity of the commonly problematic dephospho-CoA kinase and, therefore, offers an improved method for substrate loading onto PCP domains.

Enhanced Ethanol Production from De-Ashed Paper Sludge by Simultaneous Saccharification and Fermentation and Simultaneous Saccharification and Co-Fermentation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kang, L.; Wang, W.; Pallapolu, V. R.

2011-11-01

A previous study demonstrated that paper sludges with high ash contents can be converted to ethanol by simultaneous saccharification and fermentation (SSF) or simultaneous saccharification and co-fermentation (SSCF). High ash content in the sludge, however, limited solid loading in the bioreactor, causing low product concentration. To overcome this problem, sludges were de-ashed before SSF and SSCF. Low ash content in sludges also increased the ethanol yield to the extent that the enzyme dosage required to achieve 70% yield in the fermentation process was reduced by 30%. High solid loading in SSF and SSCF decreased the ethanol yield. High agitation andmore » de-ashing of the sludges were able to restore the part of the yield loss caused by high solid loading. Substitution of the laboratory fermentation medium (peptone and yeast extract) with corn steep liquor did not bring about any adverse effects in the fermentation. Fed-batch operation of the SSCF and SSF using low-ash content sludges was effective in raising the ethanol concentration, achieving 47.8 g/L and 60.0 g/L, respectively.« less

Optimization of enzymatic hydrolysis and fermentation conditions for improved bioethanol production from potato peel residues.

PubMed

Ben Taher, Imen; Fickers, Patrick; Chniti, Sofien; Hassouna, Mnasser

2017-03-01

The aim of this work was the optimization of the enzyme hydrolysis of potato peel residues (PPR) for bioethanol production. The process included a pretreatment step followed by an enzyme hydrolysis using crude enzyme system composed of cellulase, amylase and hemicellulase, produced by a mixed culture of Aspergillus niger and Trichoderma reesei. Hydrothermal, alkali and acid pretreatments were considered with regards to the enhancement of enzyme hydrolysis of potato peel residues. The obtained results showed that hydrothermal pretreatment lead to a higher enzyme hydrolysis yield compared to both acid and alkali pretreatments. Enzyme hydrolysis was also optimized for parameters such as temperature, pH, substrate loading and surfactant loading using a response surface methodology. Under optimized conditions, 77 g L -1 of reducing sugars were obtained. Yeast fermentation of the released reducing sugars led to an ethanol titer of 30 g L -1 after supplementation of the culture medium with ammonium sulfate. Moreover, a comparative study between acid and enzyme hydrolysis of potato peel residues was investigated. Results showed that enzyme hydrolysis offers higher yield of bioethanol production than acid hydrolysis. These results highlight the potential of second generation bioethanol production from potato peel residues treated with onsite produced hydrolytic enzymes. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:397-406, 2017. © 2017 American Institute of Chemical Engineers.

Enzymatic saccharification of liquid hot water and dilute sulfuric acid pretreated oil palm empty fruit bunch and sugarcane bagasse

NASA Astrophysics Data System (ADS)

Risanto, L.; Fitria; Fajriutami, T.; Hermiati, E.

2018-03-01

Oil palm empty fruit bunch (OPEFB) and sugarcane bagasse (SB) are potential feedstocks for the production of bioethanol. In this study OPEFB and SB were pretreated by liquid hot water and dilute sulfuric acid (3% H2SO4), and continued with enzymatic saccharification. Heating treatment for both methods was conducted in an autoclave at 121 °C for 1 hr. The saccharification was performed up to 72 hours with cellulase enzyme loading of 10, 20, and 30 FPU per g biomass. Results showed that OPEFB and SB pretreated with H2SO4 produced higher reducing sugars than those pretreated by liquid hot water. Higher enzyme loading also resulted in higher reducing sugars. Reducing sugars obtained from enzymatic saccharification of OPEFB were higher than those obtained from SB. The highest total reducing sugars (50.48 g/100 g biomass) was obtained from OPEFB pretreated with 3% H2SO4 at enzyme loading of 30 FPU per g biomass.

Mathematical model for internal pH control in immobilized enzyme particles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liou, J.K.; Rousseau, I.

A mathematical model has been developed for the internal pH control in immobilized enzyme particles. This model describes the kinetics of a coupled system of two enzymes, immobilized in particles of either planar, cylindrical, or spherical shape. The enzyme kinetics are assumed to be of a mixed type, including Michaelis-Menten kinetics, uncompetitive substrate inhibition, and competitive and noncompetitive product inhibition. In a case study we have considered the enzyme combination urease and penicillin acylase, whose kinetics are coupled through the pH dependence of the kinetic parameters. The hydrolysis of urea by urease yields ammonia and carbon dioxide, whereas benzylpenicillin (Pen-G)more » is converted to 6-animo penicillanic acid and phenyl acetic acid by penicillin acylase. The production of acids by the latter enzyme will cause a decrease in pH. Because of the presence of the ammonia-carbon dioxide system, however, the pH may be kept under control. In order to obtain information about the optimum performance of this enzymatic pH controller, we have computed the effectiveness factor and the conversion in a CSTR at different enzyme loadings. The results of the computer simulations indicate that a high conversion of Pen-G may be achieved (80-90%) at bulk pH values of about 7.5 - 8. 27 references.« less

Lignin Peroxidase from Streptomyces viridosporus T7A: Enzyme Concentration Using Ultrafiltration

NASA Astrophysics Data System (ADS)

Gottschalk, Leda M. F.; Bon, Elba P. S.; Nobrega, Ronaldo

It is well known that lignin degradation is a key step in the natural process of biomass decay whereby oxidative enzymes such as laccases and high redox potential ligninolytic peroxidases and oxidases play a central role. More recently, the importance of these enzymes has increased because of their prospective industrial use for the degradation of the biomass lignin to increase the accessibility of the cellulose and hemicellulose moieties to be used as renewable material for the production of fuels and chemicals. These biocatalysts also present potential application on environmental biocatalysis for the degradation of xenobiotics and recalcitrant pollutants. However, the cost for these enzymes production, separation, and concentration must be low to permit its industrial use. This work studied the concentration of lignin peroxidase (LiP), produced by Streptomyces viridosporus T7A, by ultrafiltration, in a laboratory-stirred cell, loaded with polysulfone (PS) or cellulose acetate (CA) membranes with molecular weight cutoffs (MWCO) of 10, 20, and 50 KDa. Experiments were carried out at 25 °C and pH 7.0 in accordance to the enzyme stability profile. The best process conditions and enzyme yield were obtained using a PS membrane with 10 KDa MWCO, whereby it was observed a tenfold LiP activity increase, reaching 1,000 U/L and 90% enzyme activity upholding.

Combined enzyme mediated fermentation of cellulous and xylose to ethanol by Schizosaccharoyces pombe, cellulase, .beta.-glucosidase, and xylose isomerase

DOEpatents

Lastick, Stanley M.; Mohagheghi, Ali; Tucker, Melvin P.; Grohmann, Karel

1994-01-01

A process for producing ethanol from mixed sugar streams from pretreated biomass comprising xylose and cellulose using enzymes to convert these substrates to fermentable sugars; selecting and isolating a yeast Schizosaccharomyces pombe ATCC No. 2476, having the ability to ferment these sugars as they are being formed to produce ethanol; loading the substrates with the fermentation mix composed of yeast, enzymes and substrates; fermenting the loaded substrates and enzymes under anaerobic conditions at a pH range of between about 5.0 to about 6.0 and at a temperature range of between about 35.degree. C. to about 40.degree. C. until the fermentation is completed, the xylose being isomerized to xylulose, the cellulose being converted to glucose, and these sugars being concurrently converted to ethanol by yeast through means of the anaerobic fermentation; and recovering the ethanol.

Combined enzyme mediated fermentation of cellulose and xylose to ethanol by Schizosaccharomyces pombe, cellulase, [beta]-glucosidase, and xylose isomerase

DOEpatents

Lastick, S.M.; Mohagheghi, A.; Tucker, M.P.; Grohmann, K.

1994-12-13

A process for producing ethanol from mixed sugar streams from pretreated biomass comprising xylose and cellulose using enzymes to convert these substrates to fermentable sugars; selecting and isolating a yeast Schizosaccharomyces pombe ATCC No. 2476, having the ability to ferment these sugars as they are being formed to produce ethanol; loading the substrates with the fermentation mix composed of yeast, enzymes and substrates; fermenting the loaded substrates and enzymes under anaerobic conditions at a pH range of between about 5.0 to about 6.0 and at a temperature range of between about 35 C to about 40 C until the fermentation is completed, the xylose being isomerized to xylulose, the cellulose being converted to glucose, and these sugars being concurrently converted to ethanol by yeast through means of the anaerobic fermentation; and recovering the ethanol. 2 figures.

Kinetics of enzymatic high-solid hydrolysis of lignocellulosic biomass studied by calorimetry.

PubMed

Olsen, Søren N; Lumby, Erik; McFarland, Kc; Borch, Kim; Westh, Peter

2011-03-01

Enzymatic hydrolysis of high-solid biomass (>10% w/w dry mass) has become increasingly important as a key step in the production of second-generation bioethanol. To this end, development of quantitative real-time assays is desirable both for empirical optimization and for detailed kinetic analysis. In the current work, we have investigated the application of isothermal calorimetry to study the kinetics of enzymatic hydrolysis of two substrates (pretreated corn stover and Avicel) at high-solid contents (up to 29% w/w). It was found that the calorimetric heat flow provided a true measure of the hydrolysis rate with a detection limit of about 500 pmol glucose s(-1). Hence, calorimetry is shown to be a highly sensitive real-time method, applicable for high solids, and independent on the complexity of the substrate. Dose-response experiments with a typical cellulase cocktail enabled a multidimensional analysis of the interrelationships of enzyme load and the rate, time, and extent of the reaction. The results suggest that the hydrolysis rate of pretreated corn stover is limited initially by available attack points on the substrate surface (<10% conversion) but becomes proportional to enzyme dosage (excess of attack points) at later stages (>10% conversion). This kinetic profile is interpreted as an increase in polymer end concentration (substrate for CBH) as the hydrolysis progresses, probably due to EG activity in the enzyme cocktail. Finally, irreversible enzyme inactivation did not appear to be the source of reduced hydrolysis rate over time.

Attachment of an anti-MUC1 monoclonal antibody to 5-FU loaded BSA nanoparticles for active targeting of breast cancer cells.

PubMed

Kouchakzadeh, Hasan; Shojaosadati, Seyed Abbas; Mohammadnejad, Javad; Paknejad, Malihe; Rasaee, Mohammad Javad

2012-01-01

With PR81 as a murine monoclonal antibody (mAb) that was prepared against the human breast cancer, the MUC1 receptor specific targeting is possible. In this study, PR81-conjugated bovine serum albumin (BSA) nanoparticles loaded with anticancer drug 5-fluorouracil (5-FU) were developed. Enzyme linked immunosorbant assay (ELISA) results showed high immunoreactivity of PR81 mAb conjugated to nanoparticles towards MUC1 related peptide or native cancerous MUC1 and almost no cross-reaction to non-specific proteins. In vitro experiments were performed to determine the ability of this new drug delivery system on overcoming MCF-7 breast cancer cells in comparison with four other systems. The results revealed that these cell-type specific drug loaded nanoparticles could achieve more cell death as compared to when the 5-FU was used with no carriers. Stability studies of produced drug delivery system proved high immunoreactivity of conjugated PR81 even after 11 days of storage in room temperature.

Mechanical Loading of Articular Cartilage Reduces IL-1-Induced Enzyme Expression

PubMed Central

Torzilli, P. A.; Bhargava, M.; Chen, C. T.

2011-01-01

Objective: Exposure of articular cartilage to interleukin-1 (IL-1) results in increased synthesis of matrix degrading enzymes. Previously mechanical load applied together with IL-1 stimulation was found to reduce aggrecan cleavage by ADAMTS-4 and 5 and MMP-1, -3, -9, and -13 and reduce proteoglycan loss from the extracellular matrix. To further delineate the inhibition mechanism the gene expression of ADAMTS-4 and 5; MMP-1, -3, -9, and -13; and TIMP-1, -2, and -3 were measured. Design: Mature bovine articular cartilage was stimulated with a 0.5 MPa compressive stress and 10 ng/ml of IL-1α for 3 days and then allowed to recover without stimulation for 1 additional day. The media was assayed for proteoglycan content on a daily basis, while chondrocyte gene expression (mRNA) was measured during stimulation and 1 day of recovery. Results: Mechanical load alone did not change the gene expression for ADAMTS, MMP, or TIMP. IL-1 caused an increase in gene expression for all enzymes after 1 day of stimulation while not affecting the TIMP levels. Load applied together with IL-1 decreased the expression levels of ADAMTS-4 and -5 and MMP-1 and -3 and increased TIMP-3 expression. Conclusions: A mechanical load appears to modify cartilage degradation by IL-1 at the cellular level by reducing mRNA. PMID:22039566

Separation of periportal and perivenous rat hepatocytes by fluorescence-activated cell sorting: confirmation with colloidal gold as an exogenous marker.

PubMed

Braakman, I; Keij, J; Hardonk, M J; Meijer, D K; Groothuis, G M

1991-01-01

Periportal and perivenous hepatocytes are known to display various functional differences. In this study we present a new method to separate periportal and perivenous cells: after selectively loading zone 1 or zone 3 with the fluorescent label acridine orange in an antegrade or retrograde perfusion, respectively, we separated the isolated hepatocytes on a fluorescence-activated cell sorter. The common way to check on proper separation is to estimate activities of enzymes known to exhibit a heterogeneous acinar distribution. Using enzyme histochemistry, however, we found that already on short collagenase perfusion, some enzymes displayed a more shallow gradient than in vivo, making enzyme activities less suitable as zonal markers. We therefore used colloidal gold granules (17 nm) injected intravenously (2.5 mg) into the rat 2 to 3 hr before cell isolation. The gold is taken up predominantly by perivenous hepatocytes, probably because of the efficient removal of gold granules in zone 1 by competing Kupffer cells. We compared acridine orange fluorescence, presence of gold particles and activities of six marker enzymes, three biochemically and three histochemically determined. Acridine orange and gold both pointed to a high enrichment of the fractions, whereas most enzyme activities were more randomly distributed among the cells as a result of the isolation procedure. Our separation procedure yielded fractions highly enriched in either viable periportal or perivenous cells, both from one liver. The use of colloidal gold as a marker to monitor separation is a valuable alternative to the more risky estimation of enzyme activities.

Additives enhancing enzymatic hydrolysis of lignocellulosic biomass.

PubMed

Rocha-Martín, Javier; Martinez-Bernal, Claudio; Pérez-Cobas, Yolanda; Reyes-Sosa, Francisco Manuel; García, Bruno Díez

2017-11-01

Linked to the development of cellulolytic enzyme cocktails from Myceliophthora thermophila, we studied the effect of different additives on the enzymatic hydrolysis yield. The hydrolysis of pretreated corn stover (PCS), sugar cane straw (PSCS) and microcrystalline cellulose (Avicel) was performed under industrial conditions using high solid loadings, limited mixing, and low enzyme dosages. The addition of polyethylene glycol (PEG4000) allowed to increase the glucose yields by 10%, 7.5%, and 32%, respectively in the three materials. PEG4000 did not have significant effect on the stability of the main individual enzymes but increased beta-glucosidase and endoglucanase activity by 20% and 60% respectively. Moreover, the presence of PEG4000 accelerated cellulase-catalyzed hydrolysis reducing up to 25% the liquefaction time. However, a preliminary economical assessment concludes that even with these improvements, a lower contribution of PEG4000 to the 2G bioethanol production costs would be needed to reach commercial feasibility. Copyright © 2017. Published by Elsevier Ltd.

Coupled reactions on bioparticles: Stereoselective reduction with cofactor regeneration on PhaC inclusion bodies.

PubMed

Spieler, Valerie; Valldorf, Bernhard; Maaß, Franziska; Kleinschek, Alexander; Hüttenhain, Stefan H; Kolmar, Harald

2016-07-01

Chiral alcohols are important building blocks for specialty chemicals and pharmaceuticals. The production of chiral alcohols from ketones can be carried out stereo selectively with alcohol dehydrogenases (ADHs). To establish a process for cost-effective enzyme immobilization on solid phase for application in ketone reduction, we used an established enzyme pair consisting of ADH from Rhodococcus erythropolis and formate dehydrogenase (FDH) from Candida boidinii for NADH cofactor regeneration and co-immobilized them on modified poly-p-hydroxybutyrate synthase (PhaC)-inclusion bodies that were recombinantly produced in Escherichia coli cells. After separate production of genetically engineered and recombinantly produced enzymes and particles, cell lysates were combined and enzymes endowed with a Kcoil were captured on the surface of the Ecoil presenting particles due to coiled-coil interaction. Enzyme-loaded particles could be easily purified by centrifugation. Total conversion of 4'-chloroacetophenone to (S)-4-chloro-α-methylbenzyl alcohol could be accomplished using enzyme-loaded particles, catalytic amounts of NAD(+) and formate as substrates for FDH. Chiral GC-MS analysis revealed that immobilized ADH retained enantioselectivity with 99 % enantiomeric excess. In conclusion, this strategy may become a cost-effective alternative to coupled reactions using purified enzymes. Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A novel liquid/liquid extraction process composed of surfactant and acetonitrile for purification of polygalacturonase enzyme from Durio zibethinus.

PubMed

Amid, Mehrnoush; Manap, Yazid; Azmira, Farhana; Hussin, Muhaini; Sarker, Zaidul Islam

2015-07-01

Polygalacturonase is one of the important enzymes used in various industries such as food, detergent, pharmaceutical, textile, pulp and paper. A novel liquid/liquid extraction process composed of surfactant and acetonitrile was employed for the first time to purify polygalacturonase from Durio zibethinus. The influences of different parameters such as type and concentration of surfactants, concentrations of acetonitrile and composition of surfactant/acetonitrile on partitioning behavior and recovery of polygalacturonase was investigated. Moreover, the effect of pH of system and crude load on purification fold and yield of purified polygalacturonase were studied. The results of the experiment indicated the polygalacturonase was partitioned into surfactant top rich phase with impurities being partitioned into acetonitrile bottom rich phase in the novel method of liquid/liquid process composed of 23% (w/w) Triton X-100 and 19% (w/w) acetonitrile, at 55.6% of TLL (tie line length) crude load of 25% (w/w) at pH 6.0. Recovery and recycling of components also was measured in each successive step of liquid/liquid extraction process. The enzyme was successfully recovered by the method with a high purification factor of 14.3 and yield of 97.3% while phase components were also recovered and recycled above 95%. This study demonstrated that the novel method of liquid/liquid extraction process can be used as an efficient and economical extraction method rather than the traditional methods of extraction for the purification and recovery of the valuable enzyme. Copyright © 2015 Elsevier B.V. All rights reserved.

Sensitive high-throughput screening for the detection of reducing sugars.

PubMed

Mellitzer, Andrea; Glieder, Anton; Weis, Roland; Reisinger, Christoph; Flicker, Karlheinz

2012-01-01

The exploitation of renewable resources for the production of biofuels relies on efficient processes for the enzymatic hydrolysis of lignocellulosic materials. The development of enzymes and strains for these processes requires reliable and fast activity-based screening assays. Additionally, these assays are also required to operate on the microscale and on the high-throughput level. Herein, we report the development of a highly sensitive reducing-sugar assay in a 96-well microplate screening format. The assay is based on the formation of osazones from reducing sugars and para-hydroxybenzoic acid hydrazide. By using this sensitive assay, the enzyme loads and conversion times during lignocellulose hydrolysis can be reduced, thus allowing higher throughput. The assay is about five times more sensitive than the widely applied dinitrosalicylic acid based assay and can reliably detect reducing sugars down to 10 μM. The assay-specific variation over one microplate was determined for three different lignocellulolytic enzymes and ranges from 2 to 8%. Furthermore, the assay was combined with a microscale cultivation procedure for the activity-based screening of Pichia pastoris strains expressing functional Thermomyces lanuginosus xylanase A, Trichoderma reesei β-mannanase, or T. reesei cellobiohydrolase 2. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The influence of lignin on steam pretreatment and mechanical pulping of poplar to achieve high sugar recovery and ease of enzymatic hydrolysis.

PubMed

Chandra, Richard P; Chu, QiuLu; Hu, Jinguang; Zhong, Na; Lin, Mandy; Lee, Jin-Suk; Saddler, Jack

2016-01-01

With the goal of enhancing overall carbohydrate recovery and reducing enzyme loading refiner mechanical pulping and steam pretreatment (210°C, 5 min) were used to pretreat poplar wood chips. Neutral sulphonation post-treatment indicated that, although the lignin present in the steam pretreated substrate was less reactive, the cellulose-rich, water insoluble component was more accessible to cellulases and Simons stain. This was likely due to lignin relocation as the relative surface lignin measured by X-ray photoelectron spectroscopy increased from 0.4 to 0.8. The integration of sulphite directly into steam pretreatment resulted in the solubilisation of 60% of the lignin while more than 80% of the carbohydrate present in the original substrate was recovered in the water insoluble fraction after Na2CO3 addition. More than 80% of the sugars present in the original cellulose and xylan could be recovered after 48 h using an enzyme loading of 20 mg protein/g cellulose at a 10% substrate concentration. Copyright © 2015 Elsevier Ltd. All rights reserved.

Association between polymorphisms of the insulin-degrading enzyme gene and late-onset Alzheimer disease.

PubMed

Wang, Shitao; He, Feiyan; Wang, Ying

2015-06-01

The insulin-degrading enzyme (IDE) gene is a strong positional and biological candidate for late-onset Alzheimer disease (LOAD) susceptibility, with recent studies independently demonstrating an association between IDE gene variants and LOAD. However, previous data have been controversial. To investigate the relationship between IDE gene polymorphisms and LOAD risk, a case-control association study of 406 Han Chinese participants in Xinjiang, China, was undertaken. The LOAD and control groups consisted of 202 and 204 participants, respectively. The single-nucleotide polymorphisms rs1887922 and rs1999764 of the IDE gene were linked to LOAD incidence. The presence of the CT+CC genotype of rs1999764 had a protective effect compared to the TT genotype (adjusted P=.0001; odds ratio [OR]=0.226; 95% confidence interval [CI]=0.116-0.441), while the CT+CC genotype of rs1887922 was associated with increased LOAD risk (adjusted P=.0001; OR=3.640; 95% CI=1.889-7.016). Moreover, the effects of rs1887922 and rs1999764 were associated with LOAD risk independent of the apolipoprotein E ∊4 polymorphism and were more significant in men and women, respectively. These results demonstrate that the polymorphisms rs1887922 and rs1999764 of the IDE gene are associated with LOAD susceptibility in the Xinjiang Han population. © The Author(s) 2014.

Structural Insights into the Affinity of Cel7A Carbohydrate-binding Module for Lignin*

PubMed Central

Strobel, Kathryn L.; Pfeiffer, Katherine A.; Blanch, Harvey W.; Clark, Douglas S.

2015-01-01

The high cost of hydrolytic enzymes impedes the commercial production of lignocellulosic biofuels. High enzyme loadings are required in part due to their non-productive adsorption to lignin, a major component of biomass. Despite numerous studies documenting cellulase adsorption to lignin, few attempts have been made to engineer enzymes to reduce lignin binding. In this work, we used alanine-scanning mutagenesis to elucidate the structural basis for the lignin affinity of Trichoderma reesei Cel7A carbohydrate binding module (CBM). T. reesei Cel7A CBM mutants were produced with a Talaromyces emersonii Cel7A catalytic domain and screened for their binding to cellulose and lignin. Mutation of aromatic and polar residues on the planar face of the CBM greatly decreased binding to both cellulose and lignin, supporting the hypothesis that the cellulose-binding face is also responsible for lignin affinity. Cellulose and lignin affinity of the 31 mutants were highly correlated, although several mutants displayed selective reductions in lignin or cellulose affinity. Four mutants with increased cellulose selectivity (Q2A, H4A, V18A, and P30A) did not exhibit improved hydrolysis of cellulose in the presence of lignin. Further reduction in lignin affinity while maintaining a high level of cellulose affinity is thus necessary to generate an enzyme with improved hydrolysis capability. This work provides insights into the structural underpinnings of lignin affinity, identifies residues amenable to mutation without compromising cellulose affinity, and informs engineering strategies for family one CBMs. PMID:26209638

Soluble inhibitors/deactivators of cellulase enzymes from lignocellulosic biomass.

PubMed

Kim, Youngmi; Ximenes, Eduardo; Mosier, Nathan S; Ladisch, Michael R

2011-04-07

Liquid hot water, steam explosion, and dilute acid pretreatments of lignocellulose generate soluble inhibitors which hamper enzymatic hydrolysis as well as fermentation of sugars to ethanol. Toxic and inhibitory compounds will vary with pretreatment and include soluble sugars, furan derivatives (hydroxymethyl fulfural, furfural), organic acids (acetic, formic and, levulinic acid), and phenolic compounds. Their effect is seen when an increase in the concentration of pretreated biomass in a hydrolysis slurry results in decreased cellulose conversion, even though the ratio of enzyme to cellulose is kept constant. We used lignin-free cellulose, Solka Floc, combined with mixtures of soluble components released during pretreatment of wood, to prove that the decrease in the rate and extent of cellulose hydrolysis is due to a combination of enzyme inhibition and deactivation. The causative agents were extracted from wood pretreatment liquid using PEG surfactant, activated charcoal or ethyl acetate and then desorbed, recovered, and added back to a mixture of enzyme and cellulose. At enzyme loadings of either 1 or 25mg protein/g glucan, the most inhibitory components, later identified as phenolics, decreased the rate and extent of cellulose hydrolysis by half due to both inhibition and precipitation of the enzymes. Full enzyme activity occurred when the phenols were removed. Hence detoxification of pretreated woods through phenol removal is expected to reduce enzyme loadings, and therefore reduce enzyme costs, for a given level of cellulose conversion. Copyright © 2011 Elsevier Inc. All rights reserved.

Utilization of discard bovine bone as a support for immobilization of recombinant Rhizopus oryzae lipase expressed in Pichia pastoris.

PubMed

Clementz, Adriana L; Del Peso, Gonzalo; Canet, Albert; Yori, Juan C; Valero, Francisco

2016-09-01

In this study the possibility of using discard bovine bone as support for immobilization of Rhizopus oryzae lipase expressed in Pichia pastoris was analyzed. Discard bovine bone were milled and then subjected to a chemical treatment with acetone in order to remove lipids and blood traces. Two types of supports were evaluated: bovine bone and calcined bovine bone for 2 h at 600°C. Supports were characterized by: ICP, SEM, XRD, FTIR, XPS, and N 2 adsorption isotherms. Calcined bovine bone presented appropriate characteristics for the lipase immobilization due to the removal of collagen: high porosity, large surface area and suitable porous structure. Biocatalysts were prepared with different initial enzyme load. For the equilibrium adsorption studies, the Langmuir isotherm was used to fit the data results. The immobilization occurs in monolayer to a value of 35 UA mg -1 . The activities of biocatalysts were tested in transesterification reaction of olive oil. For the enzyme load used in the test, a final yield percentage of 49.6 was achieved after six methanol additions and 180 min of reaction, similar values were obtained using Relizyme as support. Therefore, the bovine bone discard is an economical and appropriate choice for use support immobilization of enzymes. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1246-1253, 2016. © 2016 American Institute of Chemical Engineers.

A Novel Aqueous Micellar Two-Phase System Composed of Surfactant and Sorbitol for Purification of Pectinase Enzyme from Psidium guajava and Recycling Phase Components

PubMed Central

Murshid, Fara Syazana; Manap, Mohd Yazid; Hussin, Muhaini

2015-01-01

A novel aqueous two-phase system composed of a surfactant and sorbitol was employed for the first time to purify pectinase from Psidium guajava. The influences of different parameters, including the type and concentration of the surfactant and the concentration and composition of the surfactant/sorbitol ratio, on the partitioning behavior and recovery of pectinase were investigated. Moreover, the effects of system pH and the crude load on purification fold and the yield of purified pectinase were studied. The experimental results indicated that the pectinase was partitioned into surfactant-rich top phase, and the impurities were partitioned into the sorbitol-rich bottom phase with the novel method involving an ATPS composed of 26% (w/w) Triton X-100 and 23% (w/w) sorbitol at 54.2% of the TLL crude load of 20% (w/w) at pH 6.0. The enzyme was successfully recovered by this method with a high purification factor of 15.2 and a yield of 98.3%, whereas the phase components were also recovered and recycled at rates above 96%. This study demonstrated that this novel ATPS method can be used as an efficient and economical alternative to the traditional ATPS for the purification and recovery of the valuable enzyme. PMID:25756051

MICROBIAL ENZYME ACTIVITY FOR CHARACTERIZING NUTRIENT LOADING TO GREAT LAKES COASTAL WETLANDS

EPA Science Inventory

Energy and material flows in aquatic ecosystems are mediated by microbial carbon and nutrient cycling. Extracellular enzymes produced by the microbial community aid in the degradation of organic matter and the resultant acquisition of limiting nutrients. Organic carbon sequestrat...

Large-scale aerosol-assisted synthesis of biofriendly Fe2O3 yolk-shell particles: a promising support for enzyme immobilization

NASA Astrophysics Data System (ADS)

Patel, Sanjay K. S.; Choi, Seung Ho; Kang, Yun Chan; Lee, Jung-Kul

2016-03-01

Multiple-shelled Fe2O3 yolk-shell particles were synthesized using the spray drying method and intended as a suitable support for the immobilization of commercial enzymes such as glucose oxidase (GOx), horseradish peroxidase (HRP), and laccase as model enzymes. Yolk-shell particles have an average diameter of 1-3 μm with pore diameters in the range of 16 to 28 nm. The maximum immobilization of GOx, HRP, and laccase resulted in the enzyme loading of 292, 307 and 398 mg per g of support, respectively. After cross-linking of immobilized laccase by glutaraldehyde, immobilization efficiency was improved from 83.5% to 90.2%. Km and Vmax values were 41.5 μM and 1722 μmol min-1 per mg protein for cross-linked laccase and those for free laccase were 29.3 μM and 1890 μmol min-1 per mg protein, respectively. The thermal stability of the enzyme was enhanced up to 18-fold upon cross-linking, and the enzyme retained 93.1% of residual activity after ten cycles of reuse. The immobilized enzyme has shown up to 32-fold higher stability than the free enzyme towards different solvents and it showed higher efficiency than free laccase in the decolorization of dyes and degradation of bisphenol A. The synthesized yolk-shell particles have 3-fold higher enzyme loading efficiency and lower acute toxicity than the commercial Fe2O3 spherical particles. Therefore, the use of unique yolk-shell structure Fe2O3 particles with multiple-shells will be promising for the immobilization of various enzymes in biotechnological applications with improved electrochemical properties. To the best of our knowledge, this is the first report on the use of one pot synthesized Fe2O3 yolk-shell structure particles for the immobilization of enzymes.Multiple-shelled Fe2O3 yolk-shell particles were synthesized using the spray drying method and intended as a suitable support for the immobilization of commercial enzymes such as glucose oxidase (GOx), horseradish peroxidase (HRP), and laccase as model enzymes. Yolk-shell particles have an average diameter of 1-3 μm with pore diameters in the range of 16 to 28 nm. The maximum immobilization of GOx, HRP, and laccase resulted in the enzyme loading of 292, 307 and 398 mg per g of support, respectively. After cross-linking of immobilized laccase by glutaraldehyde, immobilization efficiency was improved from 83.5% to 90.2%. Km and Vmax values were 41.5 μM and 1722 μmol min-1 per mg protein for cross-linked laccase and those for free laccase were 29.3 μM and 1890 μmol min-1 per mg protein, respectively. The thermal stability of the enzyme was enhanced up to 18-fold upon cross-linking, and the enzyme retained 93.1% of residual activity after ten cycles of reuse. The immobilized enzyme has shown up to 32-fold higher stability than the free enzyme towards different solvents and it showed higher efficiency than free laccase in the decolorization of dyes and degradation of bisphenol A. The synthesized yolk-shell particles have 3-fold higher enzyme loading efficiency and lower acute toxicity than the commercial Fe2O3 spherical particles. Therefore, the use of unique yolk-shell structure Fe2O3 particles with multiple-shells will be promising for the immobilization of various enzymes in biotechnological applications with improved electrochemical properties. To the best of our knowledge, this is the first report on the use of one pot synthesized Fe2O3 yolk-shell structure particles for the immobilization of enzymes. Electronic supplementary information (ESI) available. See DOI: 10.1039/c6nr00346j

The impact of particle size and initial solid loading on thermochemical pretreatment of wheat straw for improving sugar recovery.

PubMed

Rojas-Rejón, Oscar A; Sánchez, Arturo

2014-07-01

This work studies the effect of initial solid load (4-32 %; w/v, DS) and particle size (0.41-50 mm) on monosaccharide yield of wheat straw subjected to dilute H(2)SO(4) (0.75 %, v/v) pretreatment and enzymatic saccharification. Response surface methodology (RSM) based on a full factorial design (FFD) was used for the statistical analysis of pretreatment and enzymatic hydrolysis. The highest xylose yield obtained during pretreatment (ca. 86 %; of theoretical) was achieved at 4 % (w/v, DS) and 25 mm. The solid fraction obtained from the first set of experiments was subjected to enzymatic hydrolysis at constant enzyme dosage (17 FPU/g); statistical analysis revealed that glucose yield was favored with solids pretreated at low initial solid loads and small particle sizes. Dynamic experiments showed that glucose yield did not increase after 48 h of enzymatic hydrolysis. Once established pretreatment conditions, experiments were carried out with several initial solid loading (4-24 %; w/v, DS) and enzyme dosages (5-50 FPU/g). Two straw sizes (0.41 and 50 mm) were used for verification purposes. The highest glucose yield (ca. 55 %; of theoretical) was achieved at 4 % (w/v, DS), 0.41 mm and 50 FPU/g. Statistical analysis of experiments showed that at low enzyme dosage, particle size had a remarkable effect over glucose yield and initial solid load was the main factor for glucose yield.

Automatic and integrated micro-enzyme assay (AIμEA) platform for highly sensitive thrombin analysis via an engineered fluorescence protein-functionalized monolithic capillary column.

PubMed

Lin, Lihua; Liu, Shengquan; Nie, Zhou; Chen, Yingzhuang; Lei, Chunyang; Wang, Zhen; Yin, Chao; Hu, Huiping; Huang, Yan; Yao, Shouzhuo

2015-04-21

Nowadays, large-scale screening for enzyme discovery, engineering, and drug discovery processes require simple, fast, and sensitive enzyme activity assay platforms with high integration and potential for high-throughput detection. Herein, a novel automatic and integrated micro-enzyme assay (AIμEA) platform was proposed based on a unique microreaction system fabricated by a engineered green fluorescence protein (GFP)-functionalized monolithic capillary column, with thrombin as an example. The recombinant GFP probe was rationally engineered to possess a His-tag and a substrate sequence of thrombin, which enable it to be immobilized on the monolith via metal affinity binding, and to be released after thrombin digestion. Combined with capillary electrophoresis-laser-induced fluorescence (CE-LIF), all the procedures, including thrombin injection, online enzymatic digestion in the microreaction system, and label-free detection of the released GFP, were integrated in a single electrophoretic process. By taking advantage of the ultrahigh loading capacity of the AIμEA platform and the CE automatic programming setup, one microreaction column was sufficient for many times digestion without replacement. The novel microreaction system showed significantly enhanced catalytic efficiency, about 30 fold higher than that of the equivalent bulk reaction. Accordingly, the AIμEA platform was highly sensitive with a limit of detection down to 1 pM of thrombin. Moreover, the AIμEA platform was robust and reliable to detect thrombin in human serum samples and its inhibition by hirudin. Hence, this AIμEA platform exhibits great potential for high-throughput analysis in future biological application, disease diagnostics, and drug screening.

Dual stimuli polysaccharide nanovesicles for conjugated and physically loaded doxorubicin delivery in breast cancer cells.

PubMed

Pramod, P S; Shah, Ruchira; Jayakannan, Manickam

2015-04-21

The present work reports the development of pH and enzyme dual responsive polysaccharide vesicular nano-scaffolds for the administration of doxorubicin via physical loading and polymer-drug conjugation to breast cancer cells. Dextran was suitably modified with a renewable resource 3-pentadecyl phenol unit through imine and aliphatic ester chemical linkages that acted as pH and esterase enzyme stimuli, respectively. These dual responsive polysaccharide derivatives self-organized into 200 ± 10 nm diameter nano-vesicles in water. The water soluble anticancer drug doxorubicin (DOX·HCl) was encapsulated in the hydrophilic pocket to produce core-loaded polysaccharide vesicles whereas chemical conjugation produced DOX anchored at the hydrophobic layer of the dextran nano-vesicles. In vitro studies revealed that about 70-80% of the drug was retained under circulatory conditions at pH = 7.4 and 37 °C. At a low pH of 6.0 to 5.0 and in the presence of esterase; both imine and ester linkages were cleaved instantaneously to release 100% of the loaded drugs. Cytotoxicity assays on Wild Type Mouse Embryonic Fibroblasts (WTMEFs) confirmed the non-toxicity of the newly developed dextran derivatives at up to 500 μg mL(-1) in PBS. MTT assays on fibroblast cells revealed that DOX·HCl loaded nano-vesicles exhibited better killing abilities than DOX conjugated polymer nano-vesicles. Both DOX loaded and DOX conjugated nano-vesicles were found to show significant killing in breast cancer cells (MCF 7). Confocal microscopy images confirmed the uptake of DOX loaded (or conjugated) nano-vesicles by cells compared to free DOX. Thus, the newly developed pH and enzyme dual responsive polysaccharide vesicular assemblies are potential drug vectors for the administration of DOX in both loaded and chemically conjugated forms for the efficient killing of breast cancer cells.

Process analysis and optimization of simultaneous saccharification and co-fermentation of ethylenediamine-pretreated corn stover for ethanol production.

PubMed

Qin, Lei; Zhao, Xiong; Li, Wen-Chao; Zhu, Jia-Qing; Liu, Li; Li, Bing-Zhi; Yuan, Ying-Jin

2018-01-01

Improving ethanol concentration and reducing enzyme dosage are main challenges in bioethanol refinery from lignocellulosic biomass. Ethylenediamine (EDA) pretreatment is a novel method to improve enzymatic digestibility of lignocellulose. In this study, simultaneous saccharification and co-fermentation (SSCF) process using EDA-pretreated corn stover was analyzed and optimized to verify the constraint factors on ethanol production. Highest ethanol concentration was achieved with the following optimized SSCF conditions at 6% glucan loading: 12-h pre-hydrolysis, 34 °C, pH 5.4, and inoculum size of 5 g dry cell/L. As glucan loading increased from 6 to 9%, ethanol concentration increased from 33.8 to 48.0 g/L, while ethanol yield reduced by 7%. Mass balance of SSCF showed that the reduction of ethanol yield with the increasing solid loading was mainly due to the decrease of glucan enzymatic conversion and xylose metabolism of the strain. Tween 20 and BSA increased ethanol concentration through enhancing enzymatic efficiency. The solid-recycled SSCF process reduced enzyme dosage by 40% (from 20 to 12 mg protein/g glucan) to achieve the similar ethanol concentration (~ 40 g/L) comparing to conventional SSCF. Here, we established an efficient SSCF procedure using EDA-pretreated biomass. Glucose enzymatic yield and yeast viability were regarded as the key factors affecting ethanol production at high solid loading. The extensive analysis of SSCF would be constructive to overcome the bottlenecks and improve ethanol production in cellulosic ethanol refinery.

Subangstrom Measurements of Enzyme Function Using a Biological Nanopore, SPRNT.

PubMed

Laszlo, A H; Derrrington, I M; Gundlach, J H

2017-01-01

Nanopores are emerging as new single-molecule tools in the study of enzymes. Based on the progress in nanopore sequencing of DNA, a tool called Single-molecule Picometer Resolution Nanopore Tweezers (SPRNT) was developed to measure the movement of enzymes along DNA in real time. In this new method, an enzyme is loaded onto a DNA (or RNA) molecule. A single-stranded DNA end of this complex is drawn into a nanopore by an electrostatic potential that is applied across the pore. The single-stranded DNA passes through the pore's constriction until the enzyme comes into contact with the pore. Further progression of the DNA through the pore is then controlled by the enzyme. An ion current that flows through the pore's constriction is modulated by the DNA in the constriction. Analysis of ion current changes reveals the advance of the DNA with high spatiotemporal precision, thereby providing a real-time record of the enzyme's activity. Using an engineered version of the protein nanopore MspA, SPRNT has spatial resolution as small as 40pm at millisecond timescales, while simultaneously providing the DNA's sequence within the enzyme. In this chapter, SPRNT is introduced and its extraordinary potential is exemplified using the helicase Hel308. Two distinct substates are observed for each one-nucleotide advance; one of these about half-nucleotide long steps is ATP dependent and the other is ATP independent. The spatiotemporal resolution of this low-cost single-molecule technique lifts the study of enzymes to a new level of precision, enabling exploration of hitherto unobservable enzyme dynamics in real time. © 2017 Elsevier Inc. All rights reserved.

Aspergillus niger PA2 Tyrosinase Covalently Immobilized on a Novel Eco-Friendly Bio-Composite of Chitosan-Gelatin and Its Evaluation for L-DOPA Production

PubMed Central

Agarwal, Pragati; Dubey, Swati; Singh, Mukta; Singh, Rajesh P.

2016-01-01

Tyrosinase (EC 1.14.18.1) a copper-containing monooxygenase, isolated from a fungal isolate Aspergillus niger PA2 was subjected for immobilization onto a composite consisting of chitosan and gelatin biopolymers. The homogeneity of the chitosan-gelatin biocomposite film was characterized by X-ray diffraction analyses. To evaluate immobilization efficiency, chitosan-gelatin-Tyr bio-composite films were analyzed by field emission scanning electron microscopy, atomic force microscopy and UV-spectroscopy. The rough morphology of the film led to a high loading of enzyme and it could retain its bioactivity for a longer period. The enzyme adsorbed onto the film exhibited 72% of its activity after 10 days and exhibited good repeatability for up to nine times, after intermittent storage. Moreover, the immobilized enzyme exhibited broader pH and temperature profile as compared to free counterpart. Immobilized enzyme was further evaluated for the synthesis of L-DOPA (2,4-dihydroxy phenylalanine) which is a precursor of dopamine and a potent drug for the treatment of Parkinson's disease and for myocardium neurogenic injury. PMID:28066399

Functional Layer-by-Layer Thin Films of Inducible Nitric Oxide (NO) Synthase Oxygenase and Polyethylenimine: Modulation of Enzyme Loading and NO-Release Activity.

PubMed

Gunasekera, Bhagya; Abou Diwan, Charbel; Altawallbeh, Ghaith; Kalil, Haitham; Maher, Shaimaa; Xu, Song; Bayachou, Mekki

2018-03-07

Nitric oxide (NO) release counteracts platelet aggregation and prevents the thrombosis cascade in the inner walls of blood vessels. NO-release coatings also prevent thrombus formation on the surface of blood-contacting medical devices. Our previous work has shown that inducible nitric oxide synthase (iNOS) films release NO fluxes upon enzymatic conversion of the substrate l-arginine. In this work, we report on the modulation of enzyme loading in layer-by-layer (LbL) thin films of inducible nitric oxide synthase oxygenase (iNOSoxy) on polyethylenimine (PEI). The layer of iNOSoxy is electrostatically adsorbed onto the PEI layer. The pH of the iNOSoxy solution affects the amount of enzyme adsorbed. The overall negative surface charge of iNOSoxy in solution depends on the pH and hence determines the density of adsorbed protein on the positively charged PEI layer. We used buffered iNOSoxy solutions adjusted to pHs 8.6 and 7.0, while saline PEI solution was used at pH 7.0. Atomic force microscopy imaging of the outermost layer shows higher protein adsorption with iNOSoxy at pH 8.6 than with a solution of iNOSoxy at pH 7.0. Graphite electrodes with PEI/iNOSoxy films show higher catalytic currents for nitric oxide reduction mediated by iNOSoxy. The higher enzyme loading translates into higher NO flux when the enzyme-modified surface is exposed to a solution containing the substrate and a source of electrons. Spectrophotometric assays showed higher NO fluxes with iNOSoxy/PEI films built at pH 8.6 than with films built at pH 7.0. Fourier transform infrared analysis of iNOSoxy adsorbed on PEI at pH 8.6 and 7.0 shows structural differences of iNOSoxy in films, which explains the observed changes in enzymatic activity. Our findings show that pH provides a strategy to optimize the NOS loading and enzyme activity in NOS-based LbL thin films, which enables improved NO release with minimum layers of PEI/NOS.

Effect of linoleic-acid modified carboxymethyl chitosan on bromelain immobilization onto self-assembled nanoparticles

NASA Astrophysics Data System (ADS)

Tan, Yu-long; Liu, Chen-guang; Yu, Le-jun; Chen, Xi-guang

2008-06-01

Hydrogel nanoparticles could be prepared by using linoleic acid (LA) modified carboxymethyl chitosan (CMCS) after sonication. Bromelain could be loaded onto nanoparticles of LA-CMCS. Factors affecting the activity of the immobilized enzyme, including temperature, storage etc., were investigated in this study. The results showed that the stability of bromelain for heat and storage was improved after immobilization on nanoparticles. The Michaelis constant ( K m) of the immobilized enzyme was smaller than that of free enzyme, indicating that the immobilization could promote the stability of the enzyme and strengthen the affinity of the enzyme for the substrate.

Application of arteether-loaded polyurethane nanomicelles to induce immune response in breast cancer model.

PubMed

Jabbarzadegan, Mona; Rajayi, Hajar; Mofazzal Jahromi, Mirza Ali; Yeganeh, Hamid; Yousefi, Mehdi; Muhammad Hassan, Zuhair; Majidi, Jafar

2017-06-01

To concentrate a potent anticancer drug (Arteether) in tumor microenvironment, we encapsulated it in biodegradable and pH sensitive polyurethane (PU) nanomicelles (NMs). The nanocomplex was characterized by Fourier transform infrared (FTIR), dynamic light scattering (DLS). The loading capacity and release profile in pH of 5.4 and 7.4 were considered. The cytotoxicity effect was evaluated in vitro and in vivo settings. The level of IFN-γ and IL-4 cytokines of mice splenocytes were assessed by enzyme-linked immunosorbent assay (ELISA). The nanocomplex showed negative zeta charge of -26.2 mV, size of 42.30 nm and high loading capacity (92%). Release profile showed a faster rate of drug liberation at pH 5.4 as compared to that of pH 7.4. It indicated significant inhibitory effect on the growth of 4T1 cell line and increased IFN-γ level.

Endothelial Targeting of Semi-permeable Polymer Nanocarriers for Enzyme Therapies

PubMed Central

Dziubla, Thomas D; Shuvaev, Vladimir V.; Hong, Nan Kang; Hawkins, Brian; Muniswamy, Madesh; Takano, Hajime; Simone, Eric; Nakada, Marian T.; Fisher, Aron; Albelda, Steven M.; Muzykantov, Vladimir R.

2007-01-01

The medical utility of proteins, e.g. therapeutic enzymes, is greatly restricted by their liable nature and inadequate delivery. Most therapeutic enzymes do not accumulate in their targets and are inactivated by proteases. Targeting of enzymes encapsulated into substrate-permeable Polymeric Nano-Carriers (PNC) impermeable for proteases might overcome these limitations. To test this hypothesis, we designed endothelial targeted PNC loaded with catalase, the H2O2-detoxifying enzyme, and tested if this approach protects against vascular oxidative stress, a pathological process implicated in ischemia-reperfusion and other disease conditions. Encapsulation of catalase (MW 240KD), peroxidase (MW 42kD) and xanthine oxidase (XO, MW 300 kD) into ~300nm diameter PNC composed of co-polymers of PEG-PLGA (polyethylene glycol and poly-lactic/poly-glycolic acid) was in the range ~10% for all enzymes. PNC/catalase and PNC/peroxidase were protected from external proteolysis and exerted the enzymatic activity on their PNC diffusible substrates, H2O2 and ortho-phenylendiamine, whereas activity of encapsulated XO was negligible due to polymer impermeability to the substrate. PNC targeted to platelet-endothelial cell adhesion molecule-1 delivered active encapsulated catalase to endothelial cells and protected the endothelium against oxidative stress in cell culture and animal studies. Vascular targeting of PNC-loaded detoxifying enzymes may find wide medical applications including management of oxidative stress and other toxicities. PMID:17950837

Enzymatic hydrolysis and fermentation of dilute acid pretreated cornstalk to biohydrogen

NASA Astrophysics Data System (ADS)

Pan, C. M.; Fan, Y. T.; Hou, H. W.

2010-03-01

The coupling method of acid pretreatment and enzymatic hydrolysis of cornstalk for hydrogen production was investigated in this study. Experimental results showed that temperature, pH and enzyme loading all had an individual significant influence on soluble sugar yield and Ps. The optimum condition for soluble sugar was close to that for Ps. The maximum hydrogen yield from cornstalk by anaerobic mixed microflora was 209.8 ml/g-TVS on the optimum enzymatic hydrolysis condition which was 52 °C of temperature, pH4.8 and 9.4 IU/g of enzyme loading.

Dual stimuli polysaccharide nanovesicles for conjugated and physically loaded doxorubicin delivery in breast cancer cells

NASA Astrophysics Data System (ADS)

Pramod, P. S.; Shah, Ruchira; Jayakannan, Manickam

2015-04-01

The present work reports the development of pH and enzyme dual responsive polysaccharide vesicular nano-scaffolds for the administration of doxorubicin via physical loading and polymer-drug conjugation to breast cancer cells. Dextran was suitably modified with a renewable resource 3-pentadecyl phenol unit through imine and aliphatic ester chemical linkages that acted as pH and esterase enzyme stimuli, respectively. These dual responsive polysaccharide derivatives self-organized into 200 +/- 10 nm diameter nano-vesicles in water. The water soluble anticancer drug doxorubicin (DOX.HCl) was encapsulated in the hydrophilic pocket to produce core-loaded polysaccharide vesicles whereas chemical conjugation produced DOX anchored at the hydrophobic layer of the dextran nano-vesicles. In vitro studies revealed that about 70-80% of the drug was retained under circulatory conditions at pH = 7.4 and 37 °C. At a low pH of 6.0 to 5.0 and in the presence of esterase; both imine and ester linkages were cleaved instantaneously to release 100% of the loaded drugs. Cytotoxicity assays on Wild Type Mouse Embryonic Fibroblasts (WTMEFs) confirmed the non-toxicity of the newly developed dextran derivatives at up to 500 μg mL-1 in PBS. MTT assays on fibroblast cells revealed that DOX.HCl loaded nano-vesicles exhibited better killing abilities than DOX conjugated polymer nano-vesicles. Both DOX loaded and DOX conjugated nano-vesicles were found to show significant killing in breast cancer cells (MCF 7). Confocal microscopy images confirmed the uptake of DOX loaded (or conjugated) nano-vesicles by cells compared to free DOX. Thus, the newly developed pH and enzyme dual responsive polysaccharide vesicular assemblies are potential drug vectors for the administration of DOX in both loaded and chemically conjugated forms for the efficient killing of breast cancer cells.The present work reports the development of pH and enzyme dual responsive polysaccharide vesicular nano-scaffolds for the administration of doxorubicin via physical loading and polymer-drug conjugation to breast cancer cells. Dextran was suitably modified with a renewable resource 3-pentadecyl phenol unit through imine and aliphatic ester chemical linkages that acted as pH and esterase enzyme stimuli, respectively. These dual responsive polysaccharide derivatives self-organized into 200 +/- 10 nm diameter nano-vesicles in water. The water soluble anticancer drug doxorubicin (DOX.HCl) was encapsulated in the hydrophilic pocket to produce core-loaded polysaccharide vesicles whereas chemical conjugation produced DOX anchored at the hydrophobic layer of the dextran nano-vesicles. In vitro studies revealed that about 70-80% of the drug was retained under circulatory conditions at pH = 7.4 and 37 °C. At a low pH of 6.0 to 5.0 and in the presence of esterase; both imine and ester linkages were cleaved instantaneously to release 100% of the loaded drugs. Cytotoxicity assays on Wild Type Mouse Embryonic Fibroblasts (WTMEFs) confirmed the non-toxicity of the newly developed dextran derivatives at up to 500 μg mL-1 in PBS. MTT assays on fibroblast cells revealed that DOX.HCl loaded nano-vesicles exhibited better killing abilities than DOX conjugated polymer nano-vesicles. Both DOX loaded and DOX conjugated nano-vesicles were found to show significant killing in breast cancer cells (MCF 7). Confocal microscopy images confirmed the uptake of DOX loaded (or conjugated) nano-vesicles by cells compared to free DOX. Thus, the newly developed pH and enzyme dual responsive polysaccharide vesicular assemblies are potential drug vectors for the administration of DOX in both loaded and chemically conjugated forms for the efficient killing of breast cancer cells. Electronic supplementary information (ESI) available: 13C NMR of DEX-CHO, 2D NMR spectra of DEX-CHO, 1H NMR of DEX-IM, 1H NMR of DEX-IM-DOX conjugated, absorbance spectra of DEX-IM-DOX conjugated, DLS, FE-SEM and TEM image of DEX-CHO-5, emission spectra of pyrene and Nile red with DEX-IM-10, FE-SEM image of DEX-IM-DOX loaded, FE-SEM image of acid treated DEX-IM-5, absorbance spectra of DOX released, in vitro DOX release from drug loaded and conjugated vesicles in the presence of serum (FBS), DLS data depicting stability of DEX-IM vesicles in serum (FBS), 1HNMR, 13C NMR and HR-MS spectra of all intermediates are provided. See DOI: 10.1039/c5nr00799b

Saccharification of sunflower stalks using lignocellulases from a fungal consortium comprising Pholiota adiposa and Armillaria gemina.

PubMed

Ramachandran, Priyadharshini; Kim, Tae-Su; Dhiman, Saurabh Sudha; Li, Jinglin; Park, Ji-Hyun; Choi, Joon-Ho; Kim, Jae Young; Kim, Dongwook; Lee, Jung-Kul

2015-09-01

Lignocellulases from Armillaria gemina and Pholiota adiposa are efficient in hydrolyzing aspen and poplar biomass, respectively. In the present study, lignocellulosic enzymes obtained from a fungal consortium comprising P. adiposa and A. gemina were used for the saccharification of sunflower stalks. Sunflower stalks were thermochemically pretreated using 2 % NaOH at 50 °C for 24 h. The saccharification process parameters including substrate concentration, enzyme loading, pH, and temperature were optimized using response surface methodology to improve the saccharification yield. The highest enzymatic hydrolysis (84.3 %) was obtained using the following conditions: enzyme loading 10 FPU/g-substrate, substrate 5.5 %, temperature 50 °C, and pH 4.5. The hydrolysis yield obtained using the enzymes from the fungal consortium was equivalent to that obtained using a mixture of commercial enzymes Celluclast and Novozyme β-glucosidase. Addition of up to 500 ppm of heavy metal ions (As, Cu, Fe, Mn, Ni, Pb, and Zn) during saccharification did not significantly affect the saccharification yield. Thus, the biomass grown for phytoremediation of heavy metals can be used for the production of reducing sugars followed by ethanol fermentation.

Direct measurement of adenosine release during hypoxia in the CA1 region of the rat hippocampal slice

PubMed Central

Dale, Nicholas; Pearson, Tim; Frenguelli, Bruno G

2000-01-01

We have used an enzyme-based, twin-barrelled sensor to measure adenosine release during hypoxia in the CA1 region of rat hippocampal slices in conjunction with simultaneous extracellular field recordings of excitatory synaptic transmission. When loaded with a combination of adenosine deaminase, nucleoside phosphorylase and xanthine oxidase, the sensor responded linearly to exogenous adenosine over the concentration range 10 nM to 20 μM. Without enzymes, the sensor when placed on the surface of hippocampal slices recorded a very small net signal during hypoxia of 40 ± 43 pA (mean ±s.e.m.; n = 7). Only when one barrel was loaded with the complete sequence of enzymes and the other with the last two in the cascade did the sensor record a large net difference signal during hypoxia (1226 ± 423 pA; n = 7). This signal increased progressively during the hypoxic episode, scaled with the hypoxic depression of the simultaneously recorded field excitatory postsynaptic potential and was greatly reduced (67 ± 6.5 %; n = 9) by coformycin (0.5-2 μM), a selective inhibitor of adenosine deaminase, the first enzyme in the enzymic cascade within the sensor. For 5 min hypoxic episodes, the sensor recorded a peak concentration of adenosine of 5.6 ± 1.2 μM (n = 16) with an IC50 for the depression of transmission of approximately 3 μM. In slices pre-incubated for 3-6 h in nominally Ca2+-free artificial cerebrospinal fluid, 5 min of hypoxia resulted in an approximately 9-fold greater release of adenosine (48.9 ± 17.7 μM; n = 6). High extracellular Ca2+ (4 mM) both reduced the adenosine signal recorded by the sensor during hypoxia (3.5 ± 0.6 μM; n = 4) and delayed the hypoxic depression of excitatory synaptic transmission. PMID:10878107

Enzyme actuated bioresponsive hydrogels

NASA Astrophysics Data System (ADS)

Wilson, Andrew Nolan

Bioresponsive hydrogels are emerging with technological significance in targeted drug delivery, biosensors and regenerative medicine. Conferred with the ability to respond to specific biologically derived stimuli, the design challenge is in effectively linking the conferred biospecificity with an engineered response tailored to the needs of a particular application. Moreover, the fundamental phenomena governing the response must support an appropriate dynamic range and limit of detection. The design of these systems is inherently complicated due to the high interdependency of the governing phenomena that guide the sensing, transduction, and the actuation response of hydrogels. To investigate the dynamics of these materials, model systems may be used which seek to interrogate the system dynamics by uni-variable experimentation and limit confounding phenomena such as: polymer-solute interactions, polymer swelling dynamics and biomolecular reaction-diffusion concerns. To this end, a model system, alpha-chymotrypsin (Cht) (a protease) and a cleavable peptide-chromogen (pro-drug) covalently incorporated into a hydrogel, was investigated to understand the mechanisms of covalent loading and release by enzymatic cleavage in bio-responsive delivery systems. Using EDC and Sulfo-NHS, terminal carboxyl groups of N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide, a cleavable chromogen, were conjugated to primary amines of a hydrated poly(HEMA)-based hydrogel. Hydrogel discs were incubated in buffered Cht causing enzyme-mediated cleavage of the peptide and concomitant release of the chromophore for monitoring. To investigate substrate loading and the effects of hydrogel morphology on the system, the concentration of the amino groups (5, 10, 20, and 30 mol%) and the cross-linked density (1, 5, 7, 9 and 12 mol%) were independently varied. Loading-Release Efficiency of the chromogen was shown to exhibit a positive relation to increasing amino groups (AEMA). The release rates demonstrated a negative relation to increasing cross-linked density attributed to decreasing void fractions and increasing tortuosities. The diffusion coefficient of Cht, D0, Cht, was determined to be 6.9 +/- 0.5 x 10-7 cm2 s -1, and the range of Deff of Cht for 1 to 12 mol% TEGDA was determined to 6.9 x10-8 to 0.1 x 10 -8cm2 s-1. We show how these parameters may be optimized and used to achieve programmed release rates in engineered bio-responsive systems. The field of bioresponsive hydrogels is continuing to expand as the need for such materials persists. Future work will enable more control over the loading and release of therapeutic and diagnostic moieties. Continued research regarding in enzymatically actuated hydrogels will involve pre-polymerization loading methodologies; in silico diffusion-reaction multiphysics modeling; enzyme actuated degradation of the polymer; and substation of various mediating enzyme, cleavable peptides, and release molecules.

Effects of indigestible dextrin on glucose tolerance in rats.

PubMed

Wakabayashi, S; Kishimoto, Y; Matsuoka, A

1995-03-01

A recently developed indigestible dextrin (IDex) was studied for its effects on glucose tolerance in male Sprague-Dawley rats. IDex is a low viscosity, water-soluble dietary fibre obtained by heating and enzyme treatment of potato starch. It has an average molecular weight of 1600. An oral glucose tolerance test was conducted with 8-week-old rats to evaluate the effects of IDex on the increase in plasma glucose and insulin levels after a single administration of various sugars (1.5 g/kg body weight). The increase in both plasma glucose and insulin levels following sucrose, maltose and maltodextrin loading was significantly reduced by IDex (0.15 g/kg body weight). This effect was not noted following glucose, high fructose syrup and lactose loading. To evaluate the effects of continual IDex ingestion on glucose tolerance, 5-week-old rats were kept for 8 weeks on a stock diet, a high sucrose diet or an IDex-supplemented high sucrose diet. An oral glucose (1.5 g/kg body weight) tolerance test was conducted in week 8. Increases in both plasma glucose and insulin levels following glucose loading were higher in the rats given a high sucrose diet than in the rats fed a stock diet. However, when IDex was included in the high sucrose diet, the impairment of glucose tolerance was alleviated. Moreover, IDex feeding also significantly reduced accumulation of body fat, regardless of changes in body weight. These findings suggest that IDex not only improves glucose tolerance following sucrose, maltose and maltodextrin loading but also stops progressive decrease in glucose tolerance by preventing a high sucrose diet from causing obesity.

Effects of tea saponin on glucan conversion and bonding behaviour of cellulolytic enzymes during enzymatic hydrolysis of corncob residue with high lignin content

PubMed Central

2013-01-01

Background Recently, interest in the utilization of corncob residue (CCR, with high lignin of 45.1%) as a feedstock for bioethanol has been growing. Surfactants have been one of the most popular additives intended to prevent the inhibitory effect of lignin on cellulolytic enzymes, thereby improving hydrolysis. In this study, the effects of biosurfactant tea saponin (TS) on the enzymatic hydrolysis of CCR and the bonding behavior of cellulolytic enzymes to the substrate were investigated. The surface tension in the supernatant was also detected to obtain information about the characteristics and stability of TS. Results The glucose concentration was 17.15 mg/mL at 120 hours of hydrolysis with the low loading of cellulolytic enzymes (7.0 FPU/g cellulose and 10.5 BGU/g cellulose) and 5% CCR. The optimal dosage of TS was its critical micelle concentration (cmc, 1.80 mg/mL). The glucose yield was enhanced from 34.29 to 46.28 g/100 g dry matter by TS. The results indicate that TS can promote the adsorption of cellulolytic enzymes on the substrate and mediate the release of adsorbed enzymes. Meanwhile, TS improves the recovery of the cellulolytic enzymes after a hydrolysis cycle and prevents deactivation of the enzymes during the intense shaking process. The surface tension in supernatants of digested CCR with TS remained at 50.00 mN/m during the course of hydrolysis. It is interesting to note that biosurfactant TS can maintain the surface tension in supernatants, despite its digestibility by cellulolytic enzymes. Conclusions Serving as an accelerant of lignocellulose hydrolysis, TS can also be degraded by the cellulolytic enzymes and release glucose while retaining stability, which reduces the cost of both the cellulolytic enzymes and the additive. As the glucose from the TS could be utilized by yeast, further efforts will investigate the mechanism of function and the application of TS in the production of ethanol by simultaneous saccharification and fermentation (SSF). PMID:24225035

Stochastic molecular model of enzymatic hydrolysis of cellulose for ethanol production

PubMed Central

2013-01-01

Background During cellulosic ethanol production, cellulose hydrolysis is achieved by synergistic action of cellulase enzyme complex consisting of multiple enzymes with different mode of actions. Enzymatic hydrolysis of cellulose is one of the bottlenecks in the commercialization of the process due to low hydrolysis rates and high cost of enzymes. A robust hydrolysis model that can predict hydrolysis profile under various scenarios can act as an important forecasting tool to improve the hydrolysis process. However, multiple factors affecting hydrolysis: cellulose structure and complex enzyme-substrate interactions during hydrolysis make it diffucult to develop mathematical kinetic models that can simulate hydrolysis in presence of multiple enzymes with high fidelity. In this study, a comprehensive hydrolysis model based on stochastic molecular modeling approch in which each hydrolysis event is translated into a discrete event is presented. The model captures the structural features of cellulose, enzyme properties (mode of actions, synergism, inhibition), and most importantly dynamic morphological changes in the substrate that directly affect the enzyme-substrate interactions during hydrolysis. Results Cellulose was modeled as a group of microfibrils consisting of elementary fibrils bundles, where each elementary fibril was represented as a three dimensional matrix of glucose molecules. Hydrolysis of cellulose was simulated based on Monte Carlo simulation technique. Cellulose hydrolysis results predicted by model simulations agree well with the experimental data from literature. Coefficients of determination for model predictions and experimental values were in the range of 0.75 to 0.96 for Avicel hydrolysis by CBH I action. Model was able to simulate the synergistic action of multiple enzymes during hydrolysis. The model simulations captured the important experimental observations: effect of structural properties, enzyme inhibition and enzyme loadings on the hydrolysis and degree of synergism among enzymes. Conclusions The model was effective in capturing the dynamic behavior of cellulose hydrolysis during action of individual as well as multiple cellulases. Simulations were in qualitative and quantitative agreement with experimental data. Several experimentally observed phenomena were simulated without the need for any additional assumptions or parameter changes and confirmed the validity of using the stochastic molecular modeling approach to quantitatively and qualitatively describe the cellulose hydrolysis. PMID:23638989

Evidence for cyclooxygenase-dependent sweating in young males during intermittent exercise in the heat

PubMed Central

Fujii, Naoto; McGinn, Ryan; Stapleton, Jill M; Paull, Gabrielle; Meade, Robert D; Kenny, Glen P

2014-01-01

Our recent work implicated nitric oxide (NO) in the control of sweating during intermittent exercise; however, it is unclear if cyclooxygenase (COX) is also involved. On separate days, ten healthy young (24 ± 4 years) males cycled in the heat (35°C). Two 30 min exercise bouts were performed at either a moderate (400 W, moderate heat load) or high (700 W, high heat load) rate of metabolic heat production and were followed by 20 and 40 min of recovery, respectively. Forearm sweating (ventilated capsule) was evaluated at four skin sites that were continuously perfused via intradermal microdialysis with: (1) lactated Ringer solution (Control), (2) 10 mm ketorolac (a non-selective COX inhibitor), (3) 10 mm NG-nitro-l-arginine methyl ester (l-NAME; a non-selective NO synthase inhibitor) or (4) a combination of 10 mm ketorolac + 10 mml-NAME. During the last 5 min of the first exercise at moderate heat load, forearm sweating (mg min−1 cm−2) was equivalently reduced with ketorolac (0.54 ± 0.08), l-NAME (0.55 ± 0.07) and ketorolac+l-NAME (0.56 ± 0.08) compared to Control (0.67 ± 0.06) (all P < 0.05). Similar results were obtained for the second exercise at moderate heat load (all P < 0.05). However, forearm sweating was similar between the four sites during exercise at high heat load and during recovery regardless of exercise intensity (all P > 0.05). We show that (1) although both COX and NO modulate forearm sweating during intermittent exercise bouts in the heat at a moderate heat load, the effects are not additive, and (2) the contribution of both enzymes to forearm sweating is less evident during intermittent exercise when the heat load is high and during recovery. PMID:25326453

Enzyme nanoparticle fabrication: magnetic nanoparticle synthesis and enzyme immobilization.

PubMed

Johnson, Patrick A; Park, Hee Joon; Driscoll, Ashley J

2011-01-01

Immobilized enzymes are drawing significant attention for potential commercial applications as biocatalysts by reducing operational expenses and by increasing process utilization of the enzymes. Typically, immobilized enzymes have greater thermal and operational stability at various pH values, ionic strengths and are more resistant to denaturation that the soluble native form of the enzyme. Also, immobilized enzymes can be recycled by utilizing the physical or chemical properties of the supporting material. Magnetic nanoparticles provide advantages as the supporting material for immobilized enzymes over competing materials such as: higher surface area that allows for greater enzyme loading, lower mass transfer resistance, less fouling effect, and selective, nonchemical separation from the reaction mixture by an applied a magnetic field. Various surface modifications of magnetic nanoparticles, such as silanization, carbodiimide activation, and PEG or PVA spacing, aid in the binding of single or multienzyme systems to the particles, while cross-linking using glutaraldehyde can also stabilize the attached enzymes.

Enzymatic production of xylooligosaccharides from corn stover and corn cobs treated with aqueous ammonia.

PubMed

Zhu, Yongming; Kim, Tae Hyun; Lee, Y Y; Chen, Rongfu; Elander, Richard T

2006-01-01

A novel method of producing food-grade xylooligosaccharides from corn stover and corn cobs was investigated. The process starts with pretreatment of feedstock in aqueous ammonia, which results delignified and xylan-rich substrate. The pretreated substrates are subjected to enzymatic hydrolysis of xylan using endoxylanase for production of xylooligosaccharides. The conventional enzyme-based method involves extraction of xylan with a strong alkaline solution to form a liquid intermediate containing soluble xylan. This intermediate is heavily contaminated with various extraneous components. A costly purification step is therefore required before enzymatic hydrolysis. In the present method, xylan is obtained in solid form after pretreatment. Water-washing is all that is required for enzymatic hydrolysis of this material. The complex step of purifying soluble xylan from contaminant is essentially eliminated. Refining of xylooligosaccharides to food-grade is accomplished by charcoal adsorption followed by ethanol elution. Xylanlytic hydrolysis of the pretreated corn stover yielded glucan-rich residue that is easily digestible by cellulase enzyme. The digestibility of the residue reached 86% with enzyme loading of 10 filter paper units/g-glucan. As a feedstock for xylooligosaccharides production, corn cobs are superior to corn stover because of high xylan content and high packing density. The high packing density of corn cobs reduces water input and eventually raises the product concentration.

Large pore mesoporous silica nanomaterials for application in delivery of biomolecules

NASA Astrophysics Data System (ADS)

Knežević, Nikola Ž.; Durand, Jean-Olivier

2015-01-01

Various approaches for the synthesis of mesoporous silicate nanoparticles (MSN) with large pore (LP) diameters (in the range of 3-50 nm) are reviewed in this article. The work also covers the construction of magnetic analogues of large pore-mesoporous silica nanoparticles (LPMMSN) and their biomedical applications. The constructed materials exhibit vast potential for application in the loading and delivery of large drug molecules and biomolecules. Literature reports on the application of LPMSN and LPMMSN materials for the adsorption and delivery of proteins, enzymes, antibodies, and nucleic acids are covered in depth, which exemplify their highly potent characteristics for use in drug and biomolecule delivery to diseased tissues.Various approaches for the synthesis of mesoporous silicate nanoparticles (MSN) with large pore (LP) diameters (in the range of 3-50 nm) are reviewed in this article. The work also covers the construction of magnetic analogues of large pore-mesoporous silica nanoparticles (LPMMSN) and their biomedical applications. The constructed materials exhibit vast potential for application in the loading and delivery of large drug molecules and biomolecules. Literature reports on the application of LPMSN and LPMMSN materials for the adsorption and delivery of proteins, enzymes, antibodies, and nucleic acids are covered in depth, which exemplify their highly potent characteristics for use in drug and biomolecule delivery to diseased tissues. Dedicated to Professor Jeffrey I. Zink on the occasion of his 70th birthday.

Non-complexed four cascade enzyme mixture: simple purification and synergetic co-stabilization.

PubMed

Myung, Suwan; Zhang, Y-H Percival

2013-01-01

Cell-free biosystems comprised of synthetic enzymatic pathways would be a promising biomanufacturing platform due to several advantages, such as high product yield, fast reaction rate, easy control and access, and so on. However, it was essential to produce (purified) enzymes at low costs and stabilize them for a long time so to decrease biocatalyst costs. We studied the stability of the four recombinant enzyme mixtures, all of which originated from thermophilic microorganisms: triosephosphate isomerase (TIM) from Thermus thermophiles, fructose bisphosphate aldolase (ALD) from Thermotoga maritima, fructose bisphosphatase (FBP) from T. maritima, and phosphoglucose isomerase (PGI) from Clostridium thermocellum. It was found that TIM and ALD were very stable at evaluated temperature so that they were purified by heat precipitation followed by gradient ammonia sulfate precipitation. In contrast, PGI was not stable enough for heat treatment. In addition, the stability of a low concentration PGI was enhanced by more than 25 times in the presence of 20 mg/L bovine serum albumin or the other three enzymes. At a practical enzyme loading of 1000 U/L for each enzyme, the half-life time of free PGI was prolong to 433 h in the presence of the other three enzymes, resulting in a great increase in the total turn-over number of PGI to 6.2×10(9) mole of product per mole of enzyme. This study clearly suggested that the presence of other proteins had a strong synergetic effect on the stabilization of the thermolabile enzyme PGI due to in vitro macromolecular crowding effect. Also, this result could be used to explain why not all enzymes isolated from thermophilic microorganisms are stable in vitro because of a lack of the macromolecular crowding environment.

Stability of commercial glucanase and β-glucosidase preparations under hydrolysis conditions.

PubMed

Rosales-Calderon, Oscar; Trajano, Heather L; Duff, Sheldon J B

2014-01-01

The cost of enzymes makes enzymatic hydrolysis one of the most expensive steps in the production of lignocellulosic ethanol. Diverse studies have used commercial enzyme cocktails assuming that change in total protein concentration during hydrolysis was solely due to adsorption of endo- and exoglucanases onto the substrate. Given the sensitivity of enzymes and proteins to media conditions this assumption was tested by evaluating and modeling the protein concentration of commercial cocktails at hydrolysis conditions. In the absence of solid substrate, the total protein concentration of a mixture of Celluclast 1.5 L and Novozyme 188 decreased by as much as 45% at 50 °C after 4 days. The individual cocktails as well as a mixture of both were stable at 20 °C. At 50 °C, the protein concentration of Celluclast 1.5 was relatively constant but Novozyme 188 decreased by as much as 77%. It was hypothesized that Novozyme 188 proteins suffer a structural change at 50 °C which leads to protein aggregation and precipitation. Lyophilized β-glucosidase (P-β-glucosidase) at 50 °C exhibited an aggregation rate which was successfully modeled using first order kinetics (R (2) = 0.97). By incorporating the possible presence of chaperone proteins in Novozyme 188, the protein aggregation observed for this cocktail was successfully modeled (R (2) = 0.96). To accurately model the increasing protein stability observed at high cocktail loadings, the model was modified to include the presence of additives in the cocktail (R (2) = 0.98). By combining the measurement of total protein concentration with the proposed Novozyme 188 protein aggregation model, the endo- and exoglucanases concentration in the solid and liquid phases during hydrolysis can be more accurately determined. This methodology can be applied to various systems leading to optimization of enzyme loading by minimizing the excess of endo- and exoglucanases. In addition, the monitoring of endo- and exoglucanases concentrations can be used to build mass balances of enzyme recycling processes and to techno-economically evaluate the viability of enzyme recycling.

Stability of commercial glucanase and β-glucosidase preparations under hydrolysis conditions

PubMed Central

Rosales-Calderon, Oscar; Duff, Sheldon J.B.

2014-01-01

The cost of enzymes makes enzymatic hydrolysis one of the most expensive steps in the production of lignocellulosic ethanol. Diverse studies have used commercial enzyme cocktails assuming that change in total protein concentration during hydrolysis was solely due to adsorption of endo- and exoglucanases onto the substrate. Given the sensitivity of enzymes and proteins to media conditions this assumption was tested by evaluating and modeling the protein concentration of commercial cocktails at hydrolysis conditions. In the absence of solid substrate, the total protein concentration of a mixture of Celluclast 1.5 L and Novozyme 188 decreased by as much as 45% at 50 °C after 4 days. The individual cocktails as well as a mixture of both were stable at 20 °C. At 50 °C, the protein concentration of Celluclast 1.5 was relatively constant but Novozyme 188 decreased by as much as 77%. It was hypothesized that Novozyme 188 proteins suffer a structural change at 50 °C which leads to protein aggregation and precipitation. Lyophilized β-glucosidase (P-β-glucosidase) at 50 °C exhibited an aggregation rate which was successfully modeled using first order kinetics (R2 = 0.97). By incorporating the possible presence of chaperone proteins in Novozyme 188, the protein aggregation observed for this cocktail was successfully modeled (R2 = 0.96). To accurately model the increasing protein stability observed at high cocktail loadings, the model was modified to include the presence of additives in the cocktail (R2 = 0.98). By combining the measurement of total protein concentration with the proposed Novozyme 188 protein aggregation model, the endo- and exoglucanases concentration in the solid and liquid phases during hydrolysis can be more accurately determined. This methodology can be applied to various systems leading to optimization of enzyme loading by minimizing the excess of endo- and exoglucanases. In addition, the monitoring of endo- and exoglucanases concentrations can be used to build mass balances of enzyme recycling processes and to techno-economically evaluate the viability of enzyme recycling. PMID:24949230

Comprehensive characterization of non-cellulosic recalcitrant cell wall carbohydrates in unhydrolyzed solids from AFEX-pretreated corn stover.

PubMed

Gunawan, Christa; Xue, Saisi; Pattathil, Sivakumar; da Costa Sousa, Leonardo; Dale, Bruce E; Balan, Venkatesh

2017-01-01

Inefficient carbohydrate conversion has been an unsolved problem for various lignocellulosic biomass pretreatment technologies, including AFEX, dilute acid, and ionic liquid pretreatments. Previous work has shown 22% of total carbohydrates are typically unconverted, remaining as soluble or insoluble oligomers after hydrolysis (72 h) with excess commercial enzyme loading (20 mg enzymes/g biomass). Nearly one third (7 out of 22%) of these total unconverted carbohydrates are present in unhydrolyzed solid (UHS) residues. The presence of these unconverted carbohydrates leads to a considerable sugar yield loss, which negatively impacts the overall economics of the biorefinery. Current commercial enzyme cocktails are not effective to digest specific cross-linkages in plant cell wall glycans, especially some of those present in hemicelluloses and pectins. Thus, obtaining information about the most recalcitrant non-cellulosic glycan cross-linkages becomes a key study to rationally improve commercial enzyme cocktails, by supplementing the required enzyme activities for hydrolyzing those unconverted glycans. In this work, cell wall glycans that could not be enzymatically converted to monomeric sugars from AFEX-pretreated corn stover (CS) were characterized using compositional analysis and glycome profiling tools. The pretreated CS was hydrolyzed using commercial enzyme mixtures comprising cellulase and hemicellulase at 7% glucan loading (~20% solid loading). The carbohydrates present in UHS and liquid hydrolysate were evaluated over a time period of 168 h enzymatic hydrolysis. Cell wall glycan-specific monoclonal antibodies (mAbs) were used to characterize the type and abundance of non-cellulosic polysaccharides present in UHS over the course of enzymatic hydrolysis. 4- O -methyl-d-glucuronic acid-substituted xylan and pectic-arabinogalactan were found to be the most abundant epitopes recognized by mAbs in UHS and liquid hydrolysate, suggesting that the commercial enzyme cocktails used in this work are unable to effectively target those substituted polysaccharide residues. To our knowledge, this is the first report using glycome profiling as a tool to dynamically monitor recalcitrant cell wall carbohydrates during the course of enzymatic hydrolysis. Glycome profiling of UHS and liquid hydrolysates unveiled some of the glycans that are not cleaved and enriched after enzyme hydrolysis. The major polysaccharides include 4- O -methyl-d-glucuronic acid-substituted xylan and pectic-arabinogalactan, suggesting that enzymes with glucuronidase and arabinofuranosidase activities are required to maximize monomeric sugar yields. This methodology provides a rapid tool to assist in developing new enzyme cocktails, by supplementing the existing cocktails with the required enzyme activities for achieving complete deconstruction of pretreated biomass in the future.

Trypsin coatings on electrospun and alcohol-dispersed polymer nanofibers for trypsin digestion column

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jun, Seung-Hyun; Chang, Mun Seock; Kim, Byoung Chan

2010-09-15

The construction of a trypsin reactor in a chromatography column for rapid and efficient protein digestion in proteomics is described. Electrospun and alcohol-dispersed polymer nanofibers were used for the fabrication of highly stable trypsin coating, which was prepared by a two-step process of covalent attachment and enzyme crosslinking. In a comparative study with the trypsin coatings on asspun and non-dispersed nanofibers, it has been observed that a simple step of alcohol dispersion improved not only the enzyme loading but also the performance of protein digestion. In-column digestion of enolase was successfully performed in less than twenty minutes. By applying themore » alcohol dispersion of polymer nanofibers, the bypass of samples was reduced by filling up the column with well-dispersed nanofibers, and subsequently, interactions between the protein and the enzymes were improved yielding more complete and reproducible digestions. Regardless of alcohol-dispersion or not, trypsin coating showed better digestion performance and improved performance stability under recycled uses than covalently-attached trypsin. The combination of highly stable trypsin coating and alcoholdispersion of polymer nanofibers has opened up a new potential to develop a trypsin column for on-line and automated protein digestion.« less

Hydrolysis of virgin coconut oil using immobilized lipase in a batch reactor.

PubMed

Chua, Lee Suan; Alitabarimansor, Meisam; Lee, Chew Tin; Mat, Ramli

2012-01-01

Hydrolysis of virgin coconut oil (VCO) had been carried out by using an immobilised lipase from Mucor miehei (Lipozyme) in a water-jacketed batch reactor. The kinetic of the hydrolysis was investigated by varying the parameters such as VCO concentration, enzyme loading, water content, and reaction temperature. It was found that VCO exhibited substrate inhibition at the concentration more than 40% (v/v). Lipozyme also achieved the highest production of free fatty acids, 4.56 mM at 1% (w/v) of enzyme loading. The optimum water content for VCO hydrolysis was 7% (v/v). A relatively high content of water was required because water was one of the reactants in the hydrolysis. The progress curve and the temperature profile of the enzymatic hydrolysis also showed that Lipozyme could be used for free fatty acid production at the temperature up to 50°C. However, the highest initial reaction rate and the highest yield of free fatty acid production were at 45 and 40°C, respectively. A 100 hours of initial reaction time has to be compensated in order to obtain the highest yield of free fatty acid production at 40°C.

Hydrolysis of Virgin Coconut Oil Using Immobilized Lipase in a Batch Reactor

PubMed Central

Chua, Lee Suan; Alitabarimansor, Meisam; Lee, Chew Tin; Mat, Ramli

2012-01-01

Hydrolysis of virgin coconut oil (VCO) had been carried out by using an immobilised lipase from Mucor miehei (Lipozyme) in a water-jacketed batch reactor. The kinetic of the hydrolysis was investigated by varying the parameters such as VCO concentration, enzyme loading, water content, and reaction temperature. It was found that VCO exhibited substrate inhibition at the concentration more than 40% (v/v). Lipozyme also achieved the highest production of free fatty acids, 4.56 mM at 1% (w/v) of enzyme loading. The optimum water content for VCO hydrolysis was 7% (v/v). A relatively high content of water was required because water was one of the reactants in the hydrolysis. The progress curve and the temperature profile of the enzymatic hydrolysis also showed that Lipozyme could be used for free fatty acid production at the temperature up to 50°C. However, the highest initial reaction rate and the highest yield of free fatty acid production were at 45 and 40°C, respectively. A 100 hours of initial reaction time has to be compensated in order to obtain the highest yield of free fatty acid production at 40°C. PMID:22953055

Understanding the synergistic effect and the main factors influencing the enzymatic hydrolyzability of corn stover at low enzyme loading by hydrothermal and/or ultrafine grinding pretreatment.

PubMed

Zhang, Haiyan; Li, Junbao; Huang, Guangqun; Yang, Zengling; Han, Lujia

2018-05-26

A thorough assessment of the microstructural changes and synergistic effects of hydrothermal and/or ultrafine grinding pretreatment on the subsequent enzymatic hydrolysis of corn stover was performed in this study. The mechanism of pretreatment was elucidated by characterizing the particle size, specific surface area (SSA), pore volume (PV), average pore size, cellulose crystallinity (CrI) and surface morphology of the pretreated samples. In addition, the underlying relationships between the structural parameters and final glucose yields were elucidated, and the relative significance of the factors influencing enzymatic hydrolyzability were assessed by principal component analysis (PCA). Hydrothermal pretreatment at a lower temperature (170 °C) combined with ultrafine grinding achieved a high glucose yield (80.36%) at a low enzyme loading (5 filter paper unit (FPU)/g substrate) which is favorable. The relative significance of structural parameters in enzymatic hydrolyzability was SSA > PV > average pore size > CrI/cellulose > particle size. PV and SSA exhibited logarithmic correlations with the final enzymatic hydrolysis yield. Copyright © 2018 Elsevier Ltd. All rights reserved.

Efficient in situ growth of enzyme-inorganic hybrids on paper strips for the visual detection of glucose.

PubMed

Li, WanYun; Lu, ShiYu; Bao, ShuJuan; Shi, ZhuanZhuan; Lu, Zhisong; Li, ChangMing; Yu, Ling

2018-01-15

A visual colorimetric microfluidic paper-based analytical device (μPAD) was constructed following the direct synthesis of enzyme-inorganic hybrid nanomaterials on the paper matrix. An inorganic solution of MnSO 4 and KH 2 PO 4 containing a diluted enzyme (glucose oxidase, GOx) was subsequently pipetted onto cellulose paper for the in situ growth of GOx@Mn 3 (PO 4 ) 2 hybrid functional materials. The characterization of the morphology and chemical composition validated the presence of hybrid materials roots in the paper fiber, while the Mn 3 (PO 4 ) 2 of the hybrid provided both a surface for enzyme anchoring and a higher peroxidase-like catalytic activity as compared to the Mn 3 (PO 4 ) 2 crystal that was synthesized without enzyme modulation. This new approach for the in situ growth of an enzyme-inorganic hybrid on a paper matrix eliminates centrifugation and the dry process by casting the solution on paper. The sensing material loading was highly reproducible because of the accuracy and stability of pipetting, which eventually contributed to the reliability of the μPAD. The self-assembled natural and artificial enzyme hybrid on the μPADs specifically detected glucose from a group of interferences, which shows great specificity using this method. Moreover, the colorimetric signal exhibited detection limitation for glucose is 0.01mM, which lies in the physiological range of glucose in biological samples. Copyright © 2017 Elsevier B.V. All rights reserved.

Novel catalase loaded nanocores for the treatment of inflammatory bowel diseases.

PubMed

Parihar, Arun K S; Srivastava, Shikha; Patel, Satish; Singh, Manju R; Singh, Deependra

2017-08-01

Inflammatory bowel disease (IBD) is an inflammatory disorder of the digestive tract reported to be primarily caused by oxidative stress. In this study, alginate encapsulated nanoceramic carriers were designed to deliver acid labile antioxidant enzyme catalase orally. Complete system was characterized for size, loading efficiency, in vitro antioxidant assay and in vitro release. The prepared nanoceramic system was found to be spherical with diameter of 925 ± 6.81 nm. The in vitro release data followed the Higuchi model in acidic buffer whereas in alkaline pH sustained and almost first order release of enzyme was observed up to 6 h.

The simultaneous saccharification and fermentation of pretreated woody crops to ethanol

DOE Office of Scientific and Technical Information (OSTI.GOV)

Spindler, D.D.; Wyman, C.E.; Grohmann, K.

1991-12-31

Four promising woody crops (Populus maximowiczii x nigra (NE388), P. trichocarpa x deltoides (N11), P. tremuloides, and Sweetgum Liquidambar styraciflua) were pretreated by dilute sulfuric acid and evaluated in the simultaneous saccharification and fermentation (SSF) process for ethanol production. The yeast Saccharomyces cerevisiae was used in the fermentations alone, and in mixed cultures with {beta}-glucosidase producing Brettanomyces clausenii. Commercial Genencor 150L cellulose enyme was either employed alone or supplemented with {beta}-glucosidase. All SSFs were run at 37{degrees}C for 8 d and compared to saccharifications at 45{degrees}C under the same enzyme loadings. S. cerevisiae alone achieved the highest ethanol yields andmore » rates of hydrolysis at the higher enzyme loadings, whereas the mixed culture performed better at the lower enzyme loadings without {beta}-glucosidase supplementation. The best overall rates of fermentation (3 d) and final theoretical ethanol yields (86-90%) were achieved with P. maximowiczii x nigra (NE388) and Sweetgum Liquidambar styraciflua, followed by P. tremuloides and P. trichocarpa x deltoides (N11) with slightly slower rates and lower yields. Although there were some differences in SSF performance, all these pretreated woody crops show promise as substrates for ethanol production.« less

Towards complete hydrolysis of soy flour carbohydrates by enzyme mixtures for protein enrichment: A modeling approach.

PubMed

Loman, Abdullah Al; Ju, Lu-Kwang

2016-05-01

Soy protein is a well-known nutritional supplement in proteinaceous food and animal feed. However, soybeans contain complex carbohydrate. Selective carbohydrate removal by enzymes could increase the protein content and remove the indigestibility of soy products for inclusion in animal feed. Complete hydrolysis of soy flour carbohydrates is challenging due to the presence of proteins and different types of non-structural polysaccharides. This study is designed to guide complex enzyme mixture required for hydrolysis of all types of soy flour carbohydrates. Enzyme broths from Aspergillus niger, Aspergillus aculeatus and Trichoderma reesei fermentations were evaluated in this study for soy carbohydrate hydrolysis. The resultant hydrolysate was measured for solubilized carbohydrate by both total carbohydrate and reducing sugar analyses. Conversion data attained after 48h hydrolysis were first fitted with models to determine the maximum fractions of carbohydrate hydrolyzable by each enzyme group, i.e., cellulase, xylanase, pectinase and α-galactosidase. Kinetic models were then developed to describe the increasing conversions over time under different enzyme activities and process conditions. The models showed high fidelity in predicting soy carbohydrate hydrolysis over broad ranges of soy flour loading (5-25%) and enzyme activities: per g soy flour, cellulase, 0.04-30 FPU; xylanase, 3.5-618U; pectinase, 0.03-120U; and α-galactosidase, 0.01-60U. The models are valuable in guiding the development and production of optimal enzyme mixtures toward hydrolysis of all types of carbohydrates present in soy flour and in optimizing the design and operation of hydrolysis reactor and process. Copyright © 2016 Elsevier Inc. All rights reserved.

Functional characterisation and permeation studies of lyophilised thiolated chitosan xerogels for buccal delivery of insulin.

PubMed

Boateng, Joshua S; Mitchell, John C; Pawar, Harshavardhan; Ayensu, Isaac

2014-01-01

Stable and mucoadhesive, lyophilised, thiolated chitosan xerogels, loaded with insulin for buccal mucosa deliv- ery, in place of the currently used parenteral route have been developed. The xerogels were backed with impervious ethyl- cellulose laminate to ensure unidirectional release and also loaded with enzyme inhibitor to enhance insulin permeability across the buccal mucosa. Characterisation of xerogels using(1) HNMR confirmed the degree of deacetylation of the syn- thesised thiolated chitosan. The amount of thiol groups immobilised on the modified chitosan was quantified by Ellman's reaction and molecular weight monitored by gel permeation chromatography. The stability of the secondary structure of insulin was examined by attenuated total reflectance Fourier transform infra-red spectroscopy and circular dichroism. In vitro and ex vivo permeation studies were undertaken by using EpiOral ™ and sheep buccal membrane respectively. Insu- lin released from thiolated chitosan xerogels, loaded with aprotinin (enzyme inhibitor and permeation enhancer) showed a 1.7-fold increase in permeation through EpiOral ™ buccal tissue construct compared to the pure drug. However, permea- tion was decreased for xerogels containing the enzyme inhibitor glutathione. Further, aprotinin containing xerogels en- hanced insulin permeation through sheep buccal membrane and demonstrated good linear correlation with the permeation data from the EpiOral ™ study. The results show the potential application of lyoph ilised thiolated chitosan xerogels con- taining aprotinin with improved mucoadhesion, penetration enhancing and enzyme inhibition characteristics for buccal mucosa delivery of macromolecules such as insulin.

Utilization of recombinant Trichoderma reesei expressing Aspergillus aculeatus β-glucosidase I (JN11) for a more economical production of ethanol from lignocellulosic biomass.

PubMed

Treebupachatsakul, Treesukon; Shioya, Koki; Nakazawa, Hikaru; Kawaguchi, Takashi; Morikawa, Yasushi; Shida, Yosuke; Ogasawara, Wataru; Okada, Hirofumi

2015-12-01

The capacity of Trichoderma reesei cellulase to degrade lignocellulosic biomass has been enhanced by the construction of a recombinant T. reesei strain expressing Aspergillus aculeatus β-glucosidase I. We have confirmed highly efficient ethanol production from converge-milled Japanese cedar by recombinant T. reesei expressing A. aculeatus β-glucosidase I (JN11). We investigated the ethanol productivity of JN11 and compared it with the cocktail enzyme T. reesei PC-3-7 with reinforced cellobiase activity by the commercial Novozyme 188. Results showed that the ethanol production efficiency under enzymatic hydrolysis of JN11 was comparable to the cocktail enzyme both on simultaneous saccharification and fermentation (SSF) or separate hydrolysis and fermentation (SHF) processes. Moreover, the cocktail enzyme required more protein loading for attaining similar levels of ethanol conversion as JN11. We propose that JN11 is an intrinsically economical enzyme that can eliminate the supplementation of BGL for PC-3-7, thereby reducing the cost of industrial ethanol production from lignocellulosic biomass. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Suitable technological conditions for enzymatic hydrolysis of waste paper by Novozymes® enzymes NS50013 and NS50010.

PubMed

Brummer, Vladimir; Skryja, Pavel; Jurena, Tomas; Hlavacek, Viliam; Stehlik, Petr

2014-10-01

Waste paper belongs to a group of quantitatively the most produced waste types. Enzymatic hydrolysis is becoming a suitable way to treat this type of waste and at the same time, to produce a valuable liquid biofuel, because reducing sugars solutions that are formed during the process of saccharification can be a precursor for following or simultaneous fermentation. If it will be possible to make the enzymatic hydrolysis of the waste paper economically viable, it could serve as one of the new ways to lower the dependence of the transport sector on oil in the future. Only several studies comparing the enzymatic hydrolysis of different waste papers were performed in the past; they are summarized in this manuscript. In our experimental trials, suitable technological conditions for waste paper enzymatic hydrolysis using enzymes from Novozymes® biomass kit: enzymes NS50013 and NS50010 were investigated. The following enzymatic hydrolysis parameters in laboratory scale trials were verified on high cellulose content substrates-filter paper and cellulose pulp: type of buffer, pH, temperature, concentration of the substrate, loading of the enzyme and rate of stirring.

Optimization of pectinase immobilization on grafted alginate-agar gel beads by 24 full factorial CCD and thermodynamic profiling for evaluating of operational covalent immobilization.

PubMed

Abdel Wahab, Walaa A; Karam, Eman A; Hassan, Mohamed E; Kansoh, Amany L; Esawy, Mona A; Awad, Ghada E A

2018-07-01

Pectinase produced by a honey derived from the fungus Aspergillus awamori KX943614 was covalently immobilized onto gel beads made of alginate and agar. Polyethyleneimine, glutaraldehyde, loading time and enzyme's units were optimized by 2 4 full factorial central composite design (CCD). The immobilization process increased the optimal working pH for the free pectinase from 5 to a broader range of pH4.5-5.5 and the optimum operational temperature from 55°C to a higher temperature, of 60°C, which is favored to reduce the enzyme's microbial contamination. The thermodynamics studies showed a thermal stability enhancement against high temperature for the immobilized formula. Moreover, an increase in half-lives and D-values was achieved. The thermodynamic studies proved that immobilization of pectinase made a remarkable increase in enthalpy and free energy because of enzyme stability enhancement. The reusability test revealed that 60% of pectinase's original activity was retained after 8 successive cycles. This gel formula may be convenient for immobilization of other industrial enzymes. Copyright © 2018 Elsevier B.V. All rights reserved.

Enzyme and Thermal Dual Responsive Amphiphilic Polymer Core-Shell Nanoparticle for Doxorubicin Delivery to Cancer Cells.

PubMed

Kashyap, Smita; Singh, Nitesh; Surnar, Bapurao; Jayakannan, Manickam

2016-01-11

Dual responsive polymer nanoscaffolds for administering anticancer drugs both at the tumor site and intracellular compartments are made for improving treatment in cancers. The present work reports the design and development of new thermo- and enzyme-responsive amphiphilic copolymer core-shell nanoparticles for doxorubicin delivery at extracellular and intracellular compartments, respectively. A hydrophobic acrylate monomer was tailor-made from 3-pentadecylphenol (PDP, a natural resource) and copolymerized with oligoethylene glycol acrylate (as a hydrophilic monomer) to make new classes of thermo and enzyme dual responsive polymeric amphiphiles. Both radical and reversible addition-fragmentation chain transfer (RAFT) methodologies were adapted for making the amphiphilic copolymers. These amphiphilic copolymers were self-assembled to produce spherical core-shell nanoparticles in water. Upon heating, the core-shell nanoparticles underwent segregation to produce larger sized aggregates above the lower critical solution temperature (LCST). The dual responsive polymer scaffold was found to be capable of loading water insoluble drug, such as doxorubicin (DOX), and fluorescent probe-like Nile Red. The drug release kinetics revealed that DOX was preserved in the core-shell assemblies at normal body temperature (below LCST, ≤ 37 °C). At closer to cancer tissue temperature (above LCST, ∼43 °C), the polymeric scaffold underwent burst release to deliver 90% of loaded drugs within 2 h. At the intracellular environment (pH 7.4, 37 °C) in the presence of esterase enzyme, the amphiphilic copolymer ruptured in a slow and controlled manner to release >95% of the drugs in 12 h. Thus, both burst release of cargo at the tumor microenvironment and control delivery at intracellular compartments were accomplished in a single polymer scaffold. Cytotoxicity assays of the nascent and DOX-loaded polymer were carried out in breast cancer (MCF-7) and cervical cancer (HeLa) cells. Among the two cell lines, the DOX-loaded polymers showed enhanced killing in breast cancer cells. Furthermore, the cellular uptake of the DOX was studied by confocal and fluorescence microscopes. The present investigation opens a new enzyme and thermal-responsive polymer scaffold approach for DOX delivery in cancer cells.

Comparison of microwave and conduction-convection heating autohydrolysis pretreatment for bioethanol production.

PubMed

Aguilar-Reynosa, Alejandra; Romaní, Aloia; Rodríguez-Jasso, Rosa M; Aguilar, Cristóbal N; Garrote, Gil; Ruiz, Héctor A

2017-11-01

This work describes the application of two forms of heating for autohydrolysis pretreatment on isothermal regimen: conduction-convection heating and microwave heating processing using corn stover as raw material for bioethanol production. Pretreatments were performed using different operational conditions: residence time (10-50 min) and temperature (160-200°C) for both pretreatments. Subsequently, the susceptibility of pretreated solids was studied using low enzyme loads, and high substrate loads. The highest conversion was 95.1% for microwave pretreated solids. Also solids pretreated by microwave heating processing showed better ethanol conversion in simultaneous saccharification and fermentation process (92% corresponding to 33.8g/L). Therefore, microwave heating processing is a promising technology in the pretreatment of lignocellulosic materials. Copyright © 2017 Elsevier Ltd. All rights reserved.

Evolved strains of Scheffersomyces stipitis achieving high ethanol productivity on acid- and base-pretreated biomass hydrolyzate at high solids loading

DOE Office of Scientific and Technical Information (OSTI.GOV)

Slininger, Patricia J.; Shea-Andersh, Maureen A.; Thompson, Stephanie R.

Lignocellulosic biomass is an abundant, renewable feedstock useful for the production of fuel-grade ethanol via the processing steps of pretreatment, enzyme hydrolysis, and microbial fermentation. Traditional industrial yeasts do not ferment xylose and are not able to grow, survive, or ferment in concentrated hydrolyzates that contain enough sugar to support economical ethanol recovery since they are laden with toxic byproducts generated during pretreatment. Repetitive culturing in two types of concentrated hydrolyzates was applied along with ethanol challenged xylose-fed continuous culture to force targeted evolution of the native pentose fermenting yeast Scheffersomyces (Pichia) stipitis strain NRRL Y-7124 maintained in the ARSmore » Culture Collection, Peoria, IL. Isolates collected from various enriched populations were screened and ranked based on relative xylose uptake rate and ethanol yield. Ranking on hydrolyzates with and without nutritional supplementation was used to identify those isolates with best performance across diverse conditions. Robust S. stipitis strains adapted to perform very well in enzyme hydrolyzates of high solids loading ammonia fiber expansion-pretreated corn stover (18% weight per volume solids) and dilute sulfuric acid-pretreated switchgrass (20% w/v solids) were obtained. Improved features include reduced initial lag phase preceding growth, significantly enhanced fermentation rates, improved ethanol tolerance and yield, reduced diauxic lag during glucose-xylose transition, and ability to accumulate >40 g/L ethanol in <167 h when fermenting hydrolyzate at low initial cell density of 0.5 absorbance units and pH 5 to 6.« less

Evolved strains of Scheffersomyces stipitis achieving high ethanol productivity on acid- and base-pretreated biomass hydrolyzate at high solids loading

DOE PAGES

Slininger, Patricia J.; Shea-Andersh, Maureen A.; Thompson, Stephanie R.; ...

2015-04-09

Lignocellulosic biomass is an abundant, renewable feedstock useful for the production of fuel-grade ethanol via the processing steps of pretreatment, enzyme hydrolysis, and microbial fermentation. Traditional industrial yeasts do not ferment xylose and are not able to grow, survive, or ferment in concentrated hydrolyzates that contain enough sugar to support economical ethanol recovery since they are laden with toxic byproducts generated during pretreatment. Repetitive culturing in two types of concentrated hydrolyzates was applied along with ethanol challenged xylose-fed continuous culture to force targeted evolution of the native pentose fermenting yeast Scheffersomyces (Pichia) stipitis strain NRRL Y-7124 maintained in the ARSmore » Culture Collection, Peoria, IL. Isolates collected from various enriched populations were screened and ranked based on relative xylose uptake rate and ethanol yield. Ranking on hydrolyzates with and without nutritional supplementation was used to identify those isolates with best performance across diverse conditions. Robust S. stipitis strains adapted to perform very well in enzyme hydrolyzates of high solids loading ammonia fiber expansion-pretreated corn stover (18% weight per volume solids) and dilute sulfuric acid-pretreated switchgrass (20% w/v solids) were obtained. Improved features include reduced initial lag phase preceding growth, significantly enhanced fermentation rates, improved ethanol tolerance and yield, reduced diauxic lag during glucose-xylose transition, and ability to accumulate >40 g/L ethanol in <167 h when fermenting hydrolyzate at low initial cell density of 0.5 absorbance units and pH 5 to 6.« less

Biochemical and Molecular Characterization of a Serine Keratinase from Brevibacillus brevis US575 with Promising Keratin-Biodegradation and Hide-Dehairing Activities

PubMed Central

Jaouadi, Nadia Zaraî; Rekik, Hatem; Badis, Abdelmalek; Trabelsi, Sahar; Belhoul, Mouna; Yahiaoui, Amina Benkiar; Aicha, Houda Ben; Toumi, Abdessatar; Bejar, Samir; Jaouadi, Bassem

2013-01-01

Dehairing is one of the highly polluting operations in the leather industry. The conventional lime-sulfide process used for dehairing produces large amounts of sulfide, which poses serious toxicity and disposal problems. This operation also involves hair destruction, a process that leads to increased chemical oxygen demand (COD), biological oxygen demand (BOD), and total suspended solid (TSS) loads in the effluent. With these concerns in mind, enzyme-assisted dehairing has often been proposed as an alternative method. The main enzyme preparations so far used involved keratinases. The present paper reports on the purification of an extracellular keratinase (KERUS) newly isolated from Brevibacillus brevis strain US575. Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) analysis revealed that the purified enzyme was a monomer with a molecular mass of 29121.11 Da. The sequence of the 27 N-terminal residues of KERUS showed high homology with those of Bacillus keratinases. Optimal activity was achieved at pH 8 and 40°C. Its thermoactivity and thermostability were upgraded in the presence of 5 mM Ca2+. The enzyme was completely inhibited by phenylmethanesulfonyl fluoride (PMSF) and diiodopropyl fluorophosphates (DFP), which suggests that it belongs to the serine protease family. KERUS displayed higher levels of hydrolysis, substrate specificity, and catalytic efficiency than NUE 12 MG and KOROPON® MK EG keratinases. The enzyme also exhibited powerful keratinolytic activity that made it able to accomplish the entire feather-biodegradation process on its own. The kerUS gene encoding KERUS was cloned, sequenced, and expressed in Escherichia coli. The biochemical properties of the extracellular purified recombinant enzyme (rKERUS) were similar to those of native KERUS. Overall, the findings provide strong support for the potential candidacy of this enzyme as an effective and eco-friendly alternative to the conventional chemicals used for the dehairing of rabbit, goat, sheep and bovine hides in the leather processing industry. PMID:24146914

Retinoid receptors, transporters, and metabolizers as therapeutic targets in late onset Alzheimer disease.

PubMed

Goodman, Ann B

2006-12-01

Vitamin A (retinoid) is required in the adult brain to enable cognition, learning, and memory. While brain levels of retinoid diminish over the course of normal ageing, retinoid deficit is greater in late onset Alzheimer disease (LOAD) brains than in normal-aged controls. This paper reviews recent evidence supporting these statements and further suggests that genes necessary for the synthesis, transport and function of retinoid to and within the ageing brain are appropriate targets for treatment of LOAD. These genes tend to be clustered with genes that have been proposed as candidates in LOAD, are found at chromosomal regions linked to LOAD, and suggest the possibility of an overall coordinated regulation. This phenomenon is termed Chromeron and is analogous to the operon mechanism observed in prokaryotes. Suggested treatment targets are the retinoic-acid inactivating enzymes (CYP26)s, the retinol binding and transport proteins, retinol-binding protein (RBP)4 and transthyretin (TTR), and the retinoid receptors. TTR as a LOAD target is the subject of active investigation. The retinoid receptors and the retinoid-inactivating enzymes have previously been proposed as targets. This is the first report to suggest that RBP4 is an amenable treatment target in LOAD. RBP4 is elevated in type-2 diabetes and obesity, conditions associated with increased risk for LOAD. Fenretinide, a novel synthetic retinoic acid (RA) analog lowers RBP4 in glucose intolerant obese mice. The feasibility of using fenretinide either as an adjunct to present LOAD therapies, or on its own as an early prevention strategy should be determined. (c) 2006 Wiley-Liss, Inc.

Enzymatic hydrolysis of steam-pretreated lignocellulosic materials with Trichoderma atroviride enzymes produced in-house

PubMed Central

Kovacs, Krisztina; Macrelli, Stefano; Szakacs, George; Zacchi, Guido

2009-01-01

Background Improvement of the process of cellulase production and development of more efficient lignocellulose-degrading enzymes are necessary in order to reduce the cost of enzymes required in the biomass-to-bioethanol process. Results Lignocellulolytic enzyme complexes were produced by the mutant Trichoderma atroviride TUB F-1663 on three different steam-pretreated lignocellulosic substrates, namely spruce, wheat straw and sugarcane bagasse. Filter paper activities of the enzymes produced on the three materials were very similar, while β-glucosidase and hemicellulase activities were more dependent on the nature of the substrate. Hydrolysis of the enzyme preparations investigated produced similar glucose yields. However, the enzymes produced in-house proved to degrade the xylan and the xylose oligomers less efficiently than a commercial mixture of cellulase and β-glucosidase. Furthermore, accumulation of xylose oligomers was observed when the TUB F-1663 supernatants were applied to xylan-containing substrates, probably due to the low β-xylosidase activity of the enzymes. The efficiency of the enzymes produced in-house was enhanced by supplementation with extra commercial β-glucosidase and β-xylosidase. When the hydrolytic capacities of various mixtures of a commercial cellulase and a T. atroviride supernatant produced in the lab were investigated at the same enzyme loading, the glucose yield appeared to be correlated with the β-glucosidase activity, while the xylose yield seemed to be correlated with the β-xylosidase level in the mixtures. Conclusion Enzyme supernatants produced by the mutant T. atroviride TUB F-1663 on various pretreated lignocellulosic substrates have good filter paper activity values combined with high levels of β-glucosidase activities, leading to cellulose conversion in the enzymatic hydrolysis that is as efficient as with a commercial cellulase mixture. On the other hand, in order to achieve good xylan conversion, the supernatants produced by the mutant have to be supplemented with additional β-xylosidase activity. PMID:19580644

DOE Office of Scientific and Technical Information (OSTI.GOV)

Resch, M.

Enzymatic depolymerization of polysaccharides is a key step in the production of fuels and chemicals from lignocellulosic biomass, and discovery of synergistic biomass-degrading enzyme paradigms will enable improved conversion processes. Historically, revealing insights into enzymatic saccharification mechanisms on plant cell walls has been hindered by uncharacterized substrates and low resolution imaging techniques. Also, translating findings between model substrates to intact biomass is critical for evaluating enzyme performance. Here we employ a fungal free enzyme cocktail, a complexed cellulosomal system, and a combination of the two to investigate saccharification mechanisms on cellulose I, II and III along with corn stover frommore » Clean Fractionation (CF), which is an Organosolv pretreatment. The insoluble Cellulose Enriched Fraction (CEF) from CF contains mainly cellulose with minor amounts of residual hemicellulose and lignin, the amount of which depends on the CF pretreatment severity. Enzymatic digestions at both low and high-solids loadings demonstrate that CF reduces the amount of enzyme required to depolymerize polysaccharides relative to deacetylated, dilute acid pretreated corn stover. Transmission and scanning electron microscopy of the biomass provides evidence for the different mechanisms of enzymatic deconstruction between free and complexed enzyme systems, and reveals the basis for the synergistic relationship between the two enzyme paradigms on a process-relevant substrate for the first time. These results also demonstrate that the presence of lignin, rather than cellulose morphology, is more detrimental to cellulosome action than to free cellulases. As enzyme costs are a major economic driver for biorefineries, this study provides key inputs for the evaluation of CF as a pretreatment method for biomass conversion.« less

Potential for bioremediation of agro-industrial effluents with high loads of pesticides by selected fungi.

PubMed

Karas, Panagiotis A; Perruchon, Chiara; Exarhou, Katerina; Ehaliotis, Constantinos; Karpouzas, Dimitrios G

2011-02-01

Wastewaters from the fruit packaging industry contain a high pesticide load and require treatment before their environmental discharge. We provide first evidence for the potential bioremediation of these wastewaters. Three white rot fungi (WRF) (Phanerochaete chrysosporium, Trametes versicolor, Pleurotus ostreatus) and an Aspergillus niger strain were tested in straw extract medium (StEM) and soil extract medium (SEM) for degrading the pesticides thiabendazole (TBZ), imazalil (IMZ), thiophanate methyl (TM), ortho-phenylphenol (OPP), diphenylamine (DPA) and chlorpyrifos (CHL). Peroxidase (LiP, MnP) and laccase (Lac) activity was also determined to investigate their involvement in pesticide degradation. T. versicolor and P. ostreatus were the most efficient degraders and degraded all pesticides (10 mg l⁻¹) except TBZ, with maximum efficiency in StEM. The phenolic pesticides OPP and DPA were rapidly degraded by these two fungi with a concurrent increase in MnP and Lac activity. In contrast, these enzymes were not associated with the degradation of CHL, IMZ and TM implying the involvement of other enzymes. T. versicolor degraded spillage-level pesticide concentrations (50 mg l⁻¹) either fully (DPA, OPP) or partially (TBZ, IMZ). The fungus was also able to rapidly degrade a mixture of TM/DPA (50 mg l⁻¹), whereas it failed to degrade IMZ and TBZ when supplied in a mixture with OPP. Overall, T. versicolor and P. ostreatus showed great potential for the bioremediation of wastewaters from the fruit packaging industry. However, degradation of TBZ should be also achieved before further scaling up.

Daily variation in melatonin level, antioxidant activity and general immune response of peripheral blood mononuclear cells and lymphoid tissues of Indian goat Capra hircus during summer and winter.

PubMed

Singh, Amaresh Kumar; Ghosh, Somenath; Basu, Priyoneel; Haldar, Chandana

2014-05-01

Daily variation in circulatory melatonin level, during different seasons, has been reported to influence immune system and free-radical scavenging capacity in mammals, including human beings. Similar studies have not been carried out on small ruminant viz. goats that are susceptible to opportunistic infections, increased oxidative load and sickness during free-grazing activity and frequent exposure to agro-chemicals. Therefore, daily variation in immune status, antioxidant enzyme activity and its possible correlation with circulatory melatonin level during two different seasons, summer (long day) and winter (short day) were studied in the Indian goat, Capra hircus. The clinically important immune parameters, such as total leukocyte count, % lymphocyte count and % stimulation ratio of T-lymphocytes presented a day/night rhythm prominently in the winter. The oxidative load in terms of malonedialdehyde was always low during night while antioxidant enzymes superoxide dismutase, catalase and total antioxidant status were high during nighttime (1800 to 0600 hrs). Interestingly, the studied parameters were significantly higher during the winter in both the sexes. Rhythmometric analyses showed prominent rhythmicity in above parameters. The data presented strong positive correlation between high levels of nighttime melatonin levels and immune parameters during winter. It suggests that melatonin possesses immunoenhacing as well as antioxidative property during winter. This might be a necessity for maintenance of physiological harmony in goats to protect them from winter stress.

Power Ultrasound to Process Dairy Products

NASA Astrophysics Data System (ADS)

Bermúdez-Aguirre, Daniela; Barbosa-Cánovas, Gustavo V.

Conventional methods of pasteurizing milk involve the use of heat regardless of treatment (batch, high temperature short time - HTST or ultra high temperature - UHT sterilization), and the quality of the milk is affected because of the use of high temperatures. Consequences of thermal treatment are a decrease in nutritional properties through the destruction of vitamins or denaturation of proteins, and sometimes the flavor of milk is undesirably changed. These changes are produced at the same time that the goal of the pasteurization process is achieved, which is to have a microbiological safe product, free of pathogenic bacteria, and to reduce the load of deteriorative microorganisms and enzymes, resulting in a product with a longer storage life.

Amperometric L-lysine enzyme electrodes based on carbon nanotube/redox polymer and graphene/carbon nanotube/redox polymer composites.

PubMed

Kaçar, Ceren; Erden, Pınar Esra; Kılıç, Esma

2017-04-01

Highly sensitive L-lysine enzyme electrodes were constructed by using poly(vinylferrocene)-multiwalled carbon nanotubes-gelatine (PVF/MWCNTs-GEL) and poly(vinylferrocene)-multiwalled carbon nanotubes-gelatine-graphene (PVF/MWCNTs-GEL/GR) composites as sensing interfaces and their performances were evaluated. Lysine oxidase (LO) was immobilized onto the composite modified glassy carbon electrodes (GCE) by crosslinking using glutaraldehyde and bovine serum albumin. Effects of pH value, enzyme loading, applied potential, electrode composition, and interfering substances on the amperometric response of the enzyme electrodes were discussed. The analytical characteristics of the enzyme electrodes were also investigated. The linear range, detection limit, and sensitivity of the LO/PVF/MWCNTs-GEL/GCE were 9.9 × 10 -7 -7.0 × 10 -4  M, 1.8 × 10 -7  M (S/N = 3), and 13.51 μA mM -1  cm -2 , respectively. PVF/MWCNTs-GEL/GR-based L-lysine enzyme electrode showed a short response time (<5 s) and a linear detection range from 9.9 × 10 -7 to 7.0 × 10 -4  M with good sensitivity of 17.8 μA mM -1  cm -2 and a low detection limit of 9.2 × 10 -8  M. The PVF/MWCNTs-GEL/GR composite-based L-lysine enzyme electrode exhibited about 1.3-fold higher sensitivity than its MWCNTs-based counterpart and its detection limit was superior to the MWCNTs-based one. In addition, enzyme electrodes were successfully applied to determine L-lysine in pharmaceutical sample and cheese.

Cross-linked collagen sponges loaded with plant polyphenols with inhibitory activity towards chronic wound enzymes.

PubMed

Antonio, Francesko; Guillem, Rocasalbas; Sonia, Touriño; Clara, Mattu; Piergiorgio, Gentile; Valeria, Chiono; Gianluca, Ciardelli; Tzanov, Tzanko

2011-10-01

Collagen sponges loaded with polyphenols from Hamamelis virginiana were investigated as active materials for chronic wound dressings, evaluating in vitro the inhibition of two major enzymes that impair the wound healing process - myeloperoxidase (MPO) and collagenase. Prior to polyphenols loading, collagen was cross-linked with genipin to improve its biostability. The effect of genipin cross-linking and polyphenol concentration in the development of mechanically and enzymatically stable sponges was studied. The tensile strength of the cross-linked collagen increased with the increase of the cross-linking degree, coupled to decrease in the elongation and the swelling capacity of the sponges. The stability of the sponges to collagenase digestion reached maximum when 1 mM genipin was used. However, the biostability decreased more than 10-fold after loading the sponges with polyphenols (0.5 mg/mL), nevertheless, this effect was partially overcome using higher concentration of polyphenols (1 and 2 mg/mL) to inhibit collagenase. Moreover, the polyphenols released from the sponges were sufficient for complete inhibition of MPO activity. No considerable cytotoxicity of the genipin cross-linked collagen loaded with polyphenols was observed evaluating the NIH 3T3 fibroblasts viability. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Carbon nanotube-lipase hybrid nanoflowers with enhanced enzyme activity and enantioselectivity.

PubMed

Li, Kai; Wang, Jianhua; He, Yaojia; Abdulrazaq, Miaad Adnan; Yan, Yunjun

2018-06-19

Various nanoflowers are synthesized as supports for different methods of enzyme immobilization; however, the activities of these immobilized enzymes are limited because of their confinement in the nanoflowers. In order to increase the performance of nanoflowers, in this study, different protein-phosphate hybrid nanostructures were successfully synthesized and further enhanced by carbon nanotubes (CNTs) under the same conditions. Only Cu 3 (PO 4 ) 2 complex nanostructures exhibited flower-like structures and showed excellent results after enhancement with CNTs in this framework. An esterification reaction between lauric acid and 1-dodecanol was used to test enzyme activity during immobilization, revealing that the Cu 3 (PO 4 ) 2 /CNT/protein complex exhibited 68-fold higher activity relative to free lipase and 51-fold higher than that of Cu 3 (PO 4 ) 2 /Burkholderia cepacia lipase hybrid nanoflowers in the absence of CNTs. All three hybrid nanostructures showed good performance and exhibited excellent reusability in resolution reactions between 1-phenylethanol and vinyl acetate. Additionally, the substrate enantiomeric excess (ee s ) reached 98% in only 10 min, and the corresponding Cu 3 (PO 4 ) 2 /CNT/protein complex could be recycled eight times without obvious loss of activity. This approach involving nanoflowers enhanced with CNTs will be highly beneficial for decreasing mass-transfer resistance and providing enhanced enzyme loading along with promising potential for industrial application. Copyright © 2018 Elsevier B.V. All rights reserved.

Effective Electrochemistry of Human Sulfite Oxidase Immobilized on Quantum-Dots-Modified Indium Tin Oxide Electrode.

PubMed

Zeng, Ting; Leimkühler, Silke; Koetz, Joachim; Wollenberger, Ulla

2015-09-30

The bioelectrocatalytic sulfite oxidation by human sulfite oxidase (hSO) on indium tin oxide (ITO) is reported, which is facilitated by functionalizing of the electrode surface with polyethylenimine (PEI)-entrapped CdS nanoparticles and enzyme. hSO was assembled onto the electrode with a high surface loading of electroactive enzyme. In the presence of sulfite but without additional mediators, a high bioelectrocatalytic current was generated. Reference experiments with only PEI showed direct electron transfer and catalytic activity of hSO, but these were less pronounced. The application of the polyelectrolyte-entrapped quantum dots (QDs) on ITO electrodes provides a compatible surface for enzyme binding with promotion of electron transfer. Variations of the buffer solution conditions, e.g., ionic strength, pH, viscosity, and the effect of oxygen, were studied in order to understand intramolecular and heterogeneous electron transfer from hSO to the electrode. The results are consistent with a model derived for the enzyme by using flash photolysis in solution and spectroelectrochemistry and molecular dynamic simulations of hSO on monolayer-modified gold electrodes. Moreover, for the first time a photoelectrochemical electrode involving immobilized hSO is demonstrated where photoexcitation of the CdS/hSO-modified electrode lead to an enhanced generation of bioelectrocatalytic currents upon sulfite addition. Oxidation starts already at the redox potential of the electron transfer domain of hSO and is greatly increased by application of a small overpotential to the CdS/hSO-modified ITO.

Hyaluronic Acid and Polyethylene Glycol Hybrid Hydrogel Encapsulating Nanogel with Hemostasis and Sustainable Antibacterial Property for Wound Healing.

PubMed

Zhu, Jie; Li, Faxue; Wang, Xueli; Yu, Jianyong; Wu, Dequn

2018-04-25

Immediate hemorrhage control and anti-infection play important roles in the wound management. Besides, a moist environment is also beneficial for wound healing. Hydrogels are promising materials in urgent hemostasis and drug release. However, hydrogels have the disadvantage of rapid release profiles, leading to the exposure to high drug concentrations. In this study, we constructed hybrid hydrogels with rapid hemostasis and sustainable antibacterial property combining aminoethyl methacrylate hyaluronic acid (HA-AEMA) and methacrylated methoxy polyethylene glycol (mPEG-MA) hybrid hydrogels and chlorhexidine diacetate (CHX)-loaded nanogels. The CHX-loaded nanogels (CLNs) were prepared by the enzyme degradation of CHX-loaded lysine-based hydrogels. The HA-AEMA and mPEG-MA hybrid hydrogel loaded with CLNs (labeled as Gel@CLN) displayed a three-dimensional microporous structure and exhibited excellent swelling, mechanical property, and low cytotoxicity. The Gel@CLN hydrogel showed a prolonged release period of CHX over 240 h and the antibacterial property over 10 days. The hemostasis and wound-healing properties were evaluated in vivo using a mouse model. The results showed that hydrogel had the rapid hemostasis capacity and accelerated wound healing. In summary, CLN-loaded hydrogels may be excellent candidates as hemostasis and anti-infection materials for the wound dressing application.

Proteomics and metabolomics analyses reveal the cucurbit sieve tube system as a complex metabolic space.

PubMed

Hu, Chaoyang; Ham, Byung-Kook; El-Shabrawi, Hattem M; Alexander, Danny; Zhang, Dabing; Ryals, John; Lucas, William J

2016-09-01

The plant vascular system, and specifically the phloem, plays a pivotal role in allocation of fixed carbon to developing sink organs. Although the processes involved in loading and unloading of sugars and amino acids are well characterized, little information is available regarding the nature of other metabolites in the sieve tube system (STS) at specific sites along the pathway. Here, we elucidate spatial features of metabolite composition mapped with phloem enzymes along the cucurbit STS. Phloem sap (PS) was collected from the loading (source), unloading (apical sink region) and shoot-root junction regions of cucumber, watermelon and pumpkin. Our PS analyses revealed significant differences in the metabolic and proteomic profiles both along the source-sink pathway and between the STSs of these three cucurbits. In addition, metabolite profiles established for PS and vascular tissue indicated the presence of distinct compositions, consistent with the operation of the STS as a unique symplasmic domain. In this regard, at various locations along the STS we could map metabolites and their related enzymes to specific metabolic pathways. These findings are discussed with regard to the function of the STS as a unique and highly complex metabolic space within the plant vascular system. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

Pretreatment of corn stover using wet oxidation to enhance enzymatic digestibility.

PubMed

Varga, Eniko; Schmidt, Anette S; Réczey, Kati; Thomsen, Anne Belinda

2003-01-01

Corn stover is an abundant, promising raw material for fuel ethanol production. Although it has a high cellulose content, without pretreatment it resists enzymatic hydrolysis, like most lignocellulosic materials. Wet oxidation (water, oxygen, mild alkali or acid, elevated temperature and pressure) was investigated to enhance the enzymatic digestibility of corn stover. Six different combinations of reaction temperature, time, and pH were applied. The best conditions (60 g/L of corn stover, 195 degrees C, 15 min, 12 bar O2, 2 g/L of Na2CO3) increased the enzymatic conversion of corn stover four times, compared to untreated material. Under these conditions 60% of hemicellulose and 30% of lignin were solubilized, whereas 90% of cellulose remained in the solid fraction. After 24-h hydrolysis at 50 degrees C using 25 filter paper units (FPU)/g of drymatter (DM) biomass, the achieved conversion of cellulose to glucose was about 85%. Decreasing the hydrolysis temperature to 40 degrees C increased hydrolysis time from 24 to 72 h. Decreasing the enzyme loading to 5 FPU/g of DM biomass slightly decreased the enzymatic conversion from 83.4 to 71%. Thus, enzyme loading can be reduced without significantly affecting the efficiency of hydrolysis, an important economical aspect.

Influence of aspect ratio and surface defect density on hydrothermally grown ZnO nanorods towards amperometric glucose biosensing applications

NASA Astrophysics Data System (ADS)

Shukla, Mayoorika; Pramila; Dixit, Tejendra; Prakash, Rajiv; Palani, I. A.; Singh, Vipul

2017-11-01

In this work, hydrothermally grown ZnO Nanorods Array (ZNA) has been synthesized over Platinum (Pt) coated glass substrate, for biosensing applications. In-situ addition of strong oxidizing agent viz KMnO4 during hydrothermal growth was found to have profound effect on the physical properties of ZNA. Glucose oxidase (GOx) was later immobilized over ZNA by means of physical adsorption process. Further influence of varying aspect ratio, enzyme loading and surface defects on amperometric glucose biosensor has been analyzed. Significant variation in biosensor performance was observed by varying the amount of KMnO4 addition during the growth. Moreover, investigations revealed that the suppression of surface defects and aspect ratio variation of the ZNA played key role towards the observed improvement in the biosensor performance, thereby significantly affecting the sensitivity and response time of the fabricated biosensor. Among different biosensors fabricated having varied aspect ratio and surface defect density of ZNA, the best electrode resulted into sensitivity and response time to be 18.7 mA cm-2 M-1 and <5 s respectively. The observed results revealed that apart from high aspect ratio nanostructures and the extent of enzyme loading, surface defect density also hold a key towards ZnO nanostructures based bio-sensing applications.

Metal-organic coordination-enabled layer-by-layer self-assembly to prepare hybrid microcapsules for efficient enzyme immobilization.

PubMed

Wang, Xiaoli; Jiang, Zhongyi; Shi, Jiafu; Liang, Yanpeng; Zhang, Chunhong; Wu, Hong

2012-07-25

A novel layer-by-layer self-assembly approach enabled by metal-organic coordination was developed to prepare polymer-inorganic hybrid microcapsules. Alginate was first activated via N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxy succinimide (NHS) coupling chemistry, and subsequently reacted with dopamine. Afterward, the dopamine modified alginate (Alg-DA) and titanium(IV) bis(ammonium lactato) dihydroxide (Ti(IV)) were alternatively deposited onto CaCO3 templates. The coordination reaction between the catechol groups of Alg-DA and the Ti(IV) allowed the alternative assembly to form a series of multilayers. After removing the templates, the alginate-titanium hybrid microcapsules were obtained. The high mechanical stability of hybrid microcapsules was demonstrated by osmotic pressure experiment. Furthermore, the hybrid microcapsules displayed superior thermal stability due to Ti(IV) coordination. Catalase (CAT) was used as model enzyme, either encapsulated inside or covalently attached on the surface of the resultant microcapsules. No CAT leakage from the microcapsules was detected after incubation for 48 h. The encapsulated CAT, with a loading capacity of 450-500 mg g(-1) microcapsules, exhibited desirable long-term storage stability, whereas the covalently attached CAT, with a loading capacity of 100-150 mg g(-1) microcapsules, showed desirable operational stability.

Transcriptional and Physiological Responses to Nutrient Loading on Toxin Formation and Photosynthesis in Microcystis Aeruginosa FACHB-905

PubMed Central

Peng, Guotao; Lin, Sijie; Fan, Zhengqiu; Wang, Xiangrong

2017-01-01

An important goal of understanding harmful algae blooms is to determine how environmental factors affect the growth and toxin formation of toxin-producing species. In this study, we investigated the transcriptional responses of toxin formation gene (mcyB) and key photosynthesis genes (psaB, psbD and rbcL) of Microcystis aeruginosa FACHB-905 in different nutrient loading conditions using real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR). Three physio-biochemical parameters (malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione (GSH)) were also evaluated to provide insight into the physiological responses of Microcystis cells. We observed an upregulation of mcyB gene in nutrient-deficient conditions, especially in nitrogen (N) limitation condition, and the transcript abundance declined after the nutrient were resupplied. Differently, high transcription levels were seen in phosphorus (P) deficient treatments for key photosynthesis genes throughout the culture period, while those in N-deficient cells varied with time, suggesting an adaptive regulation of Microsystis cells to nutrient stress. Increased contents of antioxidant enzymes (SOD and GSH) were seen in both N and P-deficient conditions, suggesting the presence of excess amount of free radical generation caused by nutrient stress. The amount of SOD and GSH continued to increase even after the nutrient was reintroduced and a strong correlation was seen between the MDA and enzyme activities, indicating the robust effort of rebalancing the redox system in Microcystis cells. Based on these transcriptional and physiological responses of M. aeruginosa to nutrient loading, these results could provide more insight into Microcystis blooms management and toxin formation regulation. PMID:28513574

Transcriptional and Physiological Responses to Nutrient Loading on Toxin Formation and Photosynthesis in Microcystis Aeruginosa FACHB-905.

PubMed

Peng, Guotao; Lin, Sijie; Fan, Zhengqiu; Wang, Xiangrong

2017-05-17

An important goal of understanding harmful algae blooms is to determine how environmental factors affect the growth and toxin formation of toxin-producing species. In this study, we investigated the transcriptional responses of toxin formation gene ( mcyB ) and key photosynthesis genes ( psaB , psbD and rbcL) of Microcystis aeruginosa FACHB-905 in different nutrient loading conditions using real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR). Three physio-biochemical parameters (malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione (GSH)) were also evaluated to provide insight into the physiological responses of Microcystis cells. We observed an upregulation of mcyB gene in nutrient-deficient conditions, especially in nitrogen (N) limitation condition, and the transcript abundance declined after the nutrient were resupplied. Differently, high transcription levels were seen in phosphorus (P) deficient treatments for key photosynthesis genes throughout the culture period, while those in N-deficient cells varied with time, suggesting an adaptive regulation of Microsystis cells to nutrient stress. Increased contents of antioxidant enzymes (SOD and GSH) were seen in both N and P-deficient conditions, suggesting the presence of excess amount of free radical generation caused by nutrient stress. The amount of SOD and GSH continued to increase even after the nutrient was reintroduced and a strong correlation was seen between the MDA and enzyme activities, indicating the robust effort of rebalancing the redox system in Microcystis cells. Based on these transcriptional and physiological responses of M. aeruginosa to nutrient loading, these results could provide more insight into Microcystis blooms management and toxin formation regulation.

Optimizing the Mass-Specific Activity of Bilirubin Oxidase Adlayers through Combined Electrochemical Quartz Crystal Microbalance and Dual Polarization Interferometry Analyses.

PubMed

McArdle, Trevor; McNamara, Thomas P; Fei, Fan; Singh, Kulveer; Blanford, Christopher F

2015-11-18

Two surface analysis techniques, dual polarization interferometry (DPI) and analysis by an electrochemical quartz crystal microbalance with dissipation capability (E-QCM-D), were paired to find the deposition conditions that give the highest and most stable electrocatalytic activity per adsorbed mass of enzyme. Layers were formed by adsorption from buffered solutions of bilirubin oxidase from Myrothecium verrucaria at pH 6.0 to planar surfaces, under high enzyme loading (≥1 mg mL(-1)) for contact periods of up to 2 min. Both unmodified and carboxylate-functionalized gold-coated sensors showed that a deposition solution concentration of 10-25 mg mL(-1) gave the highest activity per mass of adsorbed enzyme with an effective catalytic rate constant (k(cat)) of about 60 s(-1). The densification of adsorbed layers observed by DPI correlated with reduced bioactivity observed by parallel E-QCM-D measurements. Postadsorption changes in thickness and density observed by DPI were incorporated into Kelvin-Voigt models of the QCM-D response. The modeled response matched experimental observations when the adlayer viscosity tripled after adsorption.

Enhancing enzymatic hydrolysis of sugarcane bagasse by ferric chloride catalyzed organosolv pretreatment and Tween 80.

PubMed

Zhang, Hongdan; Fan, Meishan; Li, Xin; Zhang, Aiping; Xie, Jun

2018-06-01

In this work, a FeCl 3 -catalyzed organosolv pretreatment was employed at 160 °C to remove hemicellulose and lignin in sugarcane bagasse leaving the cellulose-enriched residue for enzymatic hydrolysis to sugars. The solubilized hemicellulose fractions consisted more monomer xylose than oligomer xylose. The FeCl 3 -catalyzed organosolv pretreatment significantly improved the enzymatic hydrolysis, nearly 100% of cellulose components were converted to glucose after pretreatment with 0.05 M FeCl 3 . Structural analysis was employed to reveal how pretreatment affected the enzymatic hydrolysis. With the addition of Tween 80, the same level of glucose was obtained with 50% reduction of enzyme dosage after 24 h. Furthermore, the influence of Tween 80 on different pretreatment systems was investigated, indicating that the improvement was increased as the lignin content increased, decreased with high enzyme loading and extending hydrolysis time. This work suggested that the addition of Tween 80 could improve the enzymatic hydrolysis, reduce the hydrolysis time and enzyme dosage. Copyright © 2018 Elsevier Ltd. All rights reserved.

Cost evaluation of cellulase enzyme for industrial-scale cellulosic ethanol production based on rigorous Aspen Plus modeling.

PubMed

Liu, Gang; Zhang, Jian; Bao, Jie

2016-01-01

Cost reduction on cellulase enzyme usage has been the central effort in the commercialization of fuel ethanol production from lignocellulose biomass. Therefore, establishing an accurate evaluation method on cellulase enzyme cost is crucially important to support the health development of the future biorefinery industry. Currently, the cellulase cost evaluation methods were complicated and various controversial or even conflict results were presented. To give a reliable evaluation on this important topic, a rigorous analysis based on the Aspen Plus flowsheet simulation in the commercial scale ethanol plant was proposed in this study. The minimum ethanol selling price (MESP) was used as the indicator to show the impacts of varying enzyme supply modes, enzyme prices, process parameters, as well as enzyme loading on the enzyme cost. The results reveal that the enzyme cost drives the cellulosic ethanol price below the minimum profit point when the enzyme is purchased from the current industrial enzyme market. An innovative production of cellulase enzyme such as on-site enzyme production should be explored and tested in the industrial scale to yield an economically sound enzyme supply for the future cellulosic ethanol production.

Enzyme-activated intracellular drug delivery with tubule clay nanoformulation

PubMed Central

Dzamukova, Maria R.; Naumenko, Ekaterina A.; Lvov, Yuri M.; Fakhrullin, Rawil F.

2015-01-01

Fabrication of stimuli-triggered drug delivery vehicle s is an important milestone in treating cancer. Here we demonstrate the selective anticancer drug delivery into human cells with biocompatible 50-nm diameter halloysite nanotube carriers. Physically-adsorbed dextrin end stoppers secure the intercellular release of brilliant green. Drug-loaded nanotubes penetrate through the cellular membranes and their uptake efficiency depends on the cells growth rate. Intercellular glycosyl hydrolases-mediated decomposition of the dextrin tube-end stoppers triggers the release of the lumen-loaded brilliant green, which allowed for preferable elimination of human lung carcinoma cells (А549) as compared with hepatoma cells (Hep3b). The enzyme-activated intracellular delivery of brilliant green using dextrin-coated halloysite nanotubes is a promising platform for anticancer treatment. PMID:25976444

Anti-inflammatory effects of EMD in the presence of biomechanical loading and interleukin-1β in vitro.

PubMed

Nokhbehsaim, Marjan; Deschner, Birgit; Winter, Jochen; Bourauel, Christoph; Jäger, Andreas; Jepsen, Søren; Deschner, James

2012-02-01

Enamel matrix derivative (EMD) used to promote periodontal regeneration has been shown to exert anti-inflammatory effects. This in vitro study was performed to investigate if the anti-inflammatory actions of EMD are modulated by the local cellular environment, such as inflammation or occlusal, i.e., biomechanical, loading. Human periodontal ligament cells were seeded on BioFlex plates and incubated with EMD under normal, inflammatory, and biomechanical loading conditions for 1 and 6 days. In order to mimic inflammatory and biomechanical loading conditions in vitro, cells were stimulated with interleukin (IL)-1β and exposed to dynamic tensile strain, respectively. The gene expression of IL-1β, IL-1 receptor antagonist (IL-1RN), IL-6, IL-8, IL-10, and cyclooxygenase (COX)-2 was analyzed by real-time RT-PCR and the IL-6 protein synthesis by enzyme-linked immunoassay. For statistical analysis, Student's t test, ANOVA, and post-hoc comparison tests were applied (p < 0.05). EMD downregulated significantly the expression of IL-1β and COX-2 at 1 day and of IL-6, IL-8, and COX-2 at 6 days in normal condition. In an inflammatory environment, the anti-inflammatory actions of EMD were significantly enhanced at 6 days. In the presence of low biomechanical loading, EMD caused a downregulation of IL-1β and IL-8, whereas high biomechanical loading significantly abrogated the anti-inflammatory effects of EMD at both days. Neither IL-1RN nor IL-10 was upregulated by EMD. These data suggest that high occlusal forces may abrogate anti-inflammatory effects of EMD and should, therefore, be avoided immediately after the application of EMD to achieve best healing results.

Techno-economic comparison of centralized versus decentralized biorefineries for two alkaline pretreatment processes.

PubMed

Stoklosa, Ryan J; Del Pilar Orjuela, Andrea; da Costa Sousa, Leonardo; Uppugundla, Nirmal; Williams, Daniel L; Dale, Bruce E; Hodge, David B; Balan, Venkatesh

2017-02-01

In this work, corn stover subjected to ammonia fiber expansion (AFEX™) 1 pretreatment or alkaline pre-extraction followed by hydrogen peroxide post-treatment (AHP pretreatment) were compared for their enzymatic hydrolysis yields over a range of solids loadings, enzymes loadings, and enzyme combinations. Process techno-economic models were compared for cellulosic ethanol production for a biorefinery that handles 2000tons per day of corn stover employing a centralized biorefinery approach with AHP or a de-centralized AFEX pretreatment followed by biomass densification feeding a centralized biorefinery. A techno-economic analysis (TEA) of these scenarios shows that the AFEX process resulted in the highest capital investment but also has the lowest minimum ethanol selling price (MESP) at $2.09/gal, primarily due to good energy integration and an efficient ammonia recovery system. The economics of AHP could be made more competitive if oxidant loadings were reduced and the alkali and sugar losses were also decreased. Copyright © 2016 Elsevier Ltd. All rights reserved.

Parametric study for the optimization of ionic liquid pretreatment of corn stover

DOE Office of Scientific and Technical Information (OSTI.GOV)

Papa, Gabriella; Feldman, Taya; Sale, Kenneth L.

A parametric study of the efficacy of the ionic liquid (IL) pretreatment (PT) of corn stover (CS) using 1-ethyl-3-methylimidazolium acetate ([C 2C 1Im][OAc] ) and cholinium lysinate ([Ch][Lys] ) was conducted. The impact of 50% and 15% biomass loading for milled and non-milled CS on IL-PT was evaluated, as well the impact of 20 and 5 mg enzyme/g glucan on saccharification efficiency. The glucose and xylose released were generated from 32 conditions – 2 ionic liquids (ILs), 2 temperatures, 2 particle sizes (S), 2 solid loadings, and 2 enzyme loadings. Statistical analysis indicates that sugar yields were correlated with lignin andmore » xylan removal and depends on the factors, where S did not explain variation in sugar yields. Both ILs were effective in pretreating large particle sized CS, without compromising sugar yields. The knowledge from material and energy balances is an essential step in directing optimization of sugar recovery at desirable process conditions.« less

Production of high concentrated cellulosic ethanol by acetone/water oxidized pretreated beech wood.

PubMed

Katsimpouras, Constantinos; Kalogiannis, Konstantinos G; Kalogianni, Aggeliki; Lappas, Angelos A; Topakas, Evangelos

2017-01-01

Lignocellulosic biomass is an abundant and inexpensive resource for biofuel production. Alongside its biotechnological conversion, pretreatment is essential to enable efficient enzymatic hydrolysis by making cellulose susceptible to cellulases. Wet oxidation of biomass, such as acetone/water oxidation, that employs hot acetone, water, and oxygen, has been found to be an attractive pretreatment method for removing lignin while producing less degradation products. The remaining enriched cellulose fraction has the potential to be utilized under high gravity enzymatic saccharification and fermentation processes for the cost-competing production of bioethanol. Beech wood residual biomass was pretreated following an acetone/water oxidation process aiming at the production of high concentration of cellulosic ethanol. The effect of pressure, reaction time, temperature, and acetone-to-water ratio on the final composition of the pretreated samples was studied for the efficient utilization of the lignocellulosic feedstock. The optimal conditions were acetone/water ratio 1:1, 40 atm initial pressure of 40 vol% O 2 gas, and 64 atm at reaction temperature of 175 °C for 2 h incubation. The pretreated beech wood underwent an optimization step studying the effect of enzyme loading and solids content on the enzymatic liquefaction/saccharification prior to fermentation. In a custom designed free-fall mixer at 50 °C for either 6 or 12 h of prehydrolysis using an enzyme loading of 9 mg/g dry matter at 20 wt% initial solids content, high ethanol concentration of 75.9 g/L was obtained. The optimization of the pretreatment process allowed the efficient utilization of beech wood residual biomass for the production of high concentrations of cellulosic ethanol, while obtaining lignin that can be upgraded towards high-added-value chemicals. The threshold of 4 wt% ethanol concentration that is required for the sustainable bioethanol production was surpassed almost twofold, underpinning the efficient conversion of biomass to ethanol and bio-based chemicals on behalf of the biorefinery concept.

Comparative performance of precommercial cellulases hydrolyzing pretreated corn stover

PubMed Central

2011-01-01

Background Cellulases and related hydrolytic enzymes represent a key cost factor for biochemical conversion of cellulosic biomass feedstocks to sugars for biofuels and chemicals production. The US Department of Energy (DOE) is cost sharing projects to decrease the cost of enzymes for biomass saccharification. The performance of benchmark cellulase preparations produced by Danisco, DSM, Novozymes and Verenium to convert pretreated corn stover (PCS) cellulose to glucose was evaluated under common experimental conditions and is reported here in a non-attributed manner. Results Two hydrolysis modes were examined, enzymatic hydrolysis (EH) of PCS whole slurry or washed PCS solids at pH 5 and 50°C, and simultaneous saccharification and fermentation (SSF) of washed PCS solids at pH 5 and 38°C. Enzymes were dosed on a total protein mass basis, with protein quantified using both the bicinchoninic acid (BCA) assay and the Bradford assay. Substantial differences were observed in absolute cellulose to glucose conversion performance levels under the conditions tested. Higher cellulose conversion yields were obtained using washed solids compared to whole slurry, and estimated enzyme protein dosages required to achieve a particular cellulose conversion to glucose yield were extremely dependent on the protein assay used. All four enzyme systems achieved glucose yields of 90% of theoretical or higher in SSF mode. Glucose yields were reduced in EH mode, with all enzymes achieving glucose yields of at least 85% of theoretical on washed PCS solids and 75% in PCS whole slurry. One of the enzyme systems ('enzyme B') exhibited the best overall performance. However in attaining high conversion yields at lower total enzyme protein loadings, the relative and rank ordered performance of the enzyme systems varied significantly depending upon which hydrolysis mode and protein assay were used as the basis for comparison. Conclusions This study provides extensive information about the performance of four precommercial cellulase preparations. Though test conditions were not necessarily optimal for some of the enzymes, all were able to effectively saccharify PCS cellulose. Large differences in the estimated enzyme dosage requirements depending on the assay used to measure protein concentration highlight the need for better consensus methods to quantify enzyme protein. PMID:21899748

Simple and efficient immobilization of lipase B from Candida antarctica on porous styrene-divinylbenzene beads.

PubMed

Hernandez, Karel; Garcia-Galan, Cristina; Fernandez-Lafuente, Roberto

2011-06-10

Two commercial porous styrene-divinylbenzene beads (Diaion HP20LX and MCI GEL CHP20P) have been evaluated as supports to immobilize lipase B from Candida antarctica (CALB). MCI GEL CHP20P rapidly immobilized the enzyme, permitting a very high loading capacity: around 110mgCALB/wetg of support compared to the 50mg obtained using decaoctyl Sepabeads. Although enzyme specificity of the enzyme immobilized on different supports was quite altered by the support used in the immobilization, specific activity of the enzyme immobilized on MCI GEL CHP20P was always higher than those found using decaoctyl Sepabeads for all assayed substrates. Thus, a CALB biocatalyst having 3-8 folds (depending on the substrate) higher activity/wet gram of support than the commercial Novozym 435 was obtained. Half-live of CAL-Diaion HP20LX at 60°C was 2-3 higher than the one of Novozym 435, it was 30-40 higher in the presence of 50% acetonitrile and it was around 100 folds greater in the presence of 10M hydrogen peroxide. Results indicate that styrene-divinylbenzene supports may be promising alternatives as supports to immobilize CALB. Copyright © 2011 Elsevier Inc. All rights reserved.

Ethanol production potential from AFEX™ and steam-exploded sugarcane residues for sugarcane biorefineries.

PubMed

Mokomele, Thapelo; da Costa Sousa, Leonardo; Balan, Venkatesh; van Rensburg, Eugéne; Dale, Bruce E; Görgens, Johann F

2018-01-01

Expanding biofuel markets are challenged by the need to meet future biofuel demands and mitigate greenhouse gas emissions, while using domestically available feedstock sustainably. In the context of the sugar industry, exploiting under-utilized cane leaf matter (CLM) in addition to surplus sugarcane bagasse as supplementary feedstock for second-generation ethanol production has the potential to improve bioenergy yields per unit land. In this study, the ethanol yields and processing bottlenecks of ammonia fibre expansion (AFEX™) and steam explosion (StEx) as adopted technologies for pretreating sugarcane bagasse and CLM were experimentally measured and compared for the first time. Ethanol yields between 249 and 256 kg Mg -1 raw dry biomass (RDM) were obtained with AFEX™-pretreated sugarcane bagasse and CLM after high solids loading enzymatic hydrolysis and fermentation. In contrast, StEx-pretreated sugarcane bagasse and CLM resulted in substantially lower ethanol yields that ranged between 162 and 203 kg Mg -1 RDM. The ethanol yields from StEx-treated sugarcane residues were limited by the aggregated effect of sugar degradation during pretreatment, enzyme inhibition during enzymatic hydrolysis and microbial inhibition of S. cerevisiae 424A (LNH-ST) during fermentation. However, relatively high enzyme dosages (> 20 mg g -1 glucan) were required irrespective of pretreatment method to reach 75% carbohydrate conversion, even when optimal combinations of Cellic ® CTec3, Cellic ® HTec3 and Pectinex Ultra-SP were used. Ethanol yields per hectare sugarcane cultivation area were estimated at 4496 and 3416 L ha -1 for biorefineries using AFEX™- or StEx-treated sugarcane residues, respectively. AFEX™ proved to be a more effective pretreatment method for sugarcane residues relative to StEx due to the higher fermentable sugar recovery and enzymatic hydrolysate fermentability after high solids loading enzymatic hydrolysis and fermentation by S. cerevisiae 424A (LNH-ST). The identification of auxiliary enzyme activities, adequate process integration and the use of robust xylose-fermenting ethanologens were identified as opportunities to further improve ethanol yields from AFEX™- and StEx-treated sugarcane residues.

Effects of in vivo static compressive loading on aggrecan and type II and X collagens in the rat growth plate extracellular matrix.

PubMed

Cancel, Mathilde; Grimard, Guy; Thuillard-Crisinel, Delphine; Moldovan, Florina; Villemure, Isabelle

2009-02-01

Mechanical loads are essential to normal bone growth, but excessive loads can lead to progressive deformities. In addition, growth plate extracellular matrix remodelling is essential to regulate the normal longitudinal bone growth process and to ensure physiological bone mineralization. In order to investigate the effects of static compression on growth plate extracellular matrix using an in vivo animal model, a loading device was used to precisely apply a compressive stress of 0.2 MPa for two weeks on the seventh caudal vertebra (Cd7) of rats during the pubertal growth spurt. Control, sham and loaded groups were studied. Growth modulation was quantified based on calcein labelling, and three matrix components (type II and X collagens, and aggrecan) were assessed using immunohistochemistry/safranin-O staining. As well, extracellular matrix components and enzymes (MMP-3 and -13, ADAMTS-4 and -5) were studied by qRT-PCR. Loading reduced Cd7 growth by 29% (p<0.05) and 15% (p=0.07) when compared to controls and shams respectively. No significant change could be observed in the mRNA expression of collagens and the proteolytic enzyme MMP-13. However, MMP-3 was significantly increased in the loaded group as compared to the control group (p<0.05). No change was observed in aggrecan and ADAMTS-4 and -5 expression. Low immunostaining for type II and X collagens was observed in 83% of the loaded rats as compared to the control rats. This in vivo study shows that, during pubertal growth spurt, two-week static compression reduced caudal vertebrae growth rates; this mechanical growth modulation occurred with decreased type II and X collagen proteins in the growth plate.

Delivery of adapalene using a novel topical gel based on tea tree oil nano-emulsion: Permeation, antibacterial and safety assessments.

PubMed

Najafi-Taher, Roqya; Ghaemi, Behnaz; Amani, Amir

2018-07-30

The aim of present study was to design and optimize 0.1% adapalene loaded nano-emulsion to improve the drug efficacy and increase its user compliance. Effect of type and concentration of surfactants was studied on size of 0.1% adapalene loaded nano-emulsion. Optimized formulation was then evaluated for particle size, polydispersity index, morphology, viscosity, and pH. Subsequently, 1% carbopol® 934 was incorporated to the optimized formulation for preparation of its gel form. The efficacy and safety of 0.1% adapalene loaded nano-emulsion gel was assessed compared to marketed gel containing 0.1% adapalene. In-vitro studies showed that adapalene permeation through the skin was negligible in both adapalene loaded nano-emulsion gel and adapalene marketed gel. Furthermore, drug distribution studies in skin indicated higher retention of adapalene in the dermis in adapalene loaded nano-emulsion gel compared with adapalene marketed gel. Antibacterial activity against Propionibacterium acnes showed that adapalene loaded nano-emulsion is effective in reducing minimum inhibitory concentration of the formulation in comparison with tea tree oil nano-emulsion, and pure tea tree oil. In vivo skin irritation studies showed absence of irritancy for adapalene loaded nano-emulsion gel. Also, blood and liver absorption of the drug, histological analysis of liver and liver enzyme activity of rats after 90 days' treatment were investigated. No drug was detected in blood/liver which in addition to an absence of any adverse effect on liver and enzymes showed the potential of adapalene loaded nano-emulsion gel as a novel carrier for topical delivery of adapalene. Copyright © 2018 Elsevier B.V. All rights reserved.

Improved Activity of a Thermophilic Cellulase, Cel5A, from Thermotoga maritima on Ionic Liquid Pretreated Switchgrass

PubMed Central

Chen, Zhiwei; Pereira, Jose H.; Liu, Hanbin; Tran, Huu M.; Hsu, Nathan S. Y.; Dibble, Dean; Singh, Seema; Adams, Paul D.; Sapra, Rajat; Hadi, Masood Z.; Simmons, Blake A.; Sale, Kenneth L.

2013-01-01

Ionic liquid pretreatment of biomass has been shown to greatly reduce the recalcitrance of lignocellulosic biomass, resulting in improved sugar yields after enzymatic saccharification. However, even under these improved saccharification conditions the cost of enzymes still represents a significant proportion of the total cost of producing sugars and ultimately fuels from lignocellulosic biomass. Much of the high cost of enzymes is due to the low catalytic efficiency and stability of lignocellulolytic enzymes, especially cellulases, under conditions that include high temperatures and the presence of residual pretreatment chemicals, such as acids, organic solvents, bases, or ionic liquids. Improving the efficiency of the saccharification process on ionic liquid pretreated biomass will facilitate reduced enzyme loading and cost. Thermophilic cellulases have been shown to be stable and active in ionic liquids but their activity is typically at lower levels. Cel5A_Tma, a thermophilic endoglucanase from Thermotoga maritima, is highly active on cellulosic substrates and is stable in ionic liquid environments. Here, our motivation was to engineer mutants of Cel5A_Tma with higher activity on 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]) pretreated biomass. We developed a robotic platform to screen a random mutagenesis library of Cel5A_Tma. Twelve mutants with 25–42% improvement in specific activity on carboxymethyl cellulose and up to 30% improvement on ionic-liquid pretreated switchgrass were successfully isolated and characterized from a library of twenty thousand variants. Interestingly, most of the mutations in the improved variants are located distally to the active site on the protein surface and are not directly involved with substrate binding. PMID:24244549

Improved activity of a thermophilic cellulase, Cel5A, from Thermotoga maritima on ionic liquid pretreated switchgrass.

PubMed

Chen, Zhiwei; Pereira, Jose H; Liu, Hanbin; Tran, Huu M; Hsu, Nathan S Y; Dibble, Dean; Singh, Seema; Adams, Paul D; Sapra, Rajat; Hadi, Masood Z; Simmons, Blake A; Sale, Kenneth L

2013-01-01

Ionic liquid pretreatment of biomass has been shown to greatly reduce the recalcitrance of lignocellulosic biomass, resulting in improved sugar yields after enzymatic saccharification. However, even under these improved saccharification conditions the cost of enzymes still represents a significant proportion of the total cost of producing sugars and ultimately fuels from lignocellulosic biomass. Much of the high cost of enzymes is due to the low catalytic efficiency and stability of lignocellulolytic enzymes, especially cellulases, under conditions that include high temperatures and the presence of residual pretreatment chemicals, such as acids, organic solvents, bases, or ionic liquids. Improving the efficiency of the saccharification process on ionic liquid pretreated biomass will facilitate reduced enzyme loading and cost. Thermophilic cellulases have been shown to be stable and active in ionic liquids but their activity is typically at lower levels. Cel5A_Tma, a thermophilic endoglucanase from Thermotoga maritima, is highly active on cellulosic substrates and is stable in ionic liquid environments. Here, our motivation was to engineer mutants of Cel5A_Tma with higher activity on 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]) pretreated biomass. We developed a robotic platform to screen a random mutagenesis library of Cel5A_Tma. Twelve mutants with 25-42% improvement in specific activity on carboxymethyl cellulose and up to 30% improvement on ionic-liquid pretreated switchgrass were successfully isolated and characterized from a library of twenty thousand variants. Interestingly, most of the mutations in the improved variants are located distally to the active site on the protein surface and are not directly involved with substrate binding.

Self assembly of acetylcholinesterase on a gold nanoparticles–graphene nanosheet hybrid for organophosphate pesticide detection using polyelectrolyte as a linker

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Ying; Zhang, Sheng; Du, Dan

A nanohybrid of gold nanoparticles (Au NPs) and chemically reduced graphene oxide nanosheets (cr-Gs) was synthesized by in situ growth of Au NPs on the surface of graphene nanosheets in the presence of poly(diallyldimethylammonium chloride) (PDDA), which not only improved the dispersion of Au NPs but also stabilized cholinesterase with high activity and loading efficiency. The obtained nanohybrid was characterized by TEM, XRD, XPS, and electrochemistry. Then an enzyme nanoassembly (AChE/Au NPs/cr-Gs) was prepared by self-assembling acetylcholinesterase (AChE) on Au NP/cr-Gs nanohybrid. An electrochemical sensor based on AChE/Au NPs/cr-Gs was further developed for ultrasensitive detection of organophosphate pesticide. The resultsmore » demonstrate that the developed approach provides a promising strategy to improve the sensitivity and enzyme activity of electrochemical biosensors.« less

Construction of High Drug Loading and Enzymatic Degradable Multilayer Films for Self-Defense Drug Release and Long-Term Biofilm Inhibition.

PubMed

Wang, Bailiang; Liu, Huihua; Sun, Lin; Jin, Yingying; Ding, Xiaoxu; Li, Lingli; Ji, Jian; Chen, Hao

2018-01-08

Bacterial infections and biofilm formation on the surface of implants are important issues that greatly affect biomedical applications and even cause device failure. Construction of high drug loading systems on the surface and control of drug release on-demand is an efficient way to lower the development of resistant bacteria and biofilm formation. In the present study, (montmorillonite/hyaluronic acid-gentamicin) 10 ((MMT/HA-GS) 10 ) organic/inorganic hybrid multilayer films were alternately self-assembled on substrates. The loading dosage of GS was as high as 0.85 mg/cm 2 , which could be due the high specific surface area of MMT. The obtained multilayer film with high roughness gradually degraded in hyaluronidase (HAS) solutions or a bacterial infection microenvironment, which caused the responsive release of GS. The release of GS showed dual enzyme and bacterial infection responsiveness, which also indicated good drug retention and on-demand self-defense release properties of the multilayer films. Moreover, the GS release responsiveness to E. coli showed higher sensitivity than that to S. aureus. There was only ∼5 wt % GS release from the film in PBS after 48 h of immersion, and the amount quickly increased to 30 wt % in 10 5 CFU/mL of E. coli. Importantly, the high drug dosage, smart drug release, and film peeling from the surface contributed to the efficient antibacterial properties and long-term biofilm inhibition functions. Both in vitro and in vivo antibacterial tests indicated efficient sterilization function and good mammalian cell and tissue compatibility.

Structure of PA1221, a nonribosomal peptide synthetase containing adenylation and peptidyl carrier protein domains.

PubMed

Mitchell, Carter A; Shi, Ce; Aldrich, Courtney C; Gulick, Andrew M

2012-04-17

Many bacteria use large modular enzymes for the synthesis of polyketide and peptide natural products. These multidomain enzymes contain integrated carrier domains that deliver bound substrates to multiple catalytic domains, requiring coordination of these chemical steps. Nonribosomal peptide synthetases (NRPSs) load amino acids onto carrier domains through the activity of an upstream adenylation domain. Our lab recently determined the structure of an engineered two-domain NRPS containing fused adenylation and carrier domains. This structure adopted a domain-swapped dimer that illustrated the interface between these two domains. To continue our investigation, we now examine PA1221, a natural two-domain protein from Pseudomonas aeruginosa. We have determined the amino acid specificity of this new enzyme and used domain specific mutations to demonstrate that loading the downstream carrier domain within a single protein molecule occurs more quickly than loading of a nonfused carrier domain intermolecularly. Finally, we have determined crystal structures of both apo- and holo-PA1221 proteins, the latter using a valine-adenosine vinylsulfonamide inhibitor that traps the adenylation domain-carrier domain interaction. The protein adopts an interface similar to that seen with the prior adenylation domain-carrier protein construct. A comparison of these structures with previous structures of multidomain NRPSs suggests that a large conformational change within the NRPS adenylation domains guides the carrier domain into the active site for thioester formation.

Time-Temperature Indicator Based on Enzymatic Degradation of Dye-Loaded Polyhydroxybutyrate.

PubMed

Anbukarasu, Preetam; Sauvageau, Dominic; Elias, Anastasia L

2017-09-01

An enzyme activated time-temperature indicator (TTI) which produces a direct colour change concomitant to variations in integrated time and temperature conditions is described. This direct colour change is realised by degrading a dye-loaded polyhydroxybutyrate (PHB) film by a depolymerase enzyme. The degradation of the PHB film by the enzyme causes the release of the dye in solution, which in turn undergoes an optical transition from clear to coloured with elapsing time. Macroscopic and microscopic optical observations confirms the uniform distribution of the dye in the PHB film. The dye release kinetics, mediated by the enzymatic reaction, are tested at different temperatures ranging from 4 to 37 °C, and are used to determine the suitability of a dye-loaded PHB as a time-temperature indicator for fresh food products based on kinetic parameters previously reported. The kinetic analysis shows that the activation energy of the dye release process is 74 kJ mol -1 , and that, at 37 °C, the dye would be totally released within 6 h. However, when incubated at 4 °C, the TTI requires in the range of 168 h (7 days) to release all the dye. These kinetics values highlight the potential of the TTI for monitoring fresh food products that have optimum shelf life around 4 °C. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Immobilisation and characterisation of biocatalytic co-factor recycling enzymes, glucose dehydrogenase and NADH oxidase, on aldehyde functional ReSyn™ polymer microspheres.

PubMed

Twala, Busisiwe V; Sewell, B Trevor; Jordaan, Justin

2012-05-10

The use of enzymes in industrial applications is limited by their instability, cost and difficulty in their recovery and re-use. Immobilisation is a technique which has been shown to alleviate these limitations in biocatalysis. Here we describe the immobilisation of two biocatalytically relevant co-factor recycling enzymes, glucose dehydrogenase (GDH) and NADH oxidase (NOD) on aldehyde functional ReSyn™ polymer microspheres with varying functional group densities. The successful immobilisation of the enzymes on this new high capacity microsphere technology resulted in the maintenance of activity of ∼40% for GDH and a maximum of 15.4% for NOD. The microsphere variant with highest functional group density of ∼3500 μmol g⁻¹ displayed the highest specific activity for the immobilisation of both enzymes at 33.22 U mg⁻¹ and 6.75 U mg⁻¹ for GDH and NOD with respective loading capacities of 51% (0.51 mg mg⁻¹) and 129% (1.29 mg mg⁻¹). The immobilised GDH further displayed improved activity in the acidic pH range. Both enzymes displayed improved pH and thermal stability with the most pronounced thermal stability for GDH displayed on ReSyn™ A during temperature incubation at 65 °C with a 13.59 fold increase, and NOD with a 2.25-fold improvement at 45 °C on the same microsphere variant. An important finding is the suitability of the microspheres for stabilisation of the multimeric protein GDH. Copyright © 2012 Elsevier Inc. All rights reserved.

Biochemical and physiological parameters and estimated work output in draught horses pulling loads for long periods.

PubMed

Perez, R; Recabarren, S E; Valdes, P; Hetz, E

1992-01-01

A study was undertaken in five draught horses of 648 +/- 33 kg body weight to find the effects of continuously pulling loads on their cardiovascular, respiratory and metabolic responses. A cart equipped with an odometer, for measuring distance, and a hydraulic dynamometer, for measuring draught force, was used. Heart and respiration rates and rectal temperatures were recorded. Blood samples for measuring arterial and venous pH and blood gases, haemoglobin, glucose and lactic acid concentrations and the serum activity of the enzymes creatine phosphokinase (CK), lactate dehydrogenase, aspartate aminotransferase and alkaline phosphatase were taken before exercise and immediately after each journey (morning and afternoon) of the daily work. Draught exercise, with loads which generated forces of between 0.57 and 0.59 kN, at speeds of 1.60 to 2.11 m/s, for 8 h daily for five consecutive days, with resting intervals of 10 min each hour, was well tolerated. Exercise tolerance was evaluated from the recovery from the changes observed in the biochemical and physiological parameters induced by the work. The analysis of these showed that, when the horses were subjected to prolonged periods of resting, their loss of fitness for work was shown by significant increases in the serum activity of muscle-derived enzymes and in blood lactate concentrations during the first day of work. However, over the following days the horses adapted to the work, so that the decreases in serum enzyme activities and blood lactate concentrations were reduced. Since similar observations have been described for racehorses, the determination of blood lactate concentrations and the serum activities of muscle-derived enzymes, specifically CK, seem to be good indicators of fitness in draught horses.

Effect of Mild-to-Moderate Smoking on Viral Load, Cytokines, Oxidative Stress, and Cytochrome P450 Enzymes in HIV-Infected Individuals

PubMed Central

Ande, Anusha; McArthur, Carole; Ayuk, Leo; Awasom, Charles; Achu, Paul Ngang; Njinda, Annette; Sinha, Namita; Rao, P. S. S.; Agudelo, Marisela; Nookala, Anantha Ram; Simon, Stephen; Kumar, Anil; Kumar, Santosh

2015-01-01

Mild-to-moderate tobacco smoking is highly prevalent in HIV-infected individuals, and is known to exacerbate HIV pathogenesis. The objective of this study was to determine the specific effects of mild-to-moderate smoking on viral load, cytokine production, and oxidative stress and cytochrome P450 (CYP) pathways in HIV-infected individuals who have not yet received antiretroviral therapy (ART). Thirty-two human subjects were recruited and assigned to four different cohorts as follows: a) HIV negative non-smokers, b) HIV positive non-smokers, c) HIV negative mild-to-moderate smokers, and d) HIV positive mild-to-moderate smokers. Patients were recruited in Cameroon, Africa using strict selection criteria to exclude patients not yet eligible for ART and not receiving conventional or traditional medications. Those with active tuberculosis, hepatitis B or with a history of substance abuse were also excluded. Our results showed an increase in the viral load in the plasma of HIV positive patients who were mild-to-moderate smokers compared to individuals who did not smoke. Furthermore, although we did not observe significant changes in the levels of most pro-inflammatory cytokines, the cytokine IL-8 and MCP-1 showed a significant decrease in the plasma of HIV-infected patients and smokers compared with HIV negative non-smokers. Importantly, HIV-infected individuals and smokers showed a significant increase in oxidative stress compared with HIV negative non-smoker subjects in both plasma and monocytes. To examine the possible pathways involved in increased oxidative stress and viral load, we determined the mRNA levels of several antioxidant and cytochrome P450 enzymes in monocytes. The results showed that the levels of most antioxidants are unaltered, suggesting their inability to counter oxidative stress. While CYP2A6 was induced in smokers, CYP3A4 was induced in HIV and HIV positive smokers compared with HIV negative non-smokers. Overall, the findings suggest a possible association of oxidative stress and perhaps CYP pathway with smoking-mediated increased viral load in HIV positive individuals. PMID:25879453

Comparison of the impacts of acid and nitrogen additions on carbon fluxes in European conifer and broadleaf forests.

PubMed

Oulehle, Filip; Tahovská, Karolina; Chuman, Tomáš; Evans, Chris D; Hruška, Jakub; Růžek, Michal; Bárta, Jiří

2018-07-01

Increased reactive nitrogen (N) loadings to terrestrial ecosystems are believed to have positive effects on ecosystem carbon (C) sequestration. Global "hot spots" of N deposition are often associated with currently or formerly high deposition of sulphur (S); C fluxes in these regions might therefore not be responding solely to N loading, and could be undergoing transient change as S inputs change. In a four-year, two-forest stand (mature Norway spruce and European beech) replicated field experiment involving acidity manipulation (sulphuric acid addition), N addition (NH 4 NO 3 ) and combined treatments, we tested the extent to which altered soil solution acidity or/and soil N availability affected the concentration of soil dissolved organic carbon (DOC), soil respiration (Rs), microbial community characteristics (respiration, biomass, fungi and bacteria abundances) and enzyme activity. We demonstrated a large and consistent suppression of soil water DOC concentration driven by chemical changes associated with increased hydrogen ion concentrations under acid treatments, independent of forest type. Soil respiration was suppressed by sulphuric acid addition in the spruce forest, accompanied by reduced microbial biomass, increased fungal:bacterial ratios and increased C to N enzyme ratios. We did not observe equivalent effects of sulphuric acid treatments on Rs in the beech forest, where microbial activity appeared to be more tightly linked to N acquisition. The only changes in C cycling following N addition were increased C to N enzyme ratios, with no impact on C fluxes (either Rs or DOC). We conclude that C accumulation previously attributed solely to N deposition could be partly attributable to their simultaneous acidification. Copyright © 2018 Elsevier Ltd. All rights reserved.

High solid simultaneous saccharification and fermentation of wet oxidized corn stover to ethanol.

PubMed

Varga, Enikõ; Klinke, Helene B; Réczey, Kati; Thomsen, Anne Belinda

2004-12-05

In this study ethanol was produced from corn stover pretreated by alkaline and acidic wet oxidation (WO) (195 degrees C, 15 min, 12 bar oxygen) followed by nonisothermal simultaneous saccharification and fermentation (SSF). In the first step of the SSF, small amounts of cellulases were added at 50 degrees C, the optimal temperature of enzymes, in order to obtain better mixing condition due to some liquefaction. In the second step more cellulases were added in combination with dried baker's yeast (Saccharomyces cerevisiae) at 30 degrees C. The phenols (0.4-0.5 g/L) and carboxylic acids (4.6-5.9 g/L) were present in the hemicellulose rich hydrolyzate at subinhibitory levels, thus no detoxification was needed prior to SSF of the whole slurry. Based on the cellulose available in the WO corn stover 83% of the theoretical ethanol yield was obtained under optimized SSF conditions. This was achieved with a substrate concentration of 12% dry matter (DM) acidic WO corn stover at 30 FPU/g DM (43.5 FPU/g cellulose) enzyme loading. Even with 20 and 15 FPU/g DM (corresponding to 29 and 22 FPU/g cellulose) enzyme loading, ethanol yields of 76 and 73%, respectively, were obtained. After 120 h of SSF the highest ethanol concentration of 52 g/L (6 vol.%) was achieved, which exceeds the technical and economical limit of the industrial-scale alcohol distillation. The SSF results showed that the cellulose in pretreated corn stover can be efficiently fermented to ethanol with up to 15% DM concentration. A further increase of substrate concentration reduced the ethanol yield significant as a result of insufficient mass transfer. It was also shown that the fermentation could be followed with an easy monitoring system based on the weight loss of the produced CO2.

Rhodanese Functions as Sulfur Supplier for Key Enzymes in Sulfur Energy Metabolism

PubMed Central

Aussignargues, Clément; Giuliani, Marie-Cécile; Infossi, Pascale; Lojou, Elisabeth; Guiral, Marianne; Giudici-Orticoni, Marie-Thérèse; Ilbert, Marianne

2012-01-01

How microorganisms obtain energy is a challenging topic, and there have been numerous studies on the mechanisms involved. Here, we focus on the energy substrate traffic in the hyperthermophilic bacterium Aquifex aeolicus. This bacterium can use insoluble sulfur as an energy substrate and has an intricate sulfur energy metabolism involving several sulfur-reducing and -oxidizing supercomplexes and enzymes. We demonstrate that the cytoplasmic rhodanese SbdP participates in this sulfur energy metabolism. Rhodaneses are a widespread family of proteins known to transfer sulfur atoms. We show that SbdP has also some unusual characteristics compared with other rhodaneses; it can load a long sulfur chain, and it can interact with more than one partner. Its partners (sulfur reductase and sulfur oxygenase reductase) are key enzymes of the sulfur energy metabolism of A. aeolicus and share the capacity to use long sulfur chains as substrate. We demonstrate a positive effect of SbdP, once loaded with sulfur chains, on sulfur reductase activity, most likely by optimizing substrate uptake. Taken together, these results lead us to propose a physiological role for SbdP as a carrier and sulfur chain donor to these key enzymes, therefore enabling channeling of sulfur substrate in the cell as well as greater efficiency of the sulfur energy metabolism of A. aeolicus. PMID:22496367

Simultaneous or separated; comparison approach for saccharification and fermentation process in producing bio-ethanol from EFB

NASA Astrophysics Data System (ADS)

Bardant, Teuku Beuna; Dahnum, Deliana; Amaliyah, Nur

2017-11-01

Simultaneous Saccharification Fermentation (SSF) of palm oil (Elaeis guineensis) empty fruit bunch (EFB) pulp were investigated as a part of ethanol production process. SSF was investigated by observing the effect of substrate loading variation in range 10-20%w, cellulase loading 5-30 FPU/gr substrate and yeast addition 1-2%v to the ethanol yield. Mathematical model for describing the effects of these three variables to the ethanol yield were developed using Response Surface Methodology-Cheminformatics (RSM-CI). The model gave acceptable accuracy in predicting ethanol yield for Simultaneous Saccharification and Fermentation (SSF) with coefficient of determination (R2) 0.8899. Model validation based on data from previous study gave (R2) 0.7942 which was acceptable for using this model for trend prediction analysis. Trend prediction analysis based on model prediction yield showed that SSF gave trend for higher yield when the process was operated in high enzyme concentration and low substrate concentration. On the other hand, even SHF model showed better yield will be obtained if operated in lower substrate concentration, it still possible to operate in higher substrate concentration with slightly lower yield. Opportunity provided by SHF to operate in high loading substrate make it preferable option for application in commercial scale.

Enzyme-activated intracellular drug delivery with tubule clay nanoformulation

DOE PAGES

Dzamukova, Maria R.; Naumenko, Ekaterina A.; Lvov, Yuri M.; ...

2015-05-15

Fabrication of stimuli-triggered drug delivery vehicle is is an important milestone in treating cancer. Here we demonstrate the selective anticancer drug delivery into human cells with biocompatible 50-nm diameter halloysite nanotube carriers. Physically-adsorbed dextrin end stoppers secure the intercellular release of brilliant green. Drug-loaded nanotubes penetrate through the cellular membranes and their uptake efficiency depends on the cells growth rate. Intercellular glycosyl hydrolases-mediated decomposition of the dextrin tube-end stoppers triggers the release of the lumen-loaded brilliant green, which allowed for preferable elimination of human lung carcinoma cells (А549) as compared with hepatoma cells (Hep3b). In conclusion, the enzyme-activated intracellular deliverymore » of brilliant green using dextrin-coated halloysite nanotubes is a promising platform for anticancer treatment.« less

An Enzyme-Free Signal Amplification Technique for Ultrasensitive Colorimetric Assay of Disease Biomarkers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ye, Haihang; Yang, Kuikun; Tao, Jing

Enzyme-based colorimetric assays have been widely used in research labs and clinical diagnosis for decades. Nevertheless, as constrained by the performance of enzymes, their detection sensitivity has not been substantially improved in recent years, which inhibits many critical applications such as early detection of cancers. In this work, we demonstrate an enzyme-free signal amplification technique, based on gold vesicles encapsulated with Pd-Ir nanoparticles as peroxidase mimics, for colorimetric assay of disease biomarkers with significantly enhanced sensitivity. This technique overcomes the intrinsic limitations of enzymes, thanks to the superior catalytic efficiency of peroxidase mimics and the efficient loading and release ofmore » these mimics. Using human prostate surface antigen as a model biomarker, we demonstrated that the enzyme-free assay could reach a limit of detection at the femtogram/mL level, which is over 10 3-fold lower than that of conventional enzyme-based assay when the same antibodies and similar procedure were used.« less

An Enzyme-Free Signal Amplification Technique for Ultrasensitive Colorimetric Assay of Disease Biomarkers

DOE PAGES

Ye, Haihang; Yang, Kuikun; Tao, Jing; ...

2017-01-30

Enzyme-based colorimetric assays have been widely used in research labs and clinical diagnosis for decades. Nevertheless, as constrained by the performance of enzymes, their detection sensitivity has not been substantially improved in recent years, which inhibits many critical applications such as early detection of cancers. In this work, we demonstrate an enzyme-free signal amplification technique, based on gold vesicles encapsulated with Pd-Ir nanoparticles as peroxidase mimics, for colorimetric assay of disease biomarkers with significantly enhanced sensitivity. This technique overcomes the intrinsic limitations of enzymes, thanks to the superior catalytic efficiency of peroxidase mimics and the efficient loading and release ofmore » these mimics. Using human prostate surface antigen as a model biomarker, we demonstrated that the enzyme-free assay could reach a limit of detection at the femtogram/mL level, which is over 10 3-fold lower than that of conventional enzyme-based assay when the same antibodies and similar procedure were used.« less

Construction of an Immobilized Thermophilic Esterase on Epoxy Support for Poly(ε-caprolactone) Synthesis.

PubMed

Ren, Hui; Xing, Zhen; Yang, Jiebing; Jiang, Wei; Zhang, Gang; Tang, Jun; Li, Quanshun

2016-06-18

Developing an efficient immobilized enzyme is of great significance for improving the operational stability of enzymes in poly(ε-caprolactone) synthesis. In this paper, a thermophilic esterase AFEST from the archaeon Archaeoglobus fulgidus was successfully immobilized on the epoxy support Sepabeads EC-EP via covalent attachment, and the immobilized enzyme was then employed as a biocatalyst for poly(ε-caprolactone) synthesis. The enzyme loading and recovered activity of immobilized enzyme was measured to be 72 mg/g and 10.4 U/mg using p-nitrophenyl caprylate as the substrate at 80 °C, respectively. Through the optimization of reaction conditions (enzyme concentration, temperature, reaction time and medium), poly(ε-caprolactone) was obtained with 100% monomer conversion and low number-average molecular weight (Mn < 1300 g/mol). Further, the immobilized enzyme exhibited excellent reusability, with monomer conversion values exceeding 75% during 15 batch reactions. Finally, poly(ε-caprolactone) was enzymatically synthesized with an isolated yield of 75% and Mn value of 3005 g/mol in a gram-scale reaction.

3D Printed Programmable Release Capsules.

PubMed

Gupta, Maneesh K; Meng, Fanben; Johnson, Blake N; Kong, Yong Lin; Tian, Limei; Yeh, Yao-Wen; Masters, Nina; Singamaneni, Srikanth; McAlpine, Michael C

2015-08-12

The development of methods for achieving precise spatiotemporal control over chemical and biomolecular gradients could enable significant advances in areas such as synthetic tissue engineering, biotic-abiotic interfaces, and bionanotechnology. Living organisms guide tissue development through highly orchestrated gradients of biomolecules that direct cell growth, migration, and differentiation. While numerous methods have been developed to manipulate and implement biomolecular gradients, integrating gradients into multiplexed, three-dimensional (3D) matrices remains a critical challenge. Here we present a method to 3D print stimuli-responsive core/shell capsules for programmable release of multiplexed gradients within hydrogel matrices. These capsules are composed of an aqueous core, which can be formulated to maintain the activity of payload biomolecules, and a poly(lactic-co-glycolic) acid (PLGA, an FDA approved polymer) shell. Importantly, the shell can be loaded with plasmonic gold nanorods (AuNRs), which permits selective rupturing of the capsule when irradiated with a laser wavelength specifically determined by the lengths of the nanorods. This precise control over space, time, and selectivity allows for the ability to pattern 2D and 3D multiplexed arrays of enzyme-loaded capsules along with tunable laser-triggered rupture and release of active enzymes into a hydrogel ambient. The advantages of this 3D printing-based method include (1) highly monodisperse capsules, (2) efficient encapsulation of biomolecular payloads, (3) precise spatial patterning of capsule arrays, (4) "on the fly" programmable reconfiguration of gradients, and (5) versatility for incorporation in hierarchical architectures. Indeed, 3D printing of programmable release capsules may represent a powerful new tool to enable spatiotemporal control over biomolecular gradients.

A Potentiometric Indirect Uric Acid Sensor Based on ZnO Nanoflakes and Immobilized Uricase

PubMed Central

Usman Ali, Syed M.; Ibupoto, Zafar Hussain; Kashif, Muhammad; Hashim, Uda; Willander, Magnus

2012-01-01

In the present work zinc oxide nanoflakes (ZnO-NF) structures with a wall thickness around 50 to 100 nm were synthesized on a gold coated glass substrate using a low temperature hydrothermal method. The enzyme uricase was electrostatically immobilized in conjunction with Nafion membrane on the surface of well oriented ZnO-NFs, resulting in a sensitive, selective, stable and reproducible uric acid sensor. The electrochemical response of the ZnO-NF-based sensor vs. a Ag/AgCl reference electrode was found to be linear over a relatively wide logarithmic concentration range (500 nM to 1.5 mM). In addition, the ZnO-NF structures demonstrate vast surface area that allow high enzyme loading which results provided a higher sensitivity. The proposed ZnO-NF array-based sensor exhibited a high sensitivity of ∼66 mV/ decade in test electrolyte solutions of uric acid, with fast response time. The sensor response was unaffected by normal concentrations of common interferents such as ascorbic acid, glucose, and urea. PMID:22736977

Olive mill wastewater biodegradation potential of white-rot fungi--Mode of action of fungal culture extracts and effects of ligninolytic enzymes.

PubMed

Ntougias, Spyridon; Baldrian, Petr; Ehaliotis, Constantinos; Nerud, Frantisek; Merhautová, Věra; Zervakis, Georgios I

2015-01-01

Forty-nine white-rot strains belonging to 38 species of Basidiomycota were evaluated for olive-mill wastewater (OMW) degradation. Almost all fungi caused high total phenolics (>60%) and color (⩽ 70%) reduction, while COD and phytotoxicity decreased to a lesser extent. Culture extracts from selected Agrocybe cylindracea, Inonotus andersonii, Pleurotus ostreatus and Trametes versicolor strains showed non-altered physicochemical and enzymatic activity profiles when applied to raw OMW in the presence or absence of commercial catalase, indicating no interaction of the latter with fungal enzymes and no competition for H2O2. Hydrogen peroxide's addition resulted in drastic OMW's decolorization, with no effect on phenolic content, suggesting that oxidation affects colored components, but not necessarily phenolics. When fungal extracts were heat-treated, no phenolics decrease was observed demonstrating thus their enzymatic rather than physicochemical oxidation. Laccases added to OMW were reversibly inhibited by the effluent's high phenolic load, while peroxidases were stable and active during the entire process. Copyright © 2015 Elsevier Ltd. All rights reserved.

Visualization of a radical B 12 enzyme with its G-protein chaperone

DOE PAGES

Jost, Marco; Cracan, Valentin; Hubbard, Paul A.; ...

2015-02-09

G-protein metallochaperones ensure fidelity during cofactor assembly for a variety of metalloproteins, including adenosylcobalamin (AdoCbl)-dependent methylmalonyl-CoA mutase and hydrogenase, and thus have both medical and biofuel development applications. In this paper, we present crystal structures of IcmF, a natural fusion protein of AdoCbl-dependent isobutyryl-CoA mutase and its corresponding G-protein chaperone, which reveal the molecular architecture of a G-protein metallochaperone in complex with its target protein. These structures show that conserved G-protein elements become ordered upon target protein association, creating the molecular pathways that both sense and report on the cofactor loading state. Structures determined of both apo- and holo-forms ofmore » IcmF depict both open and closed enzyme states, in which the cofactor-binding domain is alternatively positioned for cofactor loading and for catalysis. Finally and notably, the G protein moves as a unit with the cofactor-binding domain, providing a visualization of how a chaperone assists in the sequestering of a precious cofactor inside an enzyme active site.« less

Accessibility of Enzymatically Delignified Bambusa bambos for Efficient Hydrolysis at Minimum Cellulase Loading: An Optimization Study.

PubMed

Kuila, Arindam; Mukhopadhyay, Mainak; Tuli, D K; Banerjee, Rintu

2011-01-01

In the present investigation, Bambusa bambos was used for optimization of enzymatic pretreatment and saccharification. Maximum enzymatic delignification achieved was 84%, after 8 h of incubation time. Highest reducing sugar yield from enzyme-pretreated Bambusa bambos was 818.01 mg/g dry substrate after 8 h of incubation time at a low cellulase loading (endoglucanase, β-glucosidase, exoglucanase, and xylanase were 1.63 IU/mL, 1.28 IU/mL, 0.08 IU/mL, and 47.93 IU/mL, respectively). Enzyme-treated substrate of Bambusa bambos was characterized by analytical techniques such as Fourier transformed infrared spectroscopy (FTIR), X-ray diffraction (XRD), and scanning electron microscopy (SEM). The FTIR spectrum showed that the absorption peaks of several functional groups were decreased after enzymatic pretreatment. XRD analysis indicated that cellulose crystallinity of enzyme-treated samples was increased due to the removal of amorphous lignin and hemicelluloses. SEM image showed that surface structure of Bambusa bambos was distorted after enzymatic pretreatment.

FeCl3-catalyzed ethanol pretreatment of sugarcane bagasse boosts sugar yields with low enzyme loadings and short hydrolysis time.

PubMed

Zhang, Hongdan; Zhang, Shuaishuai; Yuan, Hongyou; Lyu, Gaojin; Xie, Jun

2018-02-01

An organosolv pretreatment system consisting of 60% ethanol and 0.025 mol·L -1 FeCl 3 under various temperatures was developed in this study. During the pretreatment, the highest xylose yield was 11.4 g/100 g raw material, representing 49.8% of xylose in sugarcane bagasse. Structural features of raw material and pretreated substrates were characterized to better understand how hemicellulose removal and delignification affected subsequent enzymatic hydrolysis. The 160 °C pretreated solid presented a remarkable glucose yield of 93.8% for 72 h. Furthermore, the influence of different additives on the enzymatic hydrolysis of pretreated solid was investigated. The results indicated that the addition of Tween 80 shortened hydrolysis time to 6 h and allowed a 50% reduction of enzyme loading to achieve the same level of glucose yield. This work suggested that FeCl 3 -catalyzed organosolv pretreatment could improve the enzymatic hydrolysis significantly and reduce the hydrolysis time and enzyme dosage with the addition of Tween 80. Copyright © 2017 Elsevier Ltd. All rights reserved.

β-actin as a loading control for plasma-based Western blot analysis of major depressive disorder patients.

PubMed

Zhang, Rufang; Yang, Deyu; Zhou, Chanjuan; Cheng, Ke; Liu, Zhao; Chen, Liang; Fang, Liang; Xie, Peng

2012-08-15

Western blot analysis is a commonly used technique for determining specific protein levels in clinical samples. For normalization of protein levels in Western blot, a suitable loading control is required. On account of its relatively high and constant expression, β-actin has been widely employed in Western blot of cell cultures and tissue extracts. However, β-actin's presence in human plasma and this protein's putative role as a plasma-based loading control for Western blot analysis remain unknown. In this study, an enzyme-linked immunosorbent assay was used to determine the concentration of β-actin in human plasma, which is 6.29±0.54 ng/ml. In addition, the linearity of β-actin immunostaining and loaded protein amount was evaluated by Western blot, and a fine linearity (R²=0.974±0.012) was observed. Furthermore, the expression of plasma β-actin in major depressive disorder subjects and healthy controls was compared. The data revealed no statistically significant difference between these two groups. Moreover, the total coefficient of variation for β-actin expression in the two groups was 9.2±1.2%. These findings demonstrate that β-actin is present in human plasma and may possibly be used as a suitable loading control for plasma-based Western blot analysis in major depressive disorder. Copyright © 2012 Elsevier Inc. All rights reserved.

Mechanical loading of bovine pericardium accelerates enzymatic degradation.

PubMed

Ellsmere, J C; Khanna, R A; Lee, J M

1999-06-01

Bioprosthetic heart valves fail as the result of two simultaneous processes: structural deterioration and calcification. Leaflet deterioration and perforation have been correlated with regions of highest stress in the tissue. The failures have long been assumed to be due to simple mechanical fatigue of the collagen fibre architecture; however, we have hypothesized that local stresses-and particularly dynamic stresses-accelerate local proteolysis, leading to tissue failure. This study addresses that hypothesis. Using a novel, custom-built microtensile culture system, strips of bovine pericardium were subjected to static and dynamic loads while being exposed to solutions of microbial collagenase or trypsin (a non-specific proteolytic enzyme). The time to extend to 30% strain (defined here as time to failure) was recorded. After failure, the percentage of collagen solubilized was calculated based on the amount of hydroxyproline present in solution. All data were analyzed by analysis of variance (ANOVA). In collagenase, exposure to static load significantly decreased the time to failure (P < 0.002) due to increased mean rate of collagen solubilization. Importantly, specimens exposed to collagenase and dynamic load failed faster than those exposed to collagenase under the same average static load (P = 0.02). In trypsin, by contrast, static load never led to failure and produced only minimal degradation. Under dynamic load, however, specimens exposed to collagenase, trypsin, and even Tris/CaCl2 buffer solution, all failed. Only samples exposed to Hanks' physiological solution did not fail. Failure of the specimens exposed to trypsin and Tris/CaCl2 suggests that the non-collagenous components and the calcium-dependent proteolytic enzymes present in pericardial tissue may play roles in the pathogenesis of bioprosthetic heart valve degeneration.

Performance of non-compartmentalized enzymatic biofuel cell based on buckypaper cathode and ferrocene-containing redox polymer anode

NASA Astrophysics Data System (ADS)

Bunte, Christine; Hussein, Laith; Urban, Gerald A.

2014-02-01

Novel single compartment Glucose/O2 biofuel cells (BFCs) were developed using immobilized enzymes and the mediated electron transfer (MET) approach. The bioanode was prepared through a ferrocene-containing redox polymer crosslinked in the presence of a biocatalyst on a glassy carbon support. Here, the redox polymer can physically entrap the enzyme and prevent it from leaching. Additionally it provides a biocompatible microenvironment and thus could extend the life time of enzyme. On the other side, the mediated biocathode was prepared based on bilirubin oxidase and 2,2‧-azinobis(3-ethylbenzothiazoline-6-sulfonate) diammonium salt (ABTS2-) system which has been physically entrapped in Nafion matrix and then adsorbed directly on a highly porous, conductive and functionalized buckypaper (fBP). Both electrodes were characterized physically and electrochemically. Employing these electrodes, the resulting BFC generates an open circuit voltage (Voc) of approximately 0.550 V and a peak power density of 26 μW cm-2 at 0.2 V at 37 °C in quiescent O2-saturated physiological buffer containing 5 mM glucose. The cell sustains a load up to 225 μA cm-2. Moreover, a high short circuit current (Isc) of 300 μA cm-2 is approached. This BFC can operate in mild conditions without using any toxic materials which makes it attractive for implantable devices.

The Vital Function of Fe3O4@Au nanocomposites for Hydrolase Biosensor Design and Its Application in Detection of Methyl Parathion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhao, Yuting; Zhang, Weiying; Lin, Yuehe

A nanocomposite of gold nanoparticles (AuNPs) decorating a magnetic Fe3O4 core was synthesized using cysteamine (SH–NH2) as linker, and characterized by TEM, XPS, UV and electrochemistry. Then a hydrolase biosensor, based on self-assembly of methyl parathion hydrolase (MPH) on the Fe3O4@Au nanocomposite, was developed for sensitive and selective detection of the organophosphorus pesticide (OP) methyl parathion. The magnetic nanocomposite provides an easy way to construct the enzyme biosensor by simply exerting an external magnetic field, and also provides a simple way to renew the electrode surface by removing the magnet. Unlike inhibition-based enzyme biosensors, the hydrolase is not poisoned bymore » OPs and thus is reusable for continuous measurement. AuNPs not only provide a large surface area, high loading efficiency and fast electron transfer, but also stabilize the enzyme through electrostatic interactions. The MPH biosensor shows rapid response and high selectivity for detection of methyl parathion, with a linear range from 0.5 to 1000 ng/mL and a detection limit of 0.1 ng/mL. It also shows acceptable reproducibility and stability. The simplicity and ease of operation of the proposed method has great potential for on-site detection of P–S containing pesticides and provides a promising strategy to construct a robust biosensor.« less

Bioinspired production of magnetic laccase-biotitania particles for the removal of endocrine disrupting chemicals.

PubMed

Ardao, Inés; Magnin, Delphine; Agathos, Spiros N

2015-10-01

Microbial laccases are powerful enzymes capable of degrading lignin and other recalcitrant compounds including endocrine disrupting chemicals (EDCs). Efficient EDC removal on an industrial scale requires robust, stable, easy to handle and cost-effective immobilized biocatalysts. In this direction, magnetic biocatalysts are attractive due to their easy separation through an external magnetic field. Recently, a bioinspired immobilization technique that mimics the natural biomineralization reactions in diatoms has emerged as a fast and versatile tool for generating robust, cheap, and highly stable (nano) biocatalysts. In this work, bioinspired formation of a biotitania matrix is triggered on the surface of magnetic particles in the presence of laccase in order to produce laccase-biotitania (lac-bioTiO2 ) biocatalysts suitable for environmental applications using a novel, fast and versatile enzyme entrapment technique. Highly active lac-bioTiO2 particles have been produced and the effect of different parameters (enzyme loading, titania precursor concentration, pH, duration of the biotitania formation, and laccase adsorption steps) on the apparent activity yield of these biocatalysts were evaluated, the concentration of the titania precursor being the most influential. The lac-bioTiO2 particles were able to catalyze the removal of bisphenol A, 17α-ethinylestradiol and diclofenac in a mixture of six model EDCs and retained 90% of activity after five reaction cycles and 60% after 10 cycles. © 2015 Wiley Periodicals, Inc.

Liposomal Encapsulation Enzymes: From Medical Applications to Kinetic Characteristics.

PubMed

Jahadi, M; Khosravi-Darani, K

2017-01-01

Liposomes and nanoliposomes as small vesicles composed of phospholipid bilayer (entrapping one or more hydrophilic or lipophilic components) have recently found several potential applications in medicine and food industry. These vesicles may protect the core materials from moisture, heat and other extreme conditions. They may also provide controlled release of various bioactive agents, including food ingredients at the right place and time. Potential applications of enzyme-loaded liposomes are in the medical or biomedical field, particularly for the enzymereplacement therapy, as well as cheese industry for production of functional foods with improved health beneficial impacts on the consumer. Encapsulation process has a recondite impact on enzymes. In fact, liposome preparation techniques may alter the pH and temperature optima, affinity of the enzyme to substrate (Km), and maximum rate of reaction (Vmax). In addition, in this paper, the impact of process variables on the kinetic characteristics of enzymes encapsulated in liposomes was investigated. Also, the effects of enzyme entrapment in liposomes, prepared by different methods, on the catalytic efficiency of enzyme, as well as its kinetic properties and stability compared to native (free) enzymes has been reviewed.

Immobilization, stabilization and patterning techniques for enzyme based sensor systems.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Flounders, A.W.; Carichner, S.C.; Singh, A.K.

1997-01-01

Sandia National Laboratories has recently opened the Chemical and Radiation Detection Laboratory (CRDL) in Livermore CA to address the detection needs of a variety of government agencies (e.g., Department of Energy, Environmental Protection Agency, Department of Agriculture) as well as provide a fertile environment for the cooperative development of new industrial technologies. This laboratory consolidates a variety of existing chemical and radiation detection efforts and enables Sandia to expand into the novel area of biochemically based sensors. One aspect of this biosensor effort is further development and optimization of enzyme modified field effect transistors (EnFETs). Recent work has focused uponmore » covalent attachment of enzymes to silicon dioxide and silicon nitride surfaces for EnFET fabrication. They are also investigating methods to pattern immobilized proteins; a critical component for development of array-based sensor systems. Novel enzyme stabilization procedures are key to patterning immobilized enzyme layers while maintaining enzyme activity. Results related to maximized enzyme loading, optimized enzyme activity and fluorescent imaging of patterned surfaces will be presented.« less

Enzyme and Chemical Encapsulation in Polymeric Microcapsules,

DTIC Science & Technology

1995-01-01

Polypyrrole microcapsules (prepared via the template method) were used for immobilization of both enzymatic and chemical catalytic systems. Enzymes...immobilized Pd nanoparticles for catalysis of hydrogen peroxide decomposition. Microcapsules loaded with glucose oxidase (GOD) were found to have...from the capsules; no leakage was observed. Subtilisin was used to show that these microcapsules can be used in non-aqueous solvents. The effect of capsule wall thickness on the rate of enzymatic reaction was also explored.

Lipase-catalysed interesterification between canola oil and fully hydrogenated canola oil in contact with supercritical carbon dioxide.

PubMed

Jenab, Ehsan; Temelli, Feral; Curtis, Jonathan M

2013-12-01

The processing parameters in enzymatic reactions using CO2-expanded (CX) lipids have strong effects on the physical properties of liquid phase, degree of interesterification, and physicochemical properties of the final reaction products. CX-canola oil and fully hydrogenated canola oil (FHCO) were interesterified using Lipozyme TL IM in a high pressure stirred batch reactor. The effects of immobilised enzyme load, pressure, substrate ratio and reaction time on the formation of mixed triacylglycerols (TG) from trisaturated and triunsaturated TG were investigated. The optimal immobilised enzyme load, pressure, substrate ratio and time for the degree of interesterification to reach the highest equilibrium state were 6% (w/v) of initial substrates, 10 MPa, blend with 30% (w/w) of FHCO and 2h, respectively. The physicochemical properties of the initial blend and interesterified products with different FHCO ratios obtained at optimal reaction conditions were determined in terms of TG composition, thermal behaviour and solid fat content (SFC). The amounts of saturated and triunsaturated TG decreased while the amounts of mixed TG increased as a result of interesterification. Thus, the interesterified product had a lower melting point, and broader melting and plasticity ranges compared to the initial blends. These findings are important for better understanding of CX-lipid reactions and for optimal formulation of base-stocks of margarine and confectionary fats to meet industry demands. Copyright © 2013 Elsevier Ltd. All rights reserved.

Load capacity improvements in nucleic acid based systems using partially open feedback control.

PubMed

Kulkarni, Vishwesh; Kharisov, Evgeny; Hovakimyan, Naira; Kim, Jongmin

2014-08-15

Synthetic biology is facilitating novel methods and components to build in vivo and in vitro circuits to better understand and re-engineer biological networks. Recently, Kim and Winfree have synthesized a remarkably elegant network of transcriptional oscillators in vitro using a modular architecture of synthetic gene analogues and a few enzymes that, in turn, could be used to drive a variety of downstream circuits and nanodevices. However, these oscillators are sensitive to initial conditions and downstream load processes. Furthermore, the oscillations are not sustained since the inherently closed design suffers from enzyme deactivation, NTP fuel exhaustion, and waste product build up. In this paper, we show that a partially open architecture in which an [Symbol: see text]1 adaptive controller, implemented inside an in silico computer that resides outside the wet-lab apparatus, can ensure sustained tunable oscillations in two specific designs of the Kim-Winfree oscillator networks. We consider two broad cases of operation: (1) the oscillator network operating in isolation and (2) the oscillator network driving a DNA tweezer subject to a variable load. In both scenarios, our simulation results show a significant improvement in the tunability and robustness of these oscillator networks. Our approach can be easily adopted to improve the loading capacity of a wide range of synthetic biological devices.

L-Asparaginase encapsulated intact erythrocytes for treatment of acute lymphoblastic leukemia (ALL).

PubMed

Kwon, Young Min; Chung, Hee Sun; Moon, Cheol; Yockman, James; Park, Yoon Jeong; Gitlin, Scott D; David, Allan E; Yang, Victor C

2009-11-03

As a primary drug for the treatment of acute lymphoblastic leukemia (ALL), encapsulation of L-asparaginase (ASNase) into red blood cells (RBC) has been popular to circumvent immunogenicity from the exogenous protein. Unlike existing methods that perturbs RBC membranes, we introduce a novel method of RBC-incorporation of proteins using the membrane-translocating low molecular weight protamine (LMWP). Confocal study of fluorescence-labeled LMWP-ovalbumin, as a model protein conjugate, has shown significant fluorescence inside RBCs. Surface morphology by scanning electron microscopy of the RBCs loaded with LMWP-ASNase was indistinguishable with normal RBCs. These drug loaded RBCs also closely resembled the profile of the native erythrocytes in terms of osmotic fragility, oxygen dissociation and hematological parameters. The in vivo half-life of enzyme activity after administering 8 units of RBC/LMWP-ASNase in DBA/2 mice was prolonged to 4.5+/-0.5 days whereas that of RBCs loaded with ASNase via a hypotonic method was 2.4+/-0.7 days. Furthermore, the mean survival time of DBA/2 mice bearing mouse lymphoma cell L5178Y was improved by approximately 44% compared to the saline control group after treatment with the RBC loaded enzymes. From these data, an innovative, novel method for encapsulating proteins into intact and fully functional erythrocytes was established for potential treatment of ALL.

Mathematical modelling of metabolic pathways affected by an enzyme deficiency. Energy and redox metabolism of glucose-6-phosphate-dehydrogenase-deficient erythrocytes.

PubMed

Schuster, R; Jacobasch, G; Holzhütter, H G

1989-07-01

The effects of various forms of glucose-6-phosphate dehydrogenase deficiency on erythrocyte metabolism have been studied on the basis of a complex mathematical model which comprises the main pathways of this cell: glycolysis, pentose pathway, reactions of the glutathione and adenine nucleotide metabolism. The calculated flux rates through the oxidative pentose pathway with and without methylene blue are in good accord with experimental results. The degree of deficiency as predicted by the model on the basis of calculated upper oxidative load boundaries, as well as of maximal methylene blue stimulation, correlates with the individual clinical manifestation of the metabolic disease. Therefore, the model allows one to judge the degree of metabolic disorder in the presence of glucose-6-phosphate dehydrogenase enzymopathies if the kinetic properties of the defect enzyme are known. Experimentally accessible parameters for an assessment of the oxidative load capacity of cells in vivo are proposed. It is pointed out that the threshold of tolerance as to energetic load is drastically reduced in the case of severe glucose-6-phosphate dehydrogenase deficiency.

Optimization of Enzymatic Saccharification of Alkali Pretreated Parthenium sp. Using Response Surface Methodology

PubMed Central

Pandiyan, K.; Tiwari, Rameshwar; Singh, Surender; Nain, Pawan K. S.; Rana, Sarika; Arora, Anju; Singh, Shashi B.; Nain, Lata

2014-01-01

Parthenium sp. is a noxious weed which threatens the environment and biodiversity due to its rapid invasion. This lignocellulosic weed was investigated for its potential in biofuel production by subjecting it to mild alkali pretreatment followed by enzymatic saccharification which resulted in significant amount of fermentable sugar yield (76.6%). Optimization of enzymatic hydrolysis variables such as temperature, pH, enzyme, and substrate loading was carried out using central composite design (CCD) in response to surface methodology (RSM) to achieve the maximum saccharification yield. Data obtained from RSM was validated using ANOVA. After the optimization process, a model was proposed with predicted value of 80.08% saccharification yield under optimum conditions which was confirmed by the experimental value of 85.80%. This illustrated a good agreement between predicted and experimental response (saccharification yield). The saccharification yield was enhanced by enzyme loading and reduced by temperature and substrate loading. This study reveals that under optimized condition, sugar yield was significantly increased which was higher than earlier reports and promises the use of Parthenium sp. biomass as a feedstock for bioethanol production. PMID:24900917

Coupling alkaline pre-extraction with alkaline-oxidative post-treatment of corn stover to enhance enzymatic hydrolysis and fermentability.

PubMed

Liu, Tongjun; Williams, Daniel L; Pattathil, Sivakumar; Li, Muyang; Hahn, Michael G; Hodge, David B

2014-04-03

A two-stage chemical pretreatment of corn stover is investigated comprising an NaOH pre-extraction followed by an alkaline hydrogen peroxide (AHP) post-treatment. We propose that conventional one-stage AHP pretreatment can be improved using alkaline pre-extraction, which requires significantly less H2O2 and NaOH. To better understand the potential of this approach, this study investigates several components of this process including alkaline pre-extraction, alkaline and alkaline-oxidative post-treatment, fermentation, and the composition of alkali extracts. Mild NaOH pre-extraction of corn stover uses less than 0.1 g NaOH per g corn stover at 80°C. The resulting substrates were highly digestible by cellulolytic enzymes at relatively low enzyme loadings and had a strong susceptibility to drying-induced hydrolysis yield losses. Alkaline pre-extraction was highly selective for lignin removal over xylan removal; xylan removal was relatively minimal (~20%). During alkaline pre-extraction, up to 0.10 g of alkali was consumed per g of corn stover. AHP post-treatment at low oxidant loading (25 mg H2O2 per g pre-extracted biomass) increased glucose hydrolysis yields by 5%, which approached near-theoretical yields. ELISA screening of alkali pre-extraction liquors and the AHP post-treatment liquors demonstrated that xyloglucan and β-glucans likely remained tightly bound in the biomass whereas the majority of the soluble polymeric xylans were glucurono (arabino) xylans and potentially homoxylans. Pectic polysaccharides were depleted in the AHP post-treatment liquor relative to the alkaline pre-extraction liquor. Because the already-low inhibitor content was further decreased in the alkaline pre-extraction, the hydrolysates generated by this two-stage pretreatment were highly fermentable by Saccharomyces cerevisiae strains that were metabolically engineered and evolved for xylose fermentation. This work demonstrates that this two-stage pretreatment process is well suited for converting lignocellulose to fermentable sugars and biofuels, such as ethanol. This approach achieved high enzymatic sugars yields from pretreated corn stover using substantially lower oxidant loadings than have been reported previously in the literature. This pretreatment approach allows for many possible process configurations involving novel alkali recovery approaches and novel uses of alkaline pre-extraction liquors. Further work is required to identify the most economical configuration, including process designs using techno-economic analysis and investigating processing strategies that economize water use.

Effective L-Tyrosine Hydroxylation by Native and Immobilized Tyrosinase

PubMed Central

Lewańczuk, Marcin; Koźlecki, Tomasz; Liesiene, Jolanta; Bryjak, Jolanta

2016-01-01

Hydroxylation of L-tyrosine to 3,4-dihydroxyphenylalanine (L-DOPA) by immobilized tyrosinase in the presence of ascorbic acid (AH2), which reduces DOPA-quinone to L-DOPA, is characterized by low reaction yields that are mainly caused by the suicide inactivation of tyrosinase by L-DOPA and AH2. The main aim of this work was to compare processes with native and immobilized tyrosinase to identify the conditions that limit suicide inactivation and produce substrate conversions to L-DOPA of above 50% using HPLC analysis. It was shown that immobilized tyrosinase does not suffer from partitioning and diffusion effects, allowing a direct comparison of the reactions performed with both forms of the enzyme. In typical processes, additional aeration was applied and boron ions to produce the L-DOPA and AH2 complex and hydroxylamine to close the cycle of enzyme active center transformations. It was shown that the commonly used pH 9 buffer increased enzyme stability, with concomitant reduced reactivity of 76%, and that under these conditions, the maximal substrate conversion was approximately 25 (native) to 30% (immobilized enzyme). To increase reaction yield, the pH of the reaction mixture was reduced to 8 and 7, producing L-DOPA yields of approximately 95% (native enzyme) and 70% (immobilized). A three-fold increase in the bound enzyme load achieved 95% conversion in two successive runs, but in the third one, tyrosinase lost its activity due to strong suicide inactivation caused by L-DOPA processing. In this case, the cost of the immobilized enzyme preparation is not overcome by its reuse over time, and native tyrosinase may be more economically feasible for a single use in L-DOPA production. The practical importance of the obtained results is that highly efficient hydroxylation of monophenols by tyrosinase can be obtained by selecting the proper reaction pH and is a compromise between complexation and enzyme reactivity. PMID:27711193

A simple and sensitive enzymatic method for cholesterol quantification in macrophages and foam cells

PubMed Central

Robinet, Peggy; Wang, Zeneng; Hazen, Stanley L.; Smith, Jonathan D.

2010-01-01

A precise and sensitive method for measuring cellular free and esterified cholesterol is required in order to perform studies of macrophage cholesterol loading, metabolism, storage, and efflux. Until now, the use of an enzymatic cholesterol assay, commonly used for aqueous phase plasma cholesterol assays, has not been optimized for use with solid phase samples such as cells, due to inefficient solubilization of total cholesterol in enzyme compatible solvents. We present an efficient solubilization protocol compatible with an enzymatic cholesterol assay that does not require chemical saponification or chromatographic separation. Another issue with enzyme compatible solvents is the presence of endogenous peroxides that interfere with the enzymatic cholesterol assay. We overcame this obstacle by pretreatment of the reaction solution with the enzyme catalase, which consumed endogenous peroxides resulting in reduced background and increased sensitivity in our method. Finally, we demonstrated that this method for cholesterol quantification in macrophages yields results that are comparable to those measured by stable isotope dilution gas chromatography with mass spectrometry detection. In conclusion, we describe a sensitive, simple, and high-throughput enzymatic method to quantify cholesterol in complex matrices such as cells. PMID:20688754

Digestive kinetics determines bioavailability of pollutants. Final report, 1 July 1993--30 September 1998

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mayer, L.M.

1998-09-30

The bioavailability of sedimentary contaminants to animals in harbor sediments was addressed by studying the mechanisms by which animals solubilize contaminants during feeding and digestion. Digestive physiology work on many different animal species revealed patterns of enzymes, surfactants and dissolved organic matter that correlate with feeding mode, phyletic position, and diet. Incubation of digestive fluids to dissolve contaminants from polluted sediments was developed to provide numerical estimates of bioavailability, and showed that much higher fractions of total contaminant loading are available than predicted by currently established, aqueous equilibrium approaches. The kinetics of reactions are slow enough that variations in feedingmore » rates will influence overall bioavailability. Experimental manipulations showed mechanisms of bioavailability. Dissolved amino acids, in the form of enzyme proteins and hydrolyzed food, are responsible for solubilization of metals such as copper. At high levels, copper can inactivate digestive enzymes. Metals in sedimentary sulfide minerals were largely impervious to digestive fluid attack. Surfactants are responsible for most solubilization of polycyclic aromatic hydrocarbons (PAH), though other agents also appear to play a role. Bioavailability of both metals and PAH can be limited by saturating the digestive agents responsible for their dissolution.« less

Development of an amperometric-based glucose biosensor to measure the glucose content of fruit.

PubMed

Ang, Lee Fung; Por, Lip Yee; Yam, Mun Fei

2015-01-01

An amperometric enzyme-electrode was introduced where glucose oxidase (GOD) was immobilized on chitosan membrane via crosslinking, and then fastened on a platinum working electrode. The immobilized enzyme showed relatively high retention activity. The activity of the immobilized enzyme was influenced by its loading, being suppressed when more than 0.6 mg enzyme was used in the immobilization. The biosensor showing the highest response to glucose utilized 0.21 ml/cm2 thick chitosan membrane. The optimum experimental conditions for the biosensors in analysing glucose dissolved in 0.1 M phosphate buffer (pH 6.0) were found to be 35°C and 0.6 V applied potential. The introduced biosensor reached a steady-state current at 60 s. The apparent Michaelis-Menten constant ([Formula: see text]) of the biosensor was 14.2350 mM, and its detection limit was 0.05 mM at s/n > 3, determined experimentally. The RSD of repeatability and reproducibility of the biosensor were 2.30% and 3.70%, respectively. The biosensor was showed good stability; it retained ~36% of initial activity after two months of investigation. The performance of the biosensors was evaluated by determining the glucose content in fruit homogenates. Their accuracy was compared to that of a commercial glucose assay kit. There was no significance different between two methods, indicating the introduced biosensor is reliable.

Development of an Amperometric-Based Glucose Biosensor to Measure the Glucose Content of Fruit

PubMed Central

Ang, Lee Fung; Por, Lip Yee; Yam, Mun Fei

2015-01-01

An amperometric enzyme-electrode was introduced where glucose oxidase (GOD) was immobilized on chitosan membrane via crosslinking, and then fastened on a platinum working electrode. The immobilized enzyme showed relatively high retention activity. The activity of the immobilized enzyme was influenced by its loading, being suppressed when more than 0.6 mg enzyme was used in the immobilization. The biosensor showing the highest response to glucose utilized 0.21 ml/cm2 thick chitosan membrane. The optimum experimental conditions for the biosensors in analysing glucose dissolved in 0.1 M phosphate buffer (pH 6.0) were found to be 35°C and 0.6 V applied potential. The introduced biosensor reached a steady-state current at 60 s. The apparent Michaelis-Menten constant (KMapp) of the biosensor was 14.2350 mM, and its detection limit was 0.05 mM at s/n > 3, determined experimentally. The RSD of repeatability and reproducibility of the biosensor were 2.30% and 3.70%, respectively. The biosensor was showed good stability; it retained ~36% of initial activity after two months of investigation. The performance of the biosensors was evaluated by determining the glucose content in fruit homogenates. Their accuracy was compared to that of a commercial glucose assay kit. There was no significance different between two methods, indicating the introduced biosensor is reliable. PMID:25789757

Identification and characterization of core cellulolytic enzymes from Talaromyces cellulolyticus (formerly Acremonium cellulolyticus) critical for hydrolysis of lignocellulosic biomass

DOE PAGES

Inoue, Hiroyuki; Decker, Stephen R.; Taylor, Larry E.; ...

2014-10-09

Background: Enzymatic hydrolysis of pretreated lignocellulosic biomass is an essential process for the production of fermentable sugars for industrial use. A better understanding of fungal cellulase systems will provide clues for maximizing the hydrolysis of target biomass. Talaromyces cellulolyticus is a promising fungus for cellulase production and efficient biomass hydrolysis. Several cellulolytic enzymes purified from T. cellulolyticus were characterized in earlier studies, but the core enzymes critical for hydrolysis of lignocellulosic biomass remain unknown. Results: Six cellulolytic enzymes critical for the hydrolysis of crystalline cellulose were purified from T. cellulolyticus culture supernatant using an enzyme assay based on synergistic hydrolysismore » of Avicel. The purified enzymes were identified by their substrate specificities and analyses of trypsin-digested peptide fragments and were classified into the following glycosyl hydrolase (GH) families: GH3 (β-glucosidase, Bgl3A), GH5 (endoglucanase, Cel5A), GH6 (cellobiohydrolase II, Cel6A), GH7 (cellobiohydrolase I and endoglucanase, Cel7A and Cel7B, respectively), and GH10 (xylanase, Xyl10A). Hydrolysis of dilute acid-pretreated corn stover (PCS) with mixtures of the purified enzymes showed that Cel5A, Cel7B, and Xyl10A each had synergistic effects with a mixture of Cel6A and Cel7A. Cel5A seemed to be more effective in the synergistic hydrolysis of the PCS than Cel7B. The ratio of Cel5A, Cel6A, Cel7A, and Xyl10A was statistically optimized for the hydrolysis of PCS glucan in the presence of Bgl3A. The resultant mixture achieved higher PCS glucan hydrolysis at lower enzyme loading than a culture filtrate from T. cellulolyticus or a commercial enzyme preparation, demonstrating that the five enzymes play a role as core enzymes in the hydrolysis of PCS glucan. In Conclusion: Core cellulolytic enzymes in the T. cellulolyticus cellulase system were identified to Cel5A, Cel6A, Cel7A, Xyl10A, and Bgl3A and characterized. The optimized mixture of these five enzymes was highly effective for the hydrolysis of PCS glucan, providing a foundation for future improvement of the T. cellulolyticus cellulase system.« less

Identification and characterization of core cellulolytic enzymes from Talaromyces cellulolyticus (formerly Acremonium cellulolyticus) critical for hydrolysis of lignocellulosic biomass

DOE Office of Scientific and Technical Information (OSTI.GOV)

Inoue, Hiroyuki; Decker, Stephen R.; Taylor, Larry E.

Background: Enzymatic hydrolysis of pretreated lignocellulosic biomass is an essential process for the production of fermentable sugars for industrial use. A better understanding of fungal cellulase systems will provide clues for maximizing the hydrolysis of target biomass. Talaromyces cellulolyticus is a promising fungus for cellulase production and efficient biomass hydrolysis. Several cellulolytic enzymes purified from T. cellulolyticus were characterized in earlier studies, but the core enzymes critical for hydrolysis of lignocellulosic biomass remain unknown. Results: Six cellulolytic enzymes critical for the hydrolysis of crystalline cellulose were purified from T. cellulolyticus culture supernatant using an enzyme assay based on synergistic hydrolysismore » of Avicel. The purified enzymes were identified by their substrate specificities and analyses of trypsin-digested peptide fragments and were classified into the following glycosyl hydrolase (GH) families: GH3 (β-glucosidase, Bgl3A), GH5 (endoglucanase, Cel5A), GH6 (cellobiohydrolase II, Cel6A), GH7 (cellobiohydrolase I and endoglucanase, Cel7A and Cel7B, respectively), and GH10 (xylanase, Xyl10A). Hydrolysis of dilute acid-pretreated corn stover (PCS) with mixtures of the purified enzymes showed that Cel5A, Cel7B, and Xyl10A each had synergistic effects with a mixture of Cel6A and Cel7A. Cel5A seemed to be more effective in the synergistic hydrolysis of the PCS than Cel7B. The ratio of Cel5A, Cel6A, Cel7A, and Xyl10A was statistically optimized for the hydrolysis of PCS glucan in the presence of Bgl3A. The resultant mixture achieved higher PCS glucan hydrolysis at lower enzyme loading than a culture filtrate from T. cellulolyticus or a commercial enzyme preparation, demonstrating that the five enzymes play a role as core enzymes in the hydrolysis of PCS glucan. In Conclusion: Core cellulolytic enzymes in the T. cellulolyticus cellulase system were identified to Cel5A, Cel6A, Cel7A, Xyl10A, and Bgl3A and characterized. The optimized mixture of these five enzymes was highly effective for the hydrolysis of PCS glucan, providing a foundation for future improvement of the T. cellulolyticus cellulase system.« less

Methane gas generation from waste water extraction process of crude palm oil in experimental digesters

NASA Astrophysics Data System (ADS)

Dillon, A.; Penafiel, R.; Garzón, P. V.; Ochoa, V.

2015-12-01

Industrial processes to extract crude palm oil, generates large amounts of waste water. High concentrations of COD, ST, SV, NH4 + and low solubility of O2, make the treatment of these effluents starts with anaerobic processes. The anaerobic digestion process has several advantages over aerobic degradation: lower operating costs (not aeration), low sludge production, methane gas generation. The 4 stages of anaerobic digestion are: hydrolysis, acidogenic, acetogenesis and methanogenesis. Through the action of enzymes synthesized by microbial consortia are met. The products of each step to serve as reagents is conducted as follows. The organic load times and cell hydraulic retention, solids content, nutrient availability, pH and temperature are factors that influence directly in biodigesters. The objectives of this presentation is to; characterize the microbial inoculum and water (from palm oil wasted water) to be used in biodigestores, make specific methanogenic activity in bioassays, acclimatize the microorganisms to produce methane gas using basal mineral medium with acetate for the input power, and to determine the production of methane gas digesters high organic load.

Re-usable electrochemical glucose sensors integrated into a smartphone platform.

PubMed

Bandodkar, Amay J; Imani, Somayeh; Nuñez-Flores, Rogelio; Kumar, Rajan; Wang, Chiyi; Mohan, A M Vinu; Wang, Joseph; Mercier, Patrick P

2018-03-15

This article demonstrates a new smartphone-based reusable glucose meter. The glucose meter includes a custom-built smartphone case that houses a permanent bare sensor strip, a stylus that is loaded with enzyme-carbon composite pellets, and sensor instrumentation circuits. A custom-designed Android-based software application was developed to enable easy and clear display of measured glucose concentration. A typical test involves the user loading the software, using the stylus to dispense an enzymatic pellet on top of the bare sensor strip affixed to the case, and then introducing the sample. The electronic module then acquires and wirelessly transmits the data to the application software to be displayed on the screen. The deployed pellet is then discarded to regain the fresh bare sensor surface. Such a unique working principle allows the system to overcome challenges faced by previously reported reusable sensors, such as enzyme degradation, leaching, and hysteresis effects. Studies reveal that the enzyme loaded in the pellets are stable for up to 8 months at ambient conditions, and generate reproducible sensor signals. The work illustrates the significance of the pellet-based sensing system towards realizing a reusable, point-of-care sensor that snugly fits around a smartphone and which does not face issues usually common to reusable sensors. The versatility of this system allows it to be easily modified to detect other analytes for application in a wide range of healthcare, environmental and defense domains. Copyright © 2017 Elsevier B.V. All rights reserved.

Optimization of the Synthesis of Structured Phosphatidylcholine with Medium Chain Fatty Acid.

PubMed

Ochoa-Flores, Angélica A; Hernández-Becerra, Josafat A; Cavazos-Garduño, Adriana; Vernon-Carter, Eduardo J; García, Hugo S

2017-11-01

Structured phosphatidylcholine was successfully produced by acidolysis between phosphatidylcholine and free medium chain fatty acid, using phospholipase A 1 immobilized on Duolite A568. Response surface methodology was applied to optimize the reaction system using three process parameters: molar ratio of substrates (phosphatidylcholine to free medium chain fatty acid), enzyme loading, and reaction temperature. All parameters evaluated showed linear and quadratic significant effects on the production of modified phosphatidylcholine; molar ratio of substrates contributed positively, but temperature influenced negatively. Increased enzyme loading also led to increased production of modified phosphatidylcholine but only during the first 9 hours of the acidolysis reaction. Optimal conditions obtained from the model were a ratio of phosphatidylcholine to free medium chain fatty acid of 1:15, an enzyme loading of 12%, and a temperature of 45°C. Under these conditions a production of modified phosphatidylcholine of 52.98 % were obtained after 24 h of reaction. The prediction was confirmed from the verification experiments; the production of modified phosphatidylcholine was 53.02%, the total yield of phosphatidylcholine 64.28% and the molar incorporation of medium chain fatty acid was 42.31%. The acidolysis reaction was scaled-up in a batch reactor with a similar production of modified phosphatidylcholine, total yield of phosphatidylcholine and molar incorporation of medium chain fatty acid. Purification by column chromatography of the structured phosphatidylcholine yielded 62.53% of phosphatidylcholine enriched with 42.52% of medium chain fatty acid.

Simultaneous saccharification and viscosity reduction of cassava pulp using a multi-component starch- and cell-wall degrading enzyme for bioethanol production.

PubMed

Poonsrisawat, Aphisit; Paemanee, Atchara; Wanlapatit, Sittichoke; Piyachomkwan, Kuakoon; Eurwilaichitr, Lily; Champreda, Verawat

2017-10-01

In this study, an efficient ethanol production process using simultaneous saccharification and viscosity reduction of raw cassava pulp with no prior high temperature pre-gelatinization/liquefaction step was developed using a crude starch- and cell wall-degrading enzyme preparation from Aspergillus aculeatus BCC17849. Proteomic analysis revealed that the enzyme comprised a complex mixture of endo- and exo-acting amylases, cellulases, xylanases, and pectina ses belonging to various glycosyl hydrolase families. Enzymatic hydrolysis efficiency was dependent on the initial solid loading in the reaction. Reduction in mixture viscosity was observed with a rapid decrease in complex viscosity from 3785 to 0.45 Pa s with the enzyme dosage of 2.19 mg/g on a dried weight basis within the first 2 h, which resulted from partial destruction of the plant cell wall fiber and degradation of the released starch granules by the enzymes as shown by scanning electron microscopy. Saccharification of cassava pulp at an initial solid of 16% (w/v) in a bench-scale bioreactor resulted in 736.4 mg glucose/g, which is equivalent to 82.92% glucose yield based on the total starch and glucan in the substrate, after 96 h at 40 °C. Simultaneous saccharification and fermentation of cassava pulp by Saccharomyces cerevisiae with the uncooked enzymatic process led to a final ethanol concentration of 6.98% w/v, equivalent to 96.7% theoretical yield based on the total starch and cellulose content. The results demonstrated potential of the enzyme for low-energy processing of cassava pulp in biofuel industry.

Covalent Immobilization of Cellulase Using Magnetic Poly(ionic liquid) Support: Improvement of the Enzyme Activity and Stability.

PubMed

Hosseini, Seyed Hassan; Hosseini, Seyedeh Ameneh; Zohreh, Nasrin; Yaghoubi, Mahshid; Pourjavadi, Ali

2018-01-31

A magnetic nanocomposite was prepared by entrapment of Fe 3 O 4 nanoparticles into the cross-linked ionic liquid/epoxy type polymer. The resulting support was used for covalent immobilization of cellulase through the reaction with epoxy groups. The ionic surface of the support improved the adsorption of enzyme, and a large amount of enzyme (106.1 mg/g) was loaded onto the support surface. The effect of the presence of ionic monomer and covalent binding of enzyme was also investigated. The structure of support was characterized by various instruments such as FT-IR, TGA, VSM, XRD, TEM, SEM, and DLS. The activity and stability of immobilized cellulase were investigated in the prepared support. The results showed that the ionic surface and covalent binding of enzyme onto the support improved the activity, thermal stability, and reusability of cellulase compared to free cellulase.

Genetically Engineered Materials for Biofuels Production

NASA Astrophysics Data System (ADS)

Raab, Michael

2012-02-01

Agrivida, Inc., is an agricultural biotechnology company developing industrial crop feedstocks for the fuel and chemical industries. Agrivida's crops have improved processing traits that enable efficient, low cost conversion of the crops' cellulosic components into fermentable sugars. Currently, pretreatment and enzymatic conversion of the major cell wall components, cellulose and hemicellulose, into fermentable sugars is the most expensive processing step that prevents widespread adoption of biomass in biofuels processes. To lower production costs we are consolidating pretreatment and enzyme production within the crop. In this strategy, transgenic plants express engineered cell wall degrading enzymes in an inactive form, which can be reactivated after harvest. We have engineered protein elements that disrupt enzyme activity during normal plant growth. Upon exposure to specific processing conditions, the engineered enzymes are converted into their active forms. This mechanism significantly lowers pretreatment costs and enzyme loadings (>75% reduction) below those currently available to the industry.

Novel Biocatalytic Polymer-Based Antimicrobial Coatings as Potential Ureteral Biomaterial: Preparation and In Vitro Performance Evaluation▿

PubMed Central

Dave, Rachna N.; Joshi, Hiren M.; Venugopalan, Vayalam P.

2011-01-01

Catheters and other indwelling devices placed inside human body are prone to bacterial infection, causing serious risk to patients. Infections associated with implants are difficult to resolve, and hence the prevention of bacterial colonization of such surfaces is quite appropriate. In this context, the development of novel antimicrobial biomaterials is currently gaining momentum. We describe here the preparation and antibacterial properties of an enzyme-embedded polycaprolactone (PCL)-based coating, coimpregnated with the antibiotic gentamicin sulfate (GS). The enzyme uses PCL itself as substrate; as a result, the antibiotic gets released at a rate controlled by the degradation of the PCL base. In vitro drug release studies demonstrated sustained release of GS from the PCL film throughout its lifetime. By modulating the enzyme concentration in the PCL film, we were able to vary the lifetime of the coating from 33 h to 16 days. In the end, the polymer is completely degraded, delivering the entire load of the antibiotic. The polymer exhibited antibacterial properties against three test isolates: Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. Foley urinary catheters coated with the modified polymer exhibited sustained in vitro release of GS over a 60-h period. The results suggest that the antibiotic-plus-enzyme-loaded polymer can be used as tunable self-degrading antimicrobial biomaterial coating on catheters. PMID:21135190

Biocatalytic oxidation by chloroperoxidase from Caldariomyces fumago in polymersome nanoreactors.

PubMed

de Hoog, H M; Nallani, M; Cornelissen, J J L M; Rowan, A E; Nolte, R J M; Arends, I W C E

2009-11-21

The encapsulation of chloroperoxidase from Caldariomyces fumago (CPO) in block copolymer polymersomes is reported. Fluorescence and electron microscopy show that when the encapsulating conditions favour self-assembly of the block copolymer, the enzyme is incorporated with concentrations that are 50 times higher than the enzyme concentration before encapsulation. The oxidation of two substrates by the encapsulated enzyme was studied: i) pyrogallol, a common substrate used to assay CPO enzymatic activity and ii) thioanisole, of which the product, (R)-methyl phenyl sulfoxide, is an important pharmaceutical intermediate. The CPO-loaded polymersomes showed distinct reactivity towards these substrates. While the oxidation of pyrogallol was limited by diffusion of the substrate into the polymersome, the rate-limiting step for the oxidation of thioansiole was the turnover by the enzyme.

How Enzymes Work: A Look through the Perspective of Molecular Viscoelastic Properties

NASA Astrophysics Data System (ADS)

Qu, Hao; Zocchi, Giovanni

2013-01-01

We present nanorheology measurements on the folded state of an enzyme that show directly that the (ensemble-averaged) stress-strain relations are nonlinear and frequency dependent beyond 1-Å deformation. We argue that this frequency dependence allows for opening a nonequilibrium cycle in the force-deformation plane if the forward and backward conformational changes of the enzyme during catalysis happen at different speeds. Using a heuristic model for the experimentally established viscoelastic properties of the enzyme, we examine a number of general features of enzymatic action. We find that the proposed viscoelastic cycle is consistent with the linear decrease of the speed of motor proteins with load. We find a relation between the stall force and the maximum rate for enzymes (in general) and motors (in particular). We estimate the stall force of the motor protein kinesin from thermodynamic quantities and estimate the maximum rate of enzymes from purely mechanical quantities. We propose that the viscoelastic cycle provides a framework for considering mechanochemical coupling in enzymes on the basis of possibly universal materials properties of the folded state of proteins.

Biocatalysis: Unmasked by stretching

NASA Astrophysics Data System (ADS)

Kharlampieva, Eugenia; Tsukruk, Vladimir V.

2009-09-01

The biocatalytic activity of enzyme-loaded responsive layer-by-layer films can be switched on and off by simple mechanical stretching. Soft materials could thus be used to trigger biochemical reactions under mechanical action, with potential therapeutic applications.

Enzyme immobilisation in biocatalysis: why, what and how.

PubMed

Sheldon, Roger A; van Pelt, Sander

2013-08-07

In this tutorial review, an overview of the why, what and how of enzyme immobilisation for use in biocatalysis is presented. The importance of biocatalysis in the context of green and sustainable chemicals manufacture is discussed and the necessity for immobilisation of enzymes as a key enabling technology for practical and commercial viability is emphasised. The underlying reasons for immobilisation are the need to improve the stability and recyclability of the biocatalyst compared to the free enzyme. The lower risk of product contamination with enzyme residues and low or no allergenicity are further advantages of immobilised enzymes. Methods for immobilisation are divided into three categories: adsorption on a carrier (support), encapsulation in a carrier, and cross-linking (carrier-free). General considerations regarding immobilisation, regardless of the method used, are immobilisation yield, immobilisation efficiency, activity recovery, enzyme loading (wt% in the biocatalyst) and the physical properties, e.g. particle size and density, hydrophobicity and mechanical robustness of the immobilisate, i.e. the immobilised enzyme as a whole (enzyme + support). The choice of immobilisate is also strongly dependent on the reactor configuration used, e.g. stirred tank, fixed bed, fluidised bed, and the mode of downstream processing. Emphasis is placed on relatively recent developments, such as the use of novel supports such as mesoporous silicas, hydrogels, and smart polymers, and cross-linked enzyme aggregates (CLEAs).

Porous silicon nanoparticle as a stabilizing support for chondroitinase.

PubMed

Daneshjou, Sara; Dabirmanesh, Bahareh; Rahimi, Fereshteh; Khajeh, Khosro

2017-01-01

Chondroitinase ABCI (cABCI) from Proteus vulgaris is a drug enzyme that can be used to treat spinal cord injuries. One of the main problems of chondroitinase ABC1 is its low thermal stability. The objective of the current study was to stabilize the enzyme through entrapment within porous silicon (pSi) nanoparticles. pSi was prepared by an electrochemical etch of p-type silicon using hydrofluoric acid/ethanol. The size of nanoparticles were determined 180nm by dynamic light scattering and the mean pore diameter was in the range of 40-60nm obtained by scanning electron microscopy. Enzymes were immobilized on porouse silicon nanoparticles by entrapment. The capacity of matrix was 35μg enzyme per 1mg of silicon. The immobilized enzyme displayed lower V max values compared to the free enzyme, but Km values were the same for both enzymes. Immobilization significantly increased the enzyme stability at various temperatures (-20, 4, 25 and 37°C). For example, at 4°C, the free enzyme (in 10mM imidazole) retained 20% of its activity after 100min, while the immobilized one retained 50% of its initial activity. Nanoparticles loading capacity and the enzyme release rate showed that the selected particles could be a pharmaceutically acceptable carrier for chondroitinase. Copyright © 2016 Elsevier B.V. All rights reserved.

Mesoporous carbon nitride based biosensor for highly sensitive and selective analysis of phenol and catechol in compost bioremediation.

PubMed

Zhou, Yaoyu; Tang, Lin; Zeng, Guangming; Chen, Jun; Cai, Ye; Zhang, Yi; Yang, Guide; Liu, Yuanyuan; Zhang, Chen; Tang, Wangwang

2014-11-15

Herein, we reported here a promising biosensor by taking advantage of the unique ordered mesoporous carbon nitride material (MCN) to convert the recognition information into a detectable signal with enzyme firstly, which could realize the sensitive, especially, selective detection of catechol and phenol in compost bioremediation samples. The mechanism including the MCN based on electrochemical, biosensor assembly, enzyme immobilization, and enzyme kinetics (elucidating the lower detection limit, different linear range and sensitivity) was discussed in detail. Under optimal conditions, GCE/MCN/Tyr biosensor was evaluated by chronoamperometry measurements and the reduction current of phenol and catechol was proportional to their concentration in the range of 5.00 × 10(-8)-9.50 × 10(-6)M and 5.00 × 10(-8)-1.25 × 10(-5)M with a correlation coefficient of 0.9991 and 0.9881, respectively. The detection limits of catechol and phenol were 10.24 nM and 15.00 nM (S/N=3), respectively. Besides, the data obtained from interference experiments indicated that the biosensor had good specificity. All the results showed that this material is suitable for load enzyme and applied to the biosensor due to the proposed biosensor exhibited improved analytical performances in terms of the detection limit and specificity, provided a powerful tool for rapid, sensitive, especially, selective monitoring of catechol and phenol simultaneously. Moreover, the obtained results may open the way to other MCN-enzyme applications in the environmental field. Copyright © 2014 Elsevier B.V. All rights reserved.

Positively charged mini-protein Zbasic2 as a highly efficient silica binding module: opportunities for enzyme immobilization on unmodified silica supports.

PubMed

Bolivar, Juan M; Nidetzky, Bernd

2012-07-03

Silica is a highly attractive support material for protein immobilization in a wide range of biotechnological and biomedical-analytical applications. Without suitable derivatization, however, the silica surface is not generally usable for attachment of proteins. We show here that Z(basic2) (a three α-helix bundle mini-protein of 7 kDa size that exposes clustered positive charges from multiple arginine residues on one side) functions as highly efficient silica binding module (SBM), allowing chimeras of target protein with SBM to become very tightly attached to underivatized glass at physiological pH conditions. We used two enzymes, d-amino acid oxidase and sucrose phosphorylase, to demonstrate direct immobilization of Z(basic2) protein from complex biological samples with extremely high selectivity. Immobilized enzymes displayed full biological activity, suggesting that their binding to the glass surface had occurred in a preferred orientation via the SBM. We also show that charge complementarity was the main principle of affinity between SBM and glass surface, and Z(basic2) proteins were bound in a very strong, yet fully reversible manner, presumably through multipoint noncovalent interactions. Z(basic2) proteins were immobilized on porous glass in a loading of 30 mg protein/g support or higher, showing that attachment via the SBM combines excellent binding selectivity with a technically useful binding capacity. Therefore, Z(basic2) and silica constitute a fully orthogonal pair of binding module and insoluble support for oriented protein immobilization, and this opens up new opportunities for the application of silica-based materials in the development of supported heterogeneous biocatalysts.

D-isoascorbyl palmitate: lipase-catalyzed synthesis, structural characterization and process optimization using response surface methodology.

PubMed

Sun, Wen-Jing; Zhao, Hong-Xia; Cui, Feng-Jie; Li, Yun-Hong; Yu, Si-Lian; Zhou, Qiang; Qian, Jing-Ya; Dong, Ying

2013-07-08

Isoascorbic acid is a stereoisomer of L-ascorbic acid, and widely used as a food antioxidant. However, its highly hydrophilic behavior prevents its application in cosmetics or fats and oils-based foods. To overcome this problem, D-isoascorbyl palmitate was synthesized in the present study for improving the isoascorbic acid's oil solubility with an immobilized lipase in organic media. The structural information of synthesized product was clarified using LC-ESI-MS, FT-IR, 1H and 13C NMR analysis, and process parameters for high yield of D-isoascorbyl palmitate were optimized by using One-factor-at-a-time experiments and response surface methodology (RSM). The synthesized product had the purity of 95% and its structural characteristics were confirmed as isoascorbyl palmitate by LC-ESI-MS, FT-IR, 1H, and 13C NMR analysis. Results from "one-factor-at-a-time" experiments indicated that the enzyme load, reaction temperature and D-isoascorbic-to-palmitic acid molar ratio had a significant effect on the D-isoascorbyl palmitate conversion rate. 95.32% of conversion rate was obtained by using response surface methodology (RSM) under the the optimized condition: enzyme load of 20% (w/w), reaction temperature of 53°C and D- isoascorbic-to-palmitic acid molar ratio of 1:4 when the reaction parameters were set as: acetone 20 mL, 40 g/L of molecular sieves content, 200 rpm speed for 24-h reaction time. The findings of this study can become a reference for developing industrial processes for the preparation of isoascorbic acid ester, which might be used in food additives, cosmetic formulations and for the synthesis of other isoascorbic acid derivatives.

D-isoascorbyl palmitate: lipase-catalyzed synthesis, structural characterization and process optimization using response surface methodology

PubMed Central

2013-01-01

Background Isoascorbic acid is a stereoisomer of L-ascorbic acid, and widely used as a food antioxidant. However, its highly hydrophilic behavior prevents its application in cosmetics or fats and oils-based foods. To overcome this problem, D-isoascorbyl palmitate was synthesized in the present study for improving the isoascorbic acid’s oil solubility with an immobilized lipase in organic media. The structural information of synthesized product was clarified using LC-ESI-MS, FT-IR, 1H and 13C NMR analysis, and process parameters for high yield of D-isoascorbyl palmitate were optimized by using One–factor-at-a-time experiments and response surface methodology (RSM). Results The synthesized product had the purity of 95% and its structural characteristics were confirmed as isoascorbyl palmitate by LC-ESI-MS, FT-IR, 1H, and 13C NMR analysis. Results from “one–factor-at-a-time” experiments indicated that the enzyme load, reaction temperature and D-isoascorbic-to-palmitic acid molar ratio had a significant effect on the D-isoascorbyl palmitate conversion rate. 95.32% of conversion rate was obtained by using response surface methodology (RSM) under the the optimized condition: enzyme load of 20% (w/w), reaction temperature of 53°C and D- isoascorbic-to-palmitic acid molar ratio of 1:4 when the reaction parameters were set as: acetone 20 mL, 40 g/L of molecular sieves content, 200 rpm speed for 24-h reaction time. Conclusion The findings of this study can become a reference for developing industrial processes for the preparation of isoascorbic acid ester, which might be used in food additives, cosmetic formulations and for the synthesis of other isoascorbic acid derivatives. PMID:23835418

Effects of enzyme loading, densification, and storage on AFEX-pretreated biomass for ethanol production.

PubMed

Biersbach, Gwen; Rijal, Binod; Pryor, Scott W; Gibbons, William R

2015-12-01

Corn stover, switchgrass, and prairie cordgrass were treated with an ammonia fiber expansion (AFEX) process and a novel densification method (ComPAKco). Separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) were used to evaluate impacts of densification. ComPAKco densification is characterized by low-temperature and low-energy requirements, resulting in compacted biomass briquettes (CBB) 1-2 cm square, with a bulk density of 380-460 kg/m(3). Feedstocks were evaluated before and following AFEX pretreatment, after densification, and after storage. Two enzyme doses were tested. The low rate used 5 filter paper units (FPU) of Spezyme CP (cellulase) and 21.3 cellobiase units (CBU) of Novozyme 188 (aka NS50010 [β-glucosidase]) per gram of glucan. The high dosage rate was three times higher and resulted in 40-56 % and 33-82 % higher ethanol yields with SHF and SSF, respectively. Trials revealed no adverse effect on ethanol yield following densification or 6-month storage of densified, AFEX-pretreated feedstocks.

Density separation as a strategy to reduce the enzyme load of preharvest sprouted wheat and enhance its bread making quality.

PubMed

Olaerts, Heleen; De Bondt, Yamina; Courtin, Christophe M

2018-02-15

As preharvest sprouting of wheat impairs its use in food applications, postharvest solutions for this problem are required. Due to the high kernel to kernel variability in enzyme activity in a batch of sprouted wheat, the potential of eliminating severely sprouted kernels based on density differences in NaCl solutions was evaluated. Compared to higher density kernels, lower density kernels displayed higher α-amylase, endoxylanase, and peptidase activities as well as signs of (incipient) protein, β-glucan and arabinoxylan breakdown. By discarding lower density kernels of mildly and severely sprouted wheat batches (11% and 16%, respectively), density separation increased flour FN of the batch from 280 to 345s and from 135 to 170s and increased RVA viscosity. This in turn improved dough handling, bread crumb texture and crust color. These data indicate that density separation is a powerful technique to increase the quality of a batch of sprouted wheat. Copyright © 2017 Elsevier Ltd. All rights reserved.

Rapid saccharification for production of cellulosic biofuels.

PubMed

Lee, Dae-Seok; Wi, Seung Gon; Lee, Soo Jung; Lee, Yoon-Gyo; Kim, Yeong-Suk; Bae, Hyeun-Jong

2014-04-01

The economical production of biofuels is hindered by the recalcitrance of lignocellulose to processing, causing high consumption of processing enzymes and impeding hydrolysis of pretreated lignocellulosic biomass. We determined the major rate-limiting factor in the hydrolysis of popping pre-treated rice straw (PPRS) by examining cellulase adsorption to lignin and cellulose, amorphogenesis of PPRS, and re-hydrolysis. Based on the results, equivalence between enzyme loading and the open structural area of cellulose was required to significantly increase productive adsorption of cellulase and to accelerate enzymatic saccharification of PPRS. Amorphogenesis of PPRS by phosphoric acid treatment to expand open structural area of the cellulose fibers resulted in twofold higher cellulase adsorption and increased the yield of the first re-hydrolysis step from 13% to 46%. The total yield from PPRS was increased to 84% after 3h. These results provide evidence that cellulose structure is one of major effects on the enzymatic hydrolysis. Copyright © 2014 Elsevier Ltd. All rights reserved.

Immobilization of Pseudomonas fluorescens lipase onto magnetic nanoparticles for resolution of 2-octanol.

PubMed

Xun, Er-na; Lv, Xiao-li; Kang, Wei; Wang, Jia-xin; Zhang, Hong; Wang, Lei; Wang, Zhi

2012-10-01

The lipase from Pseudomonas fluorescens (Lipase AK, AKL) was immobilized onto the magnetic Fe(3)O(4) nanoparticles via hydrophobic interaction. Enzyme loading and immobilization yield were determined as 21.4±0.5 mg/g and 49.2±1.8 %, respectively. The immobilized AKL was successfully used for resolution of 2-octanol with vinyl acetate used as acyl donor. Effects of organic solvent, water activity, substrate ratio, and temperature were investigated. Under the optimum conditions, the preferred isomer for AKL is the (R)-2-octanol and the highest enantioselectivity (E=71.5±2.2) was obtained with a higher enzyme activity (0.197±0.01 μmol/mg/min). The results also showed that the immobilized lipase could be easily separated from reaction media by the magnetic steel and remained 89 % of its initial activity as well as the nearly unchanged enantioselectivity after five consecutive cycles, indicating a high stability in practical operation.

Single-molecule protein unfolding and translocation by an ATP-fueled proteolytic machine

PubMed Central

Aubin-Tam, Marie-Eve; Olivares, Adrian O.; Sauer, Robert T.; Baker, Tania A.; Lang, Matthew J.

2011-01-01

All cells employ ATP-powered proteases for protein-quality control and regulation. In the ClpXP protease, ClpX is a AAA+ machine that recognizes specific protein substrates, unfolds these molecules, and then translocates the denatured polypeptide through a central pore and into ClpP for degradation. Here, we use optical-trapping nanometry to probe the mechanics of enzymatic unfolding and translocation of single molecules of a multidomain substrate. Our experiments demonstrate the capacity of ClpXP and ClpX to perform mechanical work under load, reveal very fast and highly cooperative unfolding of individual substrate domains, suggest a translocation step size of 5–8 amino acids, and support a power-stroke model of denaturation in which successful enzyme-mediated unfolding of stable domains requires coincidence between mechanical pulling by the enzyme and a transient stochastic reduction in protein stability. We anticipate that single-molecule studies of the mechanical properties of other AAA+ proteolytic machines will reveal many shared features with ClpXP. PMID:21496645

Intraductal collagenase delivery into the human pancreas using syringe loading or controlled perfusion.

PubMed

Lakey, J R; Warnock, G L; Shapiro, A M; Korbutt, G S; Ao, Z; Kneteman, N M; Rajotte, R V

1999-01-01

Effective intraductal delivery of the enzyme collagenase into the pancreas is crucial to the subsequent ability to isolate viable islets. Most clinical islet transplant centers load the enzyme into the pancreas by retrograde injection using a syringe following cannulation of the pancreatic duct. An alternative approach is to perfuse the pancreas via the pancreatic duct with collagenase solution using a recirculating perfusion device system. This provides control over perfusion pressures and collagenase temperature. This study reports on our evaluation of the delivery of Liberase-HI into the pancreas of 14 consecutive adult multiorgan cadaveric donors. Alternate glands were procured and processed using an identical protocol with the exception of collagenase delivery. The first group of pancreases was loaded using the perfusion technique where cold (4 degrees C) Liberase-HI was perfused at 80 mmHg for 5 min after which the pressure was increased to 180 mmHg. The collagenase solution was then slowly warmed to 35 degrees C, transferred to the dissociation chamber and mechanically dissociated, and then purified using discontinuous gradients of Ficoll. Pancreases in the second group were loaded with collagenase (28-32 degrees C) using the syringe technique before mechanical dissociation and purification. There were no significant differences in pancreas cold ischemia, donor age, body mass index, maximum blood glucose, or serum amylase of the donors between the two groups. Mean collagenase digestion time in the digestion chamber was not different between the two groups; however, the amount of undigested tissue remaining after dissociation was significantly higher in the syringe-loaded group (15.3 +/- 2.6 g vs. 4.6 +/- 2.1 g, mean +/- SEM, p < 0.05). Postdigestion recovery of islets was 471 +/- 83 x 10(3) IE in the perfusion group compared with 391 +/- 57 x 10(3) IE for the syringe-loaded group. Postpurification recovery was higher in the perfused group (379 +/- 45 vs. 251 +/- 28 x 10(3) IE, p < 0.05, two-tailed paired t-test). No difference in in vitro islet viability was observed between the two groups following glucose perifusion with the calculated stimulation index of 4.6 +/- 0.6 for the perfusion group and 4.2 +/- 0.7 for the syringe-loaded group. Controlled perfusion via the pancreatic duct allows the effective delivery of the enzyme achieving maximal distension to all regions of the pancreas leading to an increased recovery of the islets with no detrimental effect on subsequent in vitro islet function.

Enhanced Delivery and Effects of Acid Sphingomyelinase by ICAM-1-Targeted Nanocarriers in Type B Niemann-Pick Disease Mice.

PubMed

Garnacho, Carmen; Dhami, Rajwinder; Solomon, Melani; Schuchman, Edward H; Muro, Silvia

2017-07-05

Acid sphingomyelinase deficiency in type B Niemann-Pick disease leads to lysosomal sphingomyelin storage, principally affecting lungs, liver, and spleen. Infused recombinant enzyme is beneficial, yet its delivery to the lungs is limited and requires higher dosing than liver and spleen, leading to potentially adverse reactions. Previous studies showed increased enzyme pulmonary uptake by nanocarriers targeted to ICAM-1, a protein overexpressed during inflammation. Here, using polystyrene and poly(lactic-co-glycolic acid) nanocarriers, we optimized lung delivery by varying enzyme dose and nanocarrier concentration, verified endocytosis and lysosomal trafficking in vivo, and evaluated delivered activity and effects. Raising the enzyme load of nanocarriers progressively increased absolute enzyme delivery to all lung, liver, and spleen, over the naked enzyme. Varying nanocarrier concentration inversely impacted lung versus liver and spleen uptake. Mouse intravital and postmortem examination verified endocytosis, transcytosis, and lysosomal trafficking using nanocarriers. Compared to naked enzyme, nanocarriers increased enzyme activity in organs and reduced lung sphingomyelin storage and macrophage infiltration. Although old mice with advanced disease showed reactivity (pulmonary leukocyte infiltration) to injections, including buffer without carriers, antibody, or enzyme, younger mice with mild disease did not. We conclude that anti-ICAM nanocarriers may result in effective lung enzyme therapy using low enzyme doses. Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

Alginate/polymethacrylate copolymer microparticles for the intestinal delivery of enzymes.

PubMed

Scocca, Sarah; Faustini, Massimo; Villani, Simona; Munari, Eleonora; Conte, Ubaldo; Russo, Vincenzo; Riccardi, Alessia; Vigo, Daniele; Torre, Maria Luisa

2007-04-01

Proteins administered orally must pass through the gastric environment in order to reach their site of absorption in the intestine. How to protect these exogenously administered proteins from the damaging effects of gastric acid and pepsin proteolytic activity, which often induce irreversible structural and functional alterations to the molecules, is an intriguing challenge. Another problem is the physical and chemical instability of proteins during some technological processes, which often involve the use of organic solvents or high temperatures. In this study we investigated the use of alginate microparticles containing one of two enzymes, an enteric polymer and a lyoprotectant for the intestinal delivery of proteins. The two enzymes tested in this protein delivery system were lactate dehydrogenase and alpha-amylase: the former was chosen because of its sensitivity to denaturation, the latter for its relevance in nutrition and medicine. A sodium alginate aqueous solution containing the enteric polymer, a lyoprotectant and the enzyme was either extruded or sprayed into a calcium chloride solution, with the resultant formation of beads and microspheres which were freeze-dried. About 90% of the enzyme activity was maintained during the process of loading the proteins into the microparticles and the subsequent freeze-drying process. The stability of the encapsulated enzyme in an acid medium and the enzymatic activity in an intestinal environment were then investigated by a dissolution test. This consisted of exposing the microparticles to simulated gastric fluid (pH 1.2) for 2 hours and to simulated intestinal fluid (pH 7.5+/-0.1) for 1 hour. The morphology of the microparticles did not change in the acid environment, whereas they completely dissolved within 3 min in the simulated intestinal fluid. Residual enzymatic activity after the test remained satisfactory for both enzymes. In conclusion, these microparticle systems offer promise for applications in human and veterinary medicine as well as in human and animal nutrition.

Influence of three commercial graphene derivatives on the catalytic properties of a Lactobacillus plantarum alpha-L-rhamnosidase when used as immobilization matrices.

PubMed

Antón-Millán, Noemí; Garcia-Tojal, Javier; Marty-Roda, Marta; Garroni, Sebastiano; Cuesta-López, Santiago; Tamayo-Ramos, Juan Antonio

2018-05-07

The modification of carbon nanomaterials with biological molecules paves the way towards their use in biomedical and biotechnological applications, such as next generation biocatalytic processes, development of biosensors, implantable electronic devices, or drug delivery. In this study, different commercial graphene derivatives, namely, monolayer graphene oxide (GO), graphene oxide nanocolloids (GOC), and polycarboxylate functionalized graphene nanoplatelets (GN), were compared as biomolecule carrier matrices. Detailed spectroscopic analyses showed that GO and GOC were similar in composition and functional groups content, and very different to GN, while divergent morphological characteristics were observed for each nanomaterial through microscopy analyses. The commercial alpha-L-rhamnosidase RhaB1 from the probiotic bacterium Lactobacillus plantarum, selected as a model biomolecule for its relevant role in the pharma and food industries, was directly immobilized on the different materials. The binding efficiency and biochemical properties of RhaB1-GO, RhaB1-GOC, and RhaB1-GN composites were analyzed. RhaB1-GO and RhaB1-GOC showed high binding efficiency, while the enzyme loading on GN, not tested in previous enzyme immobilization studies, was low. The enzyme showed contrasting changes when immobilized on the different material supports. The effect of pH on the activity of the three RhaB1 immobilized versions was similar to that observed for the free enzyme, while the activity-temperature profiles and the response to the presence of inhibitors varied significantly between the RhaB1 versions. In addition, the apparent Km for the immobilized and soluble enzymes did not change. Finally, the free RhaB1 and the immobilized enzyme in GOC showed the best storage and reutilization stability, keeping most of its initial activity after 8 weeks of storage at 4°C, and 10 reutilization cycles respectively. This study shows, for the first time, that distinct commercial graphene derivatives can influence differently on the catalytic properties of an enzyme during its immobilization.

H2O2 sensing using HRP modified catalyst-free ZnO nanorods synthesized by RF sputtering

NASA Astrophysics Data System (ADS)

Srivastava, Amit; Kumar, Naresh; Singh, Priti; Singh, Sunil Kumar

2017-06-01

Catalyst-free ( 00 l) oriented ZnO nanorods (NRs) -based biosensor for the H2O2 sensing has been reported. The (002) oriented ZnO NRs as confirmed by X-ray diffraction were successfully grown on indium tin oxide (ITO) coated glass substrate by radio frequency (RF) sputtering technique without using any catalyst. Horseradish peroxidase (HRP) enzyme was immobilized on ZnO NRs by physical adsorption technique to prepare the biosensor. In this HRP/ZnO NR/ITO bioelectrode, nafion solution was added to form a tight membrane on surface. The prepared bioelectrode has been used for biosensing measurements by electrochemical analyzer. The electrochemical studies reveal that the prepared HRP/ZnO NR/ITO biosensor is highly sensitive to the detection of H2O2 over a linear range of 0.250-10 μM. The ZnO NR-based biosensor showed lower value of detection limit (0.125 μM) and higher sensitivity (13.40 µA/µM cm2) towards H2O2. The observed value of higher sensitivity attributed to larger surface area of ZnO nanostructure for effective loading of HRP besides its high electron communication capability. In addition, the biosensor also shows lower value of enzyme's kinetic parameter (Michaelis-Menten constant, K m) of 0.262 μM which indicates enhanced enzyme affinity of HRP to H2O2. The reported biosensor may be useful for various applications in biosensing, clinical, food, and beverage industry.

Functionalization of paramagnetic nanoparticles for protein immobilization and purification.

PubMed

Carneiro, Lara A B C; Ward, Richard J

2018-01-01

A paramagnetic nanocomposite coated with chitosan and N-(5-Amino-1-carboxy-pentyl) iminodiacetic acid (NTA) that is suitable for protein immobilization applications has been prepared and characterized. The nanoparticle core was synthesized by controlled aggregation of Fe 3 O 4 under alkaline conditions, and Transmission Electron Microscopy revealed a size distribution of 10-50 nm. The nanoparticle core was coated with chitosan and derivatized with glutaraldehyde and NTA, as confirmed by Fourier Transform Infrared Spectroscopy. The final nanoparticles were used as a metal affinity matrix to separate a recombinant polyhistidine-tagged β-galactosidase from Bacillus subtilis directly from E. coli cell lysates with high purity (>95%). After loading with Ni 2+ , nanoparticles demonstrated a binding capacity of 250 μg of a polyhistidine-tagged β-galactosidase per milligram of support. The immobilized enzyme retained 80% activity after 9 cycles of washing, and the immobilized recombinant protein could be eluted with high purity with imidazole. The applications for these nanomagnetic composites extend beyond protein purification, and can also be used for immobilizing enzymes, where the β-galactosidase immobilized on the nanomagnetic support was used in multiple cycles of catalytic reactions with no significant loss of catalytic activity. Copyright © 2017. Published by Elsevier Inc.

Hierarchically mesostructured porous TiO2 hollow nanofibers for high performance glucose biosensing.

PubMed

Guo, Qiaohui; Liu, Lijuan; Zhang, Man; Hou, Haoqing; Song, Yonghai; Wang, Huadong; Zhong, Baoying; Wang, Li

2017-06-15

Effective immobilization of enzymes on an electrode surface is of great importance for biosensor development, but it still remains challenging because enzymes tend to denaturation and/or form close-packed structures. In this work, a free-standing TiO 2 hollow nanofibers (HNF-TiO 2 ) was successfully prepared by a simple and scalable electrospun nanofiber film template-assisted sol-gel method, and was further explored for glucose oxidase (GOD) immobilization and biosensing. This porous and nanotubular HNF-TiO 2 provides a well-defined hierarchical nanostructure for GOD loading, and the fine TiO 2 nanocrystals facilitate direct electron transfer from GOD to the electrode, also the strong interaction between GOD and HNF-TiO 2 greatly enhances the stability of the biosensor. The as-prepared glucose biosensors show good sensing performances both in O 2 -free and O 2 -containing conditions with good sensitivity, satisfactory selectivity, long-term stability and sound reliability. The novel textile formation, porous and hierarchically mesostructured nature of HNF-TiO 2 with excellent analytical performances make it a superior platform for the construction of high-performance glucose biosensors. Copyright © 2016 Elsevier B.V. All rights reserved.

BSA treatment to enhance enzymatic hydrolysis of cellulose in lignin containing substrates.

PubMed

Yang, Bin; Wyman, Charles E

2006-07-05

Cellulase and bovine serum albumin (BSA) were added to Avicel cellulose and solids containing 56% cellulose and 28% lignin from dilute sulfuric acid pretreatment of corn stover. Little BSA was adsorbed on Avicel cellulose, while pretreated corn stover solids adsorbed considerable amounts of this protein. On the other hand, cellulase was highly adsorbed on both substrates. Adding a 1% concentration of BSA to dilute acid pretreated corn stover prior to enzyme addition at 15 FPU/g cellulose enhanced filter paper activity in solution by about a factor of 2 and beta-glucosidase activity in solution by about a factor of 14. Overall, these results suggested that BSA treatment reduced adsorption of cellulase and particularly beta-glucosidase on lignin. Of particular note, BSA treatment of pretreated corn stover solids prior to enzymatic hydrolysis increased 72 h glucose yields from about 82% to about 92% at a cellulase loading of 15 FPU/g cellulose or achieved about the same yield at a loading of 7.5 FPU/g cellulose. Similar improvements were also observed for enzymatic hydrolysis of ammonia fiber explosion (AFEX) pretreated corn stover and Douglas fir treated by SO(2) steam explosion and for simultaneous saccharification and fermentation (SSF) of BSA pretreated corn stover. In addition, BSA treatment prior to hydrolysis reduced the need for beta-glucosidase supplementation of SSF. The results are consistent with non-specific competitive, irreversible adsorption of BSA on lignin and identify promising strategies to reduce enzyme requirements for cellulose hydrolysis. (c) 2006 Wiley Periodicals, Inc.

Cholesterol crystallization within hepatocyte lipid droplets and its role in murine NASH[S

PubMed Central

Ioannou, George N.; Subramanian, Savitha; Chait, Alan; Haigh, W. Geoffrey; Yeh, Matthew M.; Farrell, Geoffrey C.; Lee, Sum P.; Savard, Christopher

2017-01-01

We recently reported that cholesterol crystals form in hepatocyte lipid droplets (LDs) in human and experimental nonalcoholic steatohepatitis. Herein, we assigned WT C57BL/6J mice to a high-fat (15%) diet for 6 months, supplemented with 0%, 0.25%, 0.5%, 0.75%, or 1% dietary cholesterol. Increasing dietary cholesterol led to cholesterol loading of the liver, but not of adipose tissue, resulting in fibrosing steatohepatitis at a dietary cholesterol concentration of ≥0.5%, whereas mice on lower-cholesterol diets developed only simple steatosis. Hepatic cholesterol crystals and crown-like structures also developed at a dietary cholesterol concentration ≥0.5%. Crown-like structures consisted of activated Kupffer cells (KCs) staining positive for NLRP3 and activated caspase 1, which surrounded and processed cholesterol crystal-containing remnant LDs of dead hepatocytes. The KCs processed LDs at the center of crown-like structures in the extracellular space by lysosomal enzymes, ultimately transforming into lipid-laden foam cells. When HepG2 cells were exposed to LDL cholesterol, they developed cholesterol crystals in LD membranes, which caused activation of THP1 cells (macrophages) grown in coculture; upregulation of TNF-alpha, NLRP3, and interleukin 1beta (IL1β) mRNA; and secretion of IL-1beta. In conclusion, cholesterol crystals form on the LD membrane of hepatocytes and cause activation and cholesterol loading of KCs that surround and process these LDs by lysosomal enzymes. PMID:28404639

Engineering Cellulase Enzymes for Bioenergy

NASA Astrophysics Data System (ADS)

Atreya, Meera Elizabeth

Sustainable energy sources, such as biofuels, offer increasingly important alternatives to fossil fuels that contribute less to global climate change. The energy contained within cellulosic biofuels derives from sunlight energy stored in the form of carbon-carbon bonds comprising sugars such as glucose. Second-generation biofuels are produced from lignocellulosic biomass feedstocks, including agricultural waste products and non-food crops like Miscanthus, that contain lignin and the polysaccharides hemicellulose and cellulose. Cellulose is the most abundant biological material on Earth; it is a polymer of glucose and a structural component of plant cell walls. Accessing the sugar is challenging, as the crystalline structure of cellulose resists degradation; biochemical and thermochemical means can be used to depolymerize cellulose. Cellulase enzymes catalyze the biochemical depolymerization of cellulose into glucose. Glucose can be used as a carbon source for growth of a biofuel-producing microorganism. When it converts glucose to a hydrocarbon fuel, this microbe completes the biofuels process of transforming sunlight energy into accessible, chemical energy capable of replacing non-renewable transportation fuels. Due to strong intermolecular interactions between polymer chains, cellulose is significantly more challenging to depolymerize than starch, a more accessible polymer of glucose utilized in first-generation biofuels processes (often derived from corn). While most mammals cannot digest cellulose (dietary fiber), certain fungi and bacteria produce cellulase enzymes capable of hydrolyzing it. These organisms secrete a wide variety of glycoside hydrolase and other classes of enzymes that work in concert. Because cellulase enzymes are slow-acting and expensive to produce, my aim has been to improve the properties of these enzymes as a means to make a cellulosic biofuels process possible that is more efficient and, consequently, more economical than current methods. Protein engineering targets to improve cellulases include reducing enzyme inhibition, improving inter-enzyme synergy, and increasing enzyme thermotolerance. Ameliorating enzyme inhibition could improve catalytic activity and thus the speed of conversion from biomass to fermentable sugars. Improved enzyme synergy could reduce the enzyme loading required to achieve equivalent biomass conversion. Finally, thermostable enzymes could enable more biomass to be processed at a time, due to high temperatures decreasing the viscosity of biomass slurries. A high-temperature enzyme saccharification reaction could also decrease the risk of contamination in the resulting concentrated sugar solution. Throughout my PhD, I have explored research projects broadly across all of these topics, with the most success in addressing the issue of enzyme inhibition. Cellulase enzyme Cel7A is the most abundant cellulase employed by natural systems for cellulose hydrolysis. Cellobiohydrolase enzymes like Cel7A break down cellulose into cellobiose (two glucose molecules). Unfortunately, upon cleavage, this product molecule interferes with continued hydrolysis activity of Cel7A; the strong binding of cellobiose in the active site can obstruct the enzyme from processing down the cellulase chain. This phenomenon, known as product inhibition, is a bottleneck to efficient biomass breakdown. Using insights from computational protein modeling studies, I experimentally generated and tested mutant Cel7A enzymes for improved tolerance to cellobiose. Indeed, this strategy yielded Cel7A enzymes exhibiting reduced product inhibition, including some mutants completely impervious to cellobiose. The improvements in tolerance to cellobiose, however, resulted in an overall reduction of enzyme activity for the mutants tested. Nevertheless, my findings substantiated computational reports with experimental evidence and pinpointed an amino acid residue in the Cel7A product binding site that is of interest for follow-up mutational studies. My goal was to improve the effective catalytic activity of cellulase enzymes in industrially-relevant conditions (such as in the presence of high concentrations of cellobiose or at elevated temperatures). The insights gained from my work on enzyme inhibition may inform future efforts to address this important issue. More efficient enzymes should reduce the amount of these proteins needed to break down cellulose to glucose. This, in turn, should decrease the price of the resulting biofuel making it more cost-competitive with fossil fuels and thus encouraging adoption of renewable transportation fuels that reduce our greenhouse gas emissions.

The use of quaternised chitosan-loaded PMMA to inhibit biofilm formation and downregulate the virulence-associated gene expression of antibiotic-resistant staphylococcus.

PubMed

Tan, Honglue; Peng, Zhaoxiang; Li, Qingtian; Xu, Xiaofen; Guo, Shengrong; Tang, Tingting

2012-01-01

Biomaterial-associated infections remain a serious complication in orthopaedic surgery. Treatments, including the local use of antibiotic-loaded polymethylmethacrylate (PMMA) bone cement, are not always successful because of multiantibiotic-resistant organisms. In this study, we synthesised a new quaternised chitosan derivative (hydroxypropyltrimethyl ammonium chloride chitosan, HACC) that contains a series of substitutions of quaternary ammonium and demonstrated that HACC with a 26% degree of substitution (DS; referred to as 26%HACC) had a strong antibacterial activity and simultaneously good biocompatibility with osteogenic cells. We loaded 26%HACC at 20% by weight into PMMA bone cement to investigate whether HACC in PMMA prevents bacterial biofilm formation on the surface of bone cements. Chitosan-loaded PMMA (at the same weight ratio), gentamicin-loaded PMMA and PMMA with no antibiotic were also investigated and compared. Two clinical isolates, Staphylococcus epidermidis 389 and methicillin-resistant S. epidermidis (MRSE287), and two standard strains, S. epidermidis (ATCC35984) and methicillin-resistant Staphylococcus aureus (ATCC43300), were selected to evaluate the bacterial biofilm formation at 6, 12 and 24 h using the spread plate method, confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). The results showed that 26%HACC-loaded PMMA inhibited biofilm formation on its surface, while the PMMA control and chitosan-loaded PMMA were unable to inhibit biofilm formation. The gentamicin-loaded PMMA decreased the number of viable methicillin-resistant Staphylococcus strains, but its ability to inhibit biofilm formation was lower than 26%HACC-loaded PMMA. Real-time PCR demonstrated that 26%HACC-loaded PMMA markedly downregulated the expression of icaAD, which encodes essential enzymes for polysaccharide intercellular adhesion (PIA) biosynthesis, upregulated the expression level of icaR, which negatively mediates icaAD expression, and also downregulated the expression of MecA, which encodes membrane-bound enzymes known to be penicillin-binding proteins. Our study indicates that 26%HACC-loaded PMMA prevents biofilm formation of Staphylococcus, including antibiotic-resistant strains, on the surface of bone cement, and downregulates the virulence-associated gene expression of antibiotic-resistant staphylococcus, thus providing a promising new strategy for combating implant infections and osteomyelitis. Copyright © 2011 Elsevier Ltd. All rights reserved.

Enzymatic digestibility and ethanol fermentability of AFEX-treated starch-rich lignocellulosics such as corn silage and whole corn plant

PubMed Central

2010-01-01

Background Corn grain is an important renewable source for bioethanol production in the USA. Corn ethanol is currently produced by steam liquefaction of starch-rich grains followed by enzymatic saccharification and fermentation. Corn stover (the non-grain parts of the plant) is a potential feedstock to produce cellulosic ethanol in second-generation biorefineries. At present, corn grain is harvested by removing the grain from the living plant while leaving the stover behind on the field. Alternatively, whole corn plants can be harvested to cohydrolyze both starch and cellulose after a suitable thermochemical pretreatment to produce fermentable monomeric sugars. In this study, we used physiologically immature corn silage (CS) and matured whole corn plants (WCP) as feedstocks to produce ethanol using ammonia fiber expansion (AFEX) pretreatment followed by enzymatic hydrolysis (at low enzyme loadings) and cofermentation (for both glucose and xylose) using a cellulase-amylase-based cocktail and a recombinant Saccharomyces cerevisiae 424A (LNH-ST) strain, respectively. The effect on hydrolysis yields of AFEX pretreatment conditions and a starch/cellulose-degrading enzyme addition sequence for both substrates was also studied. Results AFEX-pretreated starch-rich substrates (for example, corn grain, soluble starch) had a 1.5-3-fold higher enzymatic hydrolysis yield compared with the untreated substrates. Sequential addition of cellulases after hydrolysis of starch within WCP resulted in 15-20% higher hydrolysis yield compared with simultaneous addition of hydrolytic enzymes. AFEX-pretreated CS gave 70% glucan conversion after 72 h of hydrolysis for 6% glucan loading (at 8 mg total enzyme loading per gram glucan). Microbial inoculation of CS before ensilation yielded a 10-15% lower glucose hydrolysis yield for the pretreated substrate, due to loss in starch content. Ethanol fermentation of AFEX-treated (at 6% w/w glucan loading) CS hydrolyzate (resulting in 28 g/L ethanol at 93% metabolic yield) and WCP (resulting in 30 g/L ethanol at 89% metabolic yield) is reported in this work. Conclusions The current results indicate the feasibility of co-utilization of whole plants (that is, starchy grains plus cellulosic residues) using an ammonia-based (AFEX) pretreatment to increase bioethanol yield and reduce overall production cost. PMID:20534126

Diagnosis of feline leukaemia virus infection by semi-quantitative real-time polymerase chain reaction.

PubMed

Pinches, Mark D G; Helps, Christopher R; Gruffydd-Jones, Tim J; Egan, Kathy; Jarrett, Oswald; Tasker, Séverine

2007-02-01

In this paper the design and use of a semi-quantitative real-time polymerase chain reaction assay (RT-PCR) for feline leukaemia virus (FeLV) provirus is described. Its performance is evaluated against established methods of FeLV diagnosis, including virus isolation and enzyme-linked immunoassay (ELISA) in a population of naturally infected cats. The RT-PCR assay is found to have both a high sensitivity (0.92) and specificity (0.99) when examined by expectation maximisation methods and is also able to detect a large number of cats with low FeLV proviral loads that were negative by other conventional test methods.

Chelation by collagen in the immobilization of Aspergillus oryzae β-galactosidase: A potential biocatalyst to hydrolyze lactose by batch processes.

PubMed

Gennari, Adriano; Mobayed, Francielle Herrmann; Volpato, Giandra; de Souza, Claucia Fernanda Volken

2018-04-01

This work is the first study of the immobilization of Aspergillus oryzae β-galactosidase (Gal) on powdered collagen (Col) that had formed a chelate with aluminum (Col-Al-Gal). Other collagen treatments, including those with acetic acid, glutaraldehyde, and a combination of aluminum and glutaraldehyde (Col-Al-Glu-Gal), were also tested. High-yield (superior to 80%) and high-efficiency (superior to 99%) immobilization was obtained for the derivatives Col-Al-Gal and Col-Al-Glu-Gal, even at high protein loads (500-1,000 mg g -1 of support). The storage stability of Gal immobilized on Col-Al and Col-Al-Glu resulted in Gal retaining approximately 60% of its initial activity after 90 days at 4 °C. The half-life values of derivatives Col-Al-Gal and Col-Al-Glu-Gal were higher than those of soluble enzyme at 65, 68, 70, and 73 °C. The derivatives Col-Al-Gal and Col-Al-Glu-Gal retained high enzyme activity in batch hydrolysis of lactose in permeate and lactose solutions for 50 and 60 cycles, respectively. Our results suggest that powdered collagen treated with aluminum, a low-cost support, is a promising support for the immobilization of β-galactosidase. Copyright © 2017 Elsevier B.V. All rights reserved.

Enhanced electrochemical sensitivity of enzyme precipitate coating (EPC)-based glucose oxidase biosensors with increased free CNT loadings.

PubMed

Kim, Jae Hyun; Jun, Sun-Ae; Kwon, Yongchai; Ha, Su; Sang, Byong-In; Kim, Jungbae

2015-02-01

Enzymatic electrodes were fabricated by using three different immobilizations of glucose oxidase (GOx): covalent enzyme attachment (CA), enzyme coating (EC), and enzyme precipitate coating (EPC), here referred to as CA-E, EC-E, and EPC-E, respectively. When additional carbon nanotubes (CNTs) were introduced from 0 to 75wt% for the EPC-E design, its initial biosensor sensitivity was improved from 2.40×10(-3) to 16.26×10(-3) A∙M(-1)∙cm(-2), while its electron charge transfer rate constant was increased from 0.33 to 1.47s(-1). When a fixed ratio of CNTs was added for three different electrode systems, EPC-E showed the best glucose sensitivity and long-term thermal stability. For example, when 75wt% of additional CNTs was added, the initial sensitivity of EPC-E was 16.26×10(-3) A∙M(-1)∙cm(-2), while those of EC-E and CA-E were only 6.42×10(-3) and 1.18×10(-3) A∙M(-1)∙cm(-2), respectively. Furthermore, EPC-E retained 63% of its initial sensitivity after thermal treatment at 40°C over 41days, while EC-E and CA-E showed only 12% and 1% of initial sensitivities, respectively. Consequently, the EPC approach with additional CNTs achieved both high sensitivity and long-term stability, which are required for continuous and accurate glucose monitoring. Copyright © 2014 Elsevier B.V. All rights reserved.

High-yield hydrogen production from biomass by in vitro metabolic engineering: Mixed sugars coutilization and kinetic modeling.

PubMed

Rollin, Joseph A; Martin del Campo, Julia; Myung, Suwan; Sun, Fangfang; You, Chun; Bakovic, Allison; Castro, Roberto; Chandrayan, Sanjeev K; Wu, Chang-Hao; Adams, Michael W W; Senger, Ryan S; Zhang, Y-H Percival

2015-04-21

The use of hydrogen (H2) as a fuel offers enhanced energy conversion efficiency and tremendous potential to decrease greenhouse gas emissions, but producing it in a distributed, carbon-neutral, low-cost manner requires new technologies. Herein we demonstrate the complete conversion of glucose and xylose from plant biomass to H2 and CO2 based on an in vitro synthetic enzymatic pathway. Glucose and xylose were simultaneously converted to H2 with a yield of two H2 per carbon, the maximum possible yield. Parameters of a nonlinear kinetic model were fitted with experimental data using a genetic algorithm, and a global sensitivity analysis was used to identify the enzymes that have the greatest impact on reaction rate and yield. After optimizing enzyme loadings using this model, volumetric H2 productivity was increased 3-fold to 32 mmol H2⋅L(-1)⋅h(-1). The productivity was further enhanced to 54 mmol H2⋅L(-1)⋅h(-1) by increasing reaction temperature, substrate, and enzyme concentrations--an increase of 67-fold compared with the initial studies using this method. The production of hydrogen from locally produced biomass is a promising means to achieve global green energy production.

High-yield hydrogen production from biomass by in vitro metabolic engineering: Mixed sugars coutilization and kinetic modeling

PubMed Central

Rollin, Joseph A.; Martin del Campo, Julia; Myung, Suwan; Sun, Fangfang; You, Chun; Bakovic, Allison; Castro, Roberto; Chandrayan, Sanjeev K.; Wu, Chang-Hao; Adams, Michael W. W.; Senger, Ryan S.; Zhang, Y.-H. Percival

2015-01-01

The use of hydrogen (H2) as a fuel offers enhanced energy conversion efficiency and tremendous potential to decrease greenhouse gas emissions, but producing it in a distributed, carbon-neutral, low-cost manner requires new technologies. Herein we demonstrate the complete conversion of glucose and xylose from plant biomass to H2 and CO2 based on an in vitro synthetic enzymatic pathway. Glucose and xylose were simultaneously converted to H2 with a yield of two H2 per carbon, the maximum possible yield. Parameters of a nonlinear kinetic model were fitted with experimental data using a genetic algorithm, and a global sensitivity analysis was used to identify the enzymes that have the greatest impact on reaction rate and yield. After optimizing enzyme loadings using this model, volumetric H2 productivity was increased 3-fold to 32 mmol H2⋅L−1⋅h−1. The productivity was further enhanced to 54 mmol H2⋅L−1⋅h−1 by increasing reaction temperature, substrate, and enzyme concentrations—an increase of 67-fold compared with the initial studies using this method. The production of hydrogen from locally produced biomass is a promising means to achieve global green energy production. PMID:25848015

High-yield hydrogen production from biomass by in vitro metabolic engineering: Mixed sugars coutilization and kinetic modeling

DOE PAGES

Rollin, Joseph A.; Martin del Campo, Julia; Myung, Suwan; ...

2015-04-06

The use of hydrogen (H 2) as a fuel offers enhanced energy conversion efficiency and tremendous potential to decrease greenhouse gas emissions, but producing it in a distributed, carbon-neutral, low-cost manner requires new technologies. Herein we demonstrate the complete conversion of glucose and xylose from plant biomass to H 2 and CO 2 based on an in vitro synthetic enzymatic pathway. Glucose and xylose were simultaneously converted to H 2 with a yield of two H 2 per carbon, the maximum possible yield. Parameters of a nonlinear kinetic model were fitted with experimental data using a genetic algorithm, and amore » global sensitivity analysis was used to identify the enzymes that have the greatest impact on reaction rate and yield. After optimizing enzyme loadings using this model, volumetric H 2 productivity was increased 3-fold to 32 mmol H 2∙L ₋1∙h ₋1. The productivity was further enhanced to 54 mmol H 2∙L ₋1∙h ₋1 by increasing reaction temperature, substrate, and enzyme concentrations—an increase of 67-fold compared with the initial studies using this method. The production of hydrogen from locally produced biomass is a promising means to achieve global green energy production.« less

Optimization of diacylglycerol production by glycerolysis of fish oil catalyzed by Lipozyme TL IM with Tween 65.

PubMed

Monte Blanco, S F M; Santos, J S; Feltes, M M C; Dors, G; Licodiedoff, S; Lerin, L A; de Oliveira, D; Ninow, J L; Furigo, A

2015-12-01

The diacylglycerols (DAG) are emulsifiers with high added value used as functional additives in food, medicine, and cosmetic industries. In glycerolysis of oils for the production of DAG, the immiscibility between the substrates (glycerol and oil phases) has to be overcome, for example, by the addition of a food grade surfactant like Tween 65. The main objective of this work was to optimize the process conditions for obtaining diacylglycerols rich in the omega-3 eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids, through the enzymatic glycerolysis of fish oil, in systems with Tween 65 and without this surfactant, using Lipozyme TL(®) IM as biocatalyst. The experiments were performed according to predetermined conditions varying the temperature, enzyme load, the oil to glycerol molar ratio and, when added, the surfactant load. After 6 h of reaction, it was possible to produce up to 20.76 and 13.76% of diacylglycerols from fish oil in the reactions with and without Tween 65, respectively.

Biochemical Characterization of β-Amino Acid Incorporation in Fluvirucin B 2 Biosynthesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Barajas, Jesus F.; Zargar, Amin; Pang, Bo

Naturally occurring lactams, such as the polyketide-derived macrolactams, provide a diverse class of natural products that could enhance existing chemically produced lactams. While β-amino acid loading in the fluvirucin B 2 polyketide pathway has been proposed by a previously identified putative biosynthetic gene cluster, biochemical characterization of the complete loading enzymes has not been described. In this paper, we elucidate the complete biosynthetic pathway of the β-amino acid loading pathway in fluvirucin B 2 biosynthesis. We demonstrate the promiscuity of the loading pathway to utilize a range of amino acids and further illustrate the ability to introduce non-native acyl transferasesmore » to selectively transfer β-amino acids onto a PKS loading platform. The results presented here provide a detailed biochemical description of β-amino acid selection and will further aid in future efforts to develop engineered lactam-producing PKS platforms.« less

Biochemical Characterization of β-Amino Acid Incorporation in Fluvirucin B 2 Biosynthesis

DOE PAGES

Barajas, Jesus F.; Zargar, Amin; Pang, Bo; ...

2018-03-30

Naturally occurring lactams, such as the polyketide-derived macrolactams, provide a diverse class of natural products that could enhance existing chemically produced lactams. While β-amino acid loading in the fluvirucin B 2 polyketide pathway has been proposed by a previously identified putative biosynthetic gene cluster, biochemical characterization of the complete loading enzymes has not been described. In this paper, we elucidate the complete biosynthetic pathway of the β-amino acid loading pathway in fluvirucin B 2 biosynthesis. We demonstrate the promiscuity of the loading pathway to utilize a range of amino acids and further illustrate the ability to introduce non-native acyl transferasesmore » to selectively transfer β-amino acids onto a PKS loading platform. The results presented here provide a detailed biochemical description of β-amino acid selection and will further aid in future efforts to develop engineered lactam-producing PKS platforms.« less

Optimization of Enzyme Co-Immobilization with Sodium Alginate and Glutaraldehyde-Activated Chitosan Beads.

PubMed

Gür, Sinem Diken; İdil, Neslihan; Aksöz, Nilüfer

2018-02-01

In this study, two different materials-alginate and glutaraldehyde-activated chitosan beads-were used for the co-immobilization of α-amylase, protease, and pectinase. Firstly, optimization of multienzyme immobilization with Na alginate beads was carried out. Optimum Na alginate and CaCl 2 concentration were found to be 2.5% and 0.1 M, respectively, and optimal enzyme loading ratio was determined as 2:1:0.02 for pectinase, protease, and α-amylase, respectively. Next, the immobilization of multiple enzymes on glutaraldehyde-activated chitosan beads was optimized (3% chitosan concentration, 0.25% glutaraldehyde with 3 h of activation and 3 h of coupling time). While co-immobilization was successfully performed with both materials, the specific activities of enzymes were found to be higher for the enzymes co-immobilized with glutaraldehyde-activated chitosan beads. In this process, glutaraldehyde was acting as a spacer arm. SEM and FTIR were used for the characterization of activated chitosan beads. Moreover, pectinase and α-amylase enzymes immobilized with chitosan beads were also found to have higher activity than their free forms. Three different enzymes were co-immobilized with these two materials for the first time in this study.

The avidity of cross-reactive virus-specific T cells for their viral and allogeneic epitopes is variable and depends on epitope expression.

PubMed

van den Heuvel, Heleen; Heutinck, Kirstin M; van der Meer-Prins, Ellen M W; Franke-van Dijk, Marry E I; van Miert, Paula P M C; Zhang, Xiaoqian; Ten Berge, Ineke J M; Claas, Frans H J

2018-01-01

Virus-specific T cells can recognize allogeneic HLA (allo-HLA) through cross-reactivity of their T-cell receptor (TCR). In a transplantation setting, such allo-HLA cross-reactivity may contribute to harmful immune responses towards the allograft, provided that the cross-reactive T cells get sufficiently activated upon recognition of the allo-HLA. An important determinant of T-cell activation is TCR avidity, which to date, has remained largely unexplored for allo-HLA-cross-reactive virus-specific T cells. For this purpose, cold target inhibition assays were performed using allo-HLA-cross-reactive virus-specific memory CD8 + T-cell clones as responders, and syngeneic cells loaded with viral peptide and allogeneic cells as hot (radioactively-labeled) and cold (non-radioactively-labeled) targets. CD8 dependency of the T-cell responses was assessed using interferon γ (IFNγ) enzyme-linked immunosorbent assay (ELISA) in the presence and absence of CD8-blocking antibodies. At high viral-peptide loading concentrations, T-cell clones consistently demonstrated lower avidity for allogeneic versus viral epitopes, but at suboptimal concentrations the opposite was observed. In line, anti-viral reactivity was CD8 independent at high, but not at suboptimal viral-peptide-loading concentrations. The avidity of allo-HLA-cross-reactive virus-specific memory CD8 + T cells is therefore highly dependent on epitope expression, and as a consequence, can be both higher and lower for allogeneic versus viral targets under different (patho)physiological conditions. Copyright © 2017 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

Virologic outcomes of HAART with concurrent use of cytochrome P450 enzyme-inducing antiepileptics: a retrospective case control study

PubMed Central

2011-01-01

Background To evaluate the efficacy of highly-active antiretroviral therapy (HAART) in individuals taking cytochrome P450 enzyme-inducing antiepileptics (EI-EADs), we evaluated the virologic response to HAART with or without concurrent antiepileptic use. Methods Participants in the US Military HIV Natural History Study were included if taking HAART for ≥6 months with concurrent use of EI-AEDs phenytoin, carbamazepine, or phenobarbital for ≥28 days. Virologic outcomes were compared to HAART-treated participants taking AEDs that are not CYP450 enzyme-inducing (NEI-AED group) as well as to a matched group of individuals not taking AEDs (non-AED group). For participants with multiple HAART regimens with AED overlap, the first 3 overlaps were studied. Results EI-AED participants (n = 19) had greater virologic failure (62.5%) compared to NEI-AED participants (n = 85; 26.7%) for the first HAART/AED overlap period (OR 4.58 [1.47-14.25]; P = 0.009). Analysis of multiple overlap periods yielded consistent results (OR 4.29 [1.51-12.21]; P = 0.006). Virologic failure was also greater in the EI-AED versus NEI-AED group with multiple HAART/AED overlaps when adjusted for both year of and viral load at HAART initiation (OR 4.19 [1.54-11.44]; P = 0.005). Compared to the non-AED group (n = 190), EI-AED participants had greater virologic failure (62.5% vs. 42.5%; P = 0.134), however this result was only significant when adjusted for viral load at HAART initiation (OR 4.30 [1.02-18.07]; P = 0.046). Conclusions Consistent with data from pharmacokinetic studies demonstrating that EI-AED use may result in subtherapeutic levels of HAART, EI-AED use is associated with greater risk of virologic failure compared to NEI-AEDs when co-administered with HAART. Concurrent use of EI-AEDs and HAART should be avoided when possible. PMID:21575228

A Novel Aqueous Two Phase System Composed of Surfactant and Xylitol for the Purification of Lipase from Pumpkin (Cucurbita moschata) Seeds and Recycling of Phase Components.

PubMed

Amid, Mehrnoush; Manap, Mohd Yazid; Hussin, Muhaini; Mustafa, Shuhaimi

2015-06-17

Lipase is one of the more important enzymes used in various industries such as the food, detergent, pharmaceutical, textile, and pulp and paper sectors. A novel aqueous two-phase system composed of surfactant and xylitol was employed for the first time to purify lipase from Cucurbita moschata. The influence of different parameters such as type and concentration of surfactants, and the composition of the surfactant/xylitol mixtures on the partitioning behavior and recovery of lipase was investigated. Moreover, the effect of system pH and crude load on the degree of purification and yield of the purified lipase were studied. The results indicated that the lipase was partitioned into the top surfactant rich phase while the impurities partitioned into the bottom xylitol-rich phase using an aqueous two phase system composed of 24% (w/w) Triton X-100 and 20% (w/w) xylitol, at 56.2% of tie line length (TLL), (TTL is one of the important parameters in this study and it is determined from a bimodal curve in which the tie-line connects two nodes on the bimodal, that represent concentration of phase components in the top and bottom phases) and a crude load of 25% (w/w) at pH 8.0. Recovery and recycling of components was also measured in each successive step process. The enzyme was successfully recovered by the proposed method with a high purification factor of 16.4 and yield of 97.4% while over 97% of the phase components were also recovered and recycled. This study demonstrated that the proposed novel aqueous two phase system method is more efficient and economical than the traditional aqueous two phase system method for the purification and recovery of the valuable enzyme lipase.

A comparison of simple rheological parameters and simulation data for Zymomonas mobilis fermentation broths with high substrate loading in a 3-L bioreactor.

PubMed

Um, Byung-Hwan; Hanley, Thomas R

2008-03-01

Traditionally, as much as 80% or more of an ethanol fermentation broth is water that must be removed. This mixture is not only costly to separate but also produces a large aqueous stream that must then be disposed of or recycled. Integrative approaches to water reduction include increasing the biomass concentration during fermentation. In this paper, experimental results are presented for the rheological behavior of high-solids enzymatic cellulose hydrolysis and ethanol fermentation for biomass conversion using Solka Floc as the model feedstock. The experimental determination of the viscosity, shear stress, and shear rate relationships of the 10 to 20% slurry concentrations with constant enzyme concentrations are performed with a variable speed rotational viscometer (2.0 to 200 rpm) at 40 degrees C. The viscosities of enzymatic suspension observed were in range of 0.0418 to 0.0144, 0.233 to 0.0348, and 0.292 to 0.0447 Pa s for shear rates up to 100 reciprocal seconds at 10, 15, and 20% initial solids (w/v), respectively. Computational fluid dynamics analysis of bioreactor mixing demonstrates the change in bioreactor mixing with increasing biomass concentration. The portion-loading method is shown to be effective for processing high-solids slurries.

Saccharification of biomass using whole solid-state fermentation medium to avoid additional separation steps.

PubMed

Pirota, Rosangela D P B; Baleeiro, Flávio C F; Farinas, Cristiane S

2013-01-01

The enzymatic hydrolysis of steam-exploded sugarcane bagasse (SESB) was investigated using enzymatic extracts (EE) and whole fermentation media (WM), produced in-house, from Aspergillus niger 3T5B8 and Trichoderma reesei Rut-C30 cultivated on wheat bran under solid-state fermentation (SSF). A detailed and quantitative comparison of the different hydrolysis conditions tested was carried out using the Chrastil approach for modeling enzymatic reactions by fitting the experimental data of total reducing sugar (TRS) released according to hydrolysis time. Conversion of SESB using A. niger enzymatic complex were up to 3.2-fold higher (in terms of TRS) than T. reesei at similar enzyme loadings, which could be correlated to the higher β-glucosidase levels (up to 35-fold higher) of A. niger enzymatic complex. Conversion yields after 72 h exceeded 40% in terms of TRS when the WM was supplemented with a low dosage of a commercial enzyme preparation. When the combination of WM (from either T. reesei or A. niger) and commercial cellulase was used, the dosage of the commercial enzyme could be reduced by half, while still providing a hydrolysis that was up to 36% more efficient. Furthermore, SESB hydrolysis using either EE or WM resulted in similar yields, indicating that the enzyme extraction/filtration steps could be eliminated from the overall process. This procedure is highly advantageous in terms of reduced enzyme and process costs, and also avoids the generation of unnecessary effluent streams. Thus, the enzymatic conversion of SESB using the WM from SSF is cost-effective and compatible with the biorefinery concept. © 2013 American Institute of Chemical Engineers.

An emerging platform for drug delivery: aerogel based systems.

PubMed

Ulker, Zeynep; Erkey, Can

2014-03-10

Over the past few decades, advances in "aerogel science" have provoked an increasing interest for these materials in pharmaceutical sciences for drug delivery applications. Because of their high surface areas, high porosities and open pore structures which can be tuned and controlled by manipulation of synthesis conditions, nanostructured aerogels represent a promising class of materials for delivery of various drugs as well as enzymes and proteins. Along with biocompatible inorganic aerogels and biodegradable organic aerogels, more complex systems such as surface functionalized aerogels, composite aerogels and layered aerogels have also been under development and possess huge potential. Emphasis is given to the details of the aerogel synthesis and drug loading methods as well as the influence of synthesis parameters and loading methods on the adsorption and release of the drugs. Owing to their ability to increase the bioavailability of low solubility drugs, to improve both their stability and their release kinetics, there are an increasing number of research articles concerning aerogels in different drug delivery applications. This review presents an up to date overview of the advances in all kinds of aerogel based drug delivery systems which are currently under investigation. Copyright © 2014 Elsevier B.V. All rights reserved.

Size exclusion chromatography for the removal of pigments from extracellular ligninolytic enzyme extracts from decayed wheat straw.

PubMed

Shukla, Dharmendra; Patel, Bhavesh; Modi, Hasmukh; Vyas, Bharat Rajiv Manuel

2011-11-01

Solid-state fermentation of wheat straw was carried out by a native white rot basidiomycete Daedaleopsis flavida strain 5A. Extract prepared from the 12-day decayed wheat straw contained extracellular ligninolytic enzymes like manganese peroxidase (MnP), manganese-independent peroxidase (MIP), lignin peroxidase (LiP) and laccase along with straw-degraded products and pigments. Sephacryl S-200 size exclusion chromatography in 16/100 column was used for the separation of these ligninolytic enzymes and straw-degraded products and pigments. Recovery of pigment-free ligninolytic enzyme activities as protein was 40% of the total proteins loaded and specific LiP activity increased 34 fold after size exclusion chromatography. Thus accurate estimation of LiP by veratryl alcohol oxidation assay was possible only after the removal of interfering pigments. The reproducibility of size exclusion chromatography is adjudged satisfactory from the consistent results obtained after seven repetitive uses of matrices.

Preparation and activity of bubbling-immobilized cellobiase within chitosan-alginate composite.

PubMed

Wang, Fang; Su, Rong-Xin; Qi, Wei; Zhang, Ming-Jia; He, Zhi-Min

2010-01-01

Cellobiase can hydrolyze cellobiose into glucose; it plays a key role in the process of cellulose hydrolysis by reducing the product inhibition. To reuse the enzyme and improve the economic value of cellulosic ethanol, cellobiase was immobilized using sodium alginate and chitosan as carriers by the bubbling method. The immobilization conditions were optimized as follows: enzyme loading of 100 U cellobiase/g carrier, 30 min immobilization, 3.5 wt% sodium alginate, 0.25 wt% chitosan, and 2 wt% calcium chloride. Compared to free enzyme, the immobilized cellobiase had a decreased apparent K(m) and the maximum activity at a lower pH, indicating its higher acidic and thermal stability. The immobilized cellobiase was further tested in the hydrolysis of cellobiose and various cellulosic substrates (microcrystalline cellulose, filter paper, and ammonia-pretreated corn cobs). Together with cellulases, the immobilized cellobiase converted the cellulosic substrates into glucose with the rate and extent similar to the free enzyme.

Slow domain reconfiguration causes power-law kinetics in a two-state enzyme.

PubMed

Grossman-Haham, Iris; Rosenblum, Gabriel; Namani, Trishool; Hofmann, Hagen

2018-01-16

Protein dynamics are typically captured well by rate equations that predict exponential decays for two-state reactions. Here, we describe a remarkable exception. The electron-transfer enzyme quiescin sulfhydryl oxidase (QSOX), a natural fusion of two functionally distinct domains, switches between open- and closed-domain arrangements with apparent power-law kinetics. Using single-molecule FRET experiments on time scales from nanoseconds to milliseconds, we show that the unusual open-close kinetics results from slow sampling of an ensemble of disordered domain orientations. While substrate accelerates the kinetics, thus suggesting a substrate-induced switch to an alternative free energy landscape of the enzyme, the power-law behavior is also preserved upon electron load. Our results show that the slow sampling of open conformers is caused by a variety of interdomain interactions that imply a rugged free energy landscape, thus providing a generic mechanism for dynamic disorder in multidomain enzymes.

Coupling alkaline pre-extraction with alkaline-oxidative post-treatment of corn stover to enhance enzymatic hydrolysis and fermentability

PubMed Central

2014-01-01

Background A two-stage chemical pretreatment of corn stover is investigated comprising an NaOH pre-extraction followed by an alkaline hydrogen peroxide (AHP) post-treatment. We propose that conventional one-stage AHP pretreatment can be improved using alkaline pre-extraction, which requires significantly less H2O2 and NaOH. To better understand the potential of this approach, this study investigates several components of this process including alkaline pre-extraction, alkaline and alkaline-oxidative post-treatment, fermentation, and the composition of alkali extracts. Results Mild NaOH pre-extraction of corn stover uses less than 0.1 g NaOH per g corn stover at 80°C. The resulting substrates were highly digestible by cellulolytic enzymes at relatively low enzyme loadings and had a strong susceptibility to drying-induced hydrolysis yield losses. Alkaline pre-extraction was highly selective for lignin removal over xylan removal; xylan removal was relatively minimal (~20%). During alkaline pre-extraction, up to 0.10 g of alkali was consumed per g of corn stover. AHP post-treatment at low oxidant loading (25 mg H2O2 per g pre-extracted biomass) increased glucose hydrolysis yields by 5%, which approached near-theoretical yields. ELISA screening of alkali pre-extraction liquors and the AHP post-treatment liquors demonstrated that xyloglucan and β-glucans likely remained tightly bound in the biomass whereas the majority of the soluble polymeric xylans were glucurono (arabino) xylans and potentially homoxylans. Pectic polysaccharides were depleted in the AHP post-treatment liquor relative to the alkaline pre-extraction liquor. Because the already-low inhibitor content was further decreased in the alkaline pre-extraction, the hydrolysates generated by this two-stage pretreatment were highly fermentable by Saccharomyces cerevisiae strains that were metabolically engineered and evolved for xylose fermentation. Conclusions This work demonstrates that this two-stage pretreatment process is well suited for converting lignocellulose to fermentable sugars and biofuels, such as ethanol. This approach achieved high enzymatic sugars yields from pretreated corn stover using substantially lower oxidant loadings than have been reported previously in the literature. This pretreatment approach allows for many possible process configurations involving novel alkali recovery approaches and novel uses of alkaline pre-extraction liquors. Further work is required to identify the most economical configuration, including process designs using techno-economic analysis and investigating processing strategies that economize water use. PMID:24693882

High-performance glucose biosensor based on chitosan-glucose oxidase immobilized polypyrrole/Nafion/functionalized multi-walled carbon nanotubes bio-nanohybrid film.

PubMed

Shrestha, Bishnu Kumar; Ahmad, Rafiq; Mousa, Hamouda M; Kim, In-Gi; Kim, Jeong In; Neupane, Madhav Prasad; Park, Chan Hee; Kim, Cheol Sang

2016-11-15

A highly electroactive bio-nanohybrid film of polypyrrole (PPy)-Nafion (Nf)-functionalized multi-walled carbon nanotubes (fMWCNTs) nanocomposite was prepared on the glassy carbon electrode (GCE) by a facile one-step electrochemical polymerization technique followed by chitosan-glucose oxidase (CH-GOx) immobilization on its surface to achieve a high-performance glucose biosensor. The as-fabricated nanohybrid composite provides high surface area for GOx immobilization and thus enhances the enzyme-loading efficiency. The structural characterization revealed that the PPy-Nf-fMWCNTs nanocomposite films were uniformly formed on GCE and after GOx immobilization, the surface porosities of the film were decreased due to enzyme encapsulation inside the bio-nanohybrid composite materials. The electrochemical behavior of the fabricated biosensor was investigated by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and amperometry measurements. The results indicated an excellent catalytic property of bio-nanohybrid film for glucose detection with improved sensitivity of 2860.3μAmM(-1)cm(-2), the linear range up to 4.7mM (R(2)=0.9992), and a low detection limit of 5μM under a signal/noise (S/N) ratio of 3. Furthermore, the resulting biosensor presented reliable selectivity, better long-term stability, good repeatability, reproducibility, and acceptable measurement of glucose concentration in real serum samples. Thus, this fabricated biosensor provides an efficient and highly sensitive platform for glucose sensing and can open up new avenues for clinical applications. Copyright © 2016 Elsevier Inc. All rights reserved.

New poly(ester urea) derived from L-leucine: electrospun scaffolds loaded with antibacterial drugs and enzymes.

PubMed

Díaz, Angélica; del Valle, Luis J; Tugushi, David; Katsarava, Ramaz; Puiggalí, Jordi

2015-01-01

Electrospun scaffolds from an amino acid containing poly(ester urea) (PEU) were developed as promising materials in the biomedical field and specifically in tissue engineering applications. The selected poly(ester urea) was obtained with a high yield and molecular weight by reaction of phosgene with a bis(α-aminoacyl)-α,ω-diol-diester monomer. The polymer having L-leucine, 1,6-hexanediol and carbonic acid units had a semicrystalline character and relatively high glass transition and melting temperatures. Furthermore it was highly soluble in most organic solvents, an interesting feature that facilitated the electrospinning process and the effective incorporation of drugs with bactericidal activity (e.g. biguanide derivatives such as clorhexidine and polyhexamethylenebiguanide) and enzymes (e.g. α-chymotrypsin) that accelerated the degradation process. Continuous micro/nanofibers were obtained under a wide range of processing conditions, being diameters of electrospun fibers dependent on the drug and solvent used. Poly(ester urea) samples were degradable in media containing lipases and proteinases but the degradation rate was highly dependent on the surface area, being specifically greater for scaffolds with respect to films. The high hydrophobicity of new scaffolds had repercussions on enzymatic degradability since different weight loss rates were found depending on how samples were exposed to the medium (e.g. forced or non-forced immersion). New scaffolds were biocompatible, as demonstrated by adhesion and proliferation assays performed with fibroblast and epithelial cells. Copyright © 2014 Elsevier B.V. All rights reserved.

A novel microfluidic microplate as the next generation assay platform for enzyme linked immunoassays (ELISA).

PubMed

Kai, Junhai; Puntambekar, Aniruddha; Santiago, Nelson; Lee, Se Hwan; Sehy, David W; Moore, Victor; Han, Jungyoup; Ahn, Chong H

2012-11-07

In this work we introduce a novel microfluidic enzyme linked immunoassays (ELISA) microplate as the next generation assay platform for unparalleled assay performances. A combination of microfluidic technology with standard SBS-configured 96-well microplate architecture, in the form of microfluidic microplate technology, allows for the improvement of ELISA workflows, conservation of samples and reagents, improved reaction kinetics, and the ability to improve the sensitivity of the assay by multiple analyte loading. This paper presents the design and characterization of the microfluidic microplate, and its application in ELISA.

Lysosomal enzyme delivery by ICAM-1-targeted nanocarriers bypassing glycosylation- and clathrin-dependent endocytosis.

PubMed

Muro, Silvia; Schuchman, Edward H; Muzykantov, Vladimir R

2006-01-01

Enzyme replacement therapy, a state-of-the-art treatment for many lysosomal storage disorders, relies on carbohydrate-mediated binding of recombinant enzymes to receptors that mediate lysosomal delivery via clathrin-dependent endocytosis. Suboptimal glycosylation of recombinant enzymes and deficiency of clathrin-mediated endocytosis in some lysosomal enzyme-deficient cells limit delivery and efficacy of enzyme replacement therapy for lysosomal disorders. We explored a novel delivery strategy utilizing nanocarriers targeted to a glycosylation- and clathrin-independent receptor, intercellular adhesion molecule (ICAM)-1, a glycoprotein expressed on diverse cell types, up-regulated and functionally involved in inflammation, a hallmark of many lysosomal disorders. We targeted recombinant human acid sphingomyelinase (ASM), deficient in types A and B Niemann-Pick disease, to ICAM-1 by loading this enzyme to nanocarriers coated with anti-ICAM. Anti-ICAM/ASM nanocarriers, but not control ASM or ASM nanocarriers, bound to ICAM-1-positive cells (activated endothelial cells and Niemann-Pick disease patient fibroblasts) via ICAM-1, in a glycosylation-independent manner. Anti-ICAM/ASM nanocarriers entered cells via CAM-mediated endocytosis, bypassing the clathrin-dependent pathway, and trafficked to lysosomes, where delivered ASM displayed stable activity and alleviated lysosomal lipid accumulation. Therefore, lysosomal enzyme targeting using nanocarriers targeted to ICAM-1 bypasses defunct pathways and may improve the efficacy of enzyme replacement therapy for lysosomal disorders, such as Niemann-Pick disease.

Nitric oxide-mediated suppression of 2,3-bisphosphoglycerate synthesis: therapeutic relevance for environmental hypoxia and sickle cell disease.

PubMed

Bertrand, R

2012-09-01

Though hemoglobin (Hb) is best known for transporting oxygen and metabolic wastes throughout the circulatory system, this erythrocyte protein also acts as a hypoxic sensor, its oxygen saturation dependent on the oxygen partial pressure (pO(2)) which varies throughout the vasculature. The production and transport of the endogenous vasodilator nitric oxide (NO) by Hb is dependent on Hb's oxygen saturation, thereby allowing the protein to auto-regulate blood flow efficiency to meet the relative demands of respiring tissues. Erythrocyte concentrations of 2,3-bisphosphoglycerate (BPG), an enhancer of oxygen off-loading from Hb, is very sensitive to changes in glycolytic rates because its synthesis by BPG synthase is dependent on the availability of the glycolytic intermediate 1,3-bisphosphoglycerate. BPG synthase, as well as some glycolytic enzymes, are also very sensitive to pH changes, and variations in BPG levels have direct consequences on the oxygen off-loading function of Hb. I hypothesize that NO may suppress BPG production by (1) inhibiting glyceraldehyde-3-phosphate dehydrogenase (G3PDH), the most critical glycolytic enzyme for the bioavailability of 1,3-bisphosphoglycerate; and to a lesser extent by (2) associated pH changes in the deoxy-Hb-catalyzed depletion of nitrite, a metabolic reservoir of NO. Both mechanisms are favored in low pO(2) environments where BPG is most needed to maximize oxygen off-loading, indicating that the auto-regulatory link between NO and Hb may have inadvertently linked Hb and BPG synthesis in an unfavorable manner. However, for reasons discussed, NO-mediated suppression of BPG may be advantageous in some circumstances; namely, for individuals living at high altitudes and those with the blood disorder sickle cell anemia. This hypothesis is thus relevant to respiratory health under both normative conditions as well as under hypoxic stress. The potential relevance of the hypothesis to comparative animal physiology and evolutionary biology is also briefly described. Copyright © 2012 Elsevier Ltd. All rights reserved.

A therapeutic HIV-1 vaccine enhances anti-HIV-1 immune responses in patients under highly active antiretroviral therapy.

PubMed

Tung, Frank Y; Tung, Jack K; Pallikkuth, Suresh; Pahwa, Savita; Fischl, Margaret A

2016-04-27

HIV-1 specific cellular immunity plays an important role in controlling viral replication. In this first-in-human therapeutic vaccination study, a replication-defective HIV-1 vaccine (HIVAX) was tested in HIV-1 infected participants undergoing highly active antiretroviral therapy (HAART) to enhance anti-HIV immunity (Clinicaltrials.gov, identifier NCT01428596). A010 was a randomized, placebo-controlled trial to evaluate the safety and the immunogenicity of a replication defective HIV-1 vaccine (HIVAX) given as a subcutaneous injection to HIV-1 infected participants who were receiving HAART with HIV-1 viral load <50 copies/ml and CD4 cell count >500 cells/mm(3). HIV-1 specific immune responses were monitored by INF-γ enzyme linked immunospot (Elispot) and intracellular cytokine staining (ICS) assay after vaccination. Following the randomized placebo-controlled vaccination phase, subjects who received HIVAX vaccine and who met eligibility underwent a 12-week analytical antiretroviral treatment interruption (ATI). Viral load was monitored throughout the study. HIVAX was well tolerated in trial participants. Transient grade 1 to 2 (mild to moderate) injection site reactions occurred in 8 of 10 vaccinated participants. HIVAX was immunogenic in all vaccinated participants. The functionality of T cells was significantly enhanced after vaccination. Median viral load (3.45 log10 copies/ml, range of 96-12,830 copies/ml) at the end of the 12-week treatment interruption in HIVAX vaccinated group was significantly lower than the pre-treatment levels. Three vaccinated participants extended ATI for up to 2 years with stable CD4 cells and low viral loads. HIVAX vaccine is generally safe, elicits strong anti-HIV-1 immune responses, and may play an important role in controlling viral load during treatment interruption in HIV-1 infected participants. Copyright © 2016 Elsevier Ltd. All rights reserved.

Tunable Enzymatic Activity and Enhanced Stability of Cellulase Immobilized in Biohybrid Nanogels.

PubMed

Peng, Huan; Rübsam, Kristin; Jakob, Felix; Schwaneberg, Ulrich; Pich, Andrij

2016-11-14

This paper reports a facile approach for encapsulation of enzymes in nanogels. Our approach is based on the use of reactive copolymers able to get conjugated with enzyme and build 3D colloidal networks or biohybrid nanogels. In a systematic study, we address the following question: how the chemical structure of nanogel network influences the biocatalytic activity of entrapped enzyme? The developed method allows precise control of the enzyme activity and improvement of enzyme resistance against harsh store conditions, chaotropic agents, and organic solvents. The nanogels were constructed via direct chemical cross-linking of water-soluble reactive copolymers poly(N-vinylpyrrolidone-co-N-methacryloxysuccinimide) with proteins such as enhanced green fluorescent protein (EGFP) and cellulase in water-in-oil emulsion. The water-soluble reactive copolymers with controlled amount of reactive succinimide groups and narrow dispersity were synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization. Poly(ethylene glycol) bis(3-aminopropyl) and branched polyethylenimine were utilized as model cross-linkers to optimize synthesis of nanogels with different architectures in the preliminary experiments. Biofluorescent nanogels with different loading amount of EGFP and varying cross-linking densities were obtained. We demonstrate that the biocatalytic activity of cellulase-conjugated nanogels (CNG) can be elegantly tuned by control of their cross-linking degrees. Circular dichroism (CD) spectra demonstrated that the secondary structures of the immobilized cellulase were changed in the aspect of α-helix contents. The secondary structures of cellulase in highly cross-linked nanogels were strongly altered compared with loosely cross-linked nanogels. The fluorescence resonance energy transfer (FRET) based study further revealed that nanogels with lower cross-linking degree enable higher substrate transport rate, providing easier access to the active site of the enzyme. The biohybrid nanogels demonstrated significantly improved stability in preserving enzymatic activity compared with free cellulase. The functional biohybrid nanogels with tunable enzymatic activity and improved stability are promising candidates for applications in biocatalysis, biomass conversion, or energy utilization fields.

The enzyme-sensitive release of prodigiosin grafted β-cyclodextrin and chitosan magnetic nanoparticles as an anticancer drug delivery system: Synthesis, characterization and cytotoxicity studies.

PubMed

Rastegari, Banafsheh; Karbalaei-Heidari, Hamid Reza; Zeinali, Sedigheh; Sheardown, Heather

2017-10-01

In present investigation, two glucose based smart tumor-targeted drug delivery systems coupled with enzyme-sensitive release strategy are introduced. Magnetic nanoparticles (Fe 3 O 4 ) were grafted with carboxymethyl chitosan (CS) and β-cyclodextrin (β-CD) as carriers. Prodigiosin (PG) was used as the model anti-tumor drug, targeting aggressive tumor cells. The morphology, properties and composition and grafting process were characterized by transmission electron microscope (TEM), Fourier transform infrared spectroscopy (FT-IR), vibration sample magnetometer (VSM), X-ray diffraction (XRD) analysis. The results revealed that the core crystal size of the nanoparticles synthesized were 14.2±2.1 and 9.8±1.4nm for β-CD and CS-MNPs respectively when measured using TEM; while dynamic light scattering (DLS) gave diameters of 121.1 and 38.2nm. The saturation magnetization (Ms) of bare magnetic nanoparticles is 50.10emucm -3 , while modification with β-CD and CS gave values of 37.48 and 65.01emucm -3 , respectively. The anticancer compound, prodigiosin (PG) was loaded into the NPs with an encapsulation efficiency of approximately 81% for the β-CD-MNPs, and 92% for the CS-MNPs. This translates to a drug loading capacity of 56.17 and 59.17mg/100mg MNPs, respectively. Measurement of in vitro release of prodigiosin from the loaded nanocarriers in the presence of the hydrolytic enzymes, alpha-amylase and chitosanase showed that 58.1 and 44.6% of the drug was released after one-hour of incubation. Cytotoxicity studies of PG-loaded nanocarriers on two cancer cell lines, MCF-7 and HepG2, and on a non-cancerous control, NIH/3T3 cells, revealed that the drug loaded nanoparticles had greater efficacy on the cancer cell lines. The selective index (SI) for free PG on MCF-7 and HepG2 cells was 1.54 and 4.42 respectively. This parameter was reduced for PG-loaded β-CD-MNPs to 1.27 and 1.85, while the SI for CS-MNPs improved considerably to 7.03 on MCF-7 cells. Complementary studies by fluorescence and confocal microscopy and flow cytometry confirm specific targeting of the nanocarriers to the cancer cells. The results suggest that CS-MNPs have higher potency and are better able to target the prodigiosin toxicity effect on cancerous cells than β-CD-MNPs. Copyright © 2017 Elsevier B.V. All rights reserved.

A novel hybrid organosolv: steam explosion method for the efficient fractionation and pretreatment of birch biomass.

PubMed

Matsakas, Leonidas; Nitsos, Christos; Raghavendran, Vijayendran; Yakimenko, Olga; Persson, Gustav; Olsson, Eva; Rova, Ulrika; Olsson, Lisbeth; Christakopoulos, Paul

2018-01-01

The main role of pretreatment is to reduce the natural biomass recalcitrance and thus enhance saccharification yield. A further prerequisite for efficient utilization of all biomass components is their efficient fractionation into well-defined process streams. Currently available pretreatment methods only partially fulfill these criteria. Steam explosion, for example, excels as a pretreatment method but has limited potential for fractionation, whereas organosolv is excellent for delignification but offers poor biomass deconstruction. In this article, a hybrid method combining the cooking and fractionation of conventional organosolv pretreatment with the implementation of an explosive discharge of the cooking mixture at the end of pretreatment was developed. The effects of various pretreatment parameters (ethanol content, duration, and addition of sulfuric acid) were evaluated. Pretreatment of birch at 200 °C with 60% v/v ethanol and 1% w/w biomass H 2 SO 4 was proven to be the most efficient pretreatment condition yielding pretreated solids with 77.9% w/w cellulose, 8.9% w/w hemicellulose, and 7.0 w/w lignin content. Under these conditions, high delignification of 86.2% was demonstrated. The recovered lignin was of high purity, with cellulose and hemicellulose contents not exceeding 0.31 and 3.25% w/w, respectively, and ash to be < 0.17% w/w in all cases, making it suitable for various applications. The pretreated solids presented high saccharification yields, reaching 68% at low enzyme load (6 FPU/g) and complete saccharification at high enzyme load (22.5 FPU/g). Finally, simultaneous saccharification and fermentation (SSF) at 20% w/w solids yielded an ethanol titer of 80 g/L after 192 h, corresponding to 90% of the theoretical maximum. The novel hybrid method developed in this study allowed for the efficient fractionation of birch biomass and production of pretreated solids with high cellulose and low lignin contents. Moreover, the explosive discharge at the end of pretreatment had a positive effect on enzymatic saccharification, resulting in high hydrolyzability of the pretreated solids and elevated ethanol titers in the following high-gravity SSF. To the best of our knowledge, the ethanol concentration obtained with this method is the highest so far for birch biomass.

Determination of the inhibitory effect of green tea extract on glucose-6-phosphate dehydrogenase based on multilayer capillary enzyme microreactor.

PubMed

Camara, Mohamed Amara; Tian, Miaomiao; Liu, Xiaoxia; Liu, Xin; Wang, Yujia; Yang, Jiqing; Yang, Li

2016-08-01

Natural herbal medicines are an important source of enzyme inhibitors for the discovery of new drugs. A number of natural extracts such as green tea have been used in prevention and treatment of diseases due to their low-cost, low toxicity and good performance. The present study reports an online assay of the activity and inhibition of the green tea extract of the Glucose 6-phosphate dehydrogenase (G6PDH) enzyme using multilayer capillary electrophoresis based immobilized enzyme microreactors (CE-IMERs). The multilayer CE-IMERs were produced with layer-by-layer electrostatic assembly, which can easily enhance the enzyme loading capacity of the microreactor. The activity of the G6PDH enzyme was determined and the enzyme inhibition by the inhibitors from green tea extract was investigated using online assay of the multilayer CE-IMERs. The Michaelis constant (Km ) of the enzyme, the IC50 and Ki values of the inhibitors were achieved and found to agree with those obtained using offline assays. The results show a competitive inhibition of green tea extract on the G6PDH enzyme. The present study provides an efficient and easy-to-operate approach for determining G6PDH enzyme reaction and the inhibition of green tea extract, which may be beneficial in research and the development of natural herbal medicines. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

MICROBIAL ENZYME ACTIVITIES IN A FRESHWATER MARSH AFTER CESSATION OF NUTRIENT LOADING. (R827641)

EPA Science Inventory

The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

Delivery of Therapeutic Proteins via Extracellular Vesicles: Review and Potential Treatments for Parkinson's Disease, Glioma, and Schwannoma.

PubMed

Hall, Justin; Prabhakar, Shilpa; Balaj, Leonora; Lai, Charles P; Cerione, Richard A; Breakefield, Xandra O

2016-04-01

Extracellular vesicles present an attractive delivery vehicle for therapeutic proteins. They intrinsically contain many proteins which can provide information to other cells. Advantages include reduced immune reactivity, especially if derived from the same host, stability in biologic fluids, and ability to target uptake. Those from mesenchymal stem cells appear to be intrinsically therapeutic, while those from cancer cells promote tumor progression. Therapeutic proteins can be loaded into vesicles by overexpression in the donor cell, with oligomerization and membrane sequences increasing their loading. Examples of protein delivery for therapeutic benefit in pre-clinical models include delivery of: catalase for Parkinson's disease to reduce oxidative stress and thus help neurons to survive; prodrug activating enzymes which can convert a prodrug which crosses the blood-brain barrier into a toxic chemotherapeutic drug for schwannomas and gliomas; and the apoptosis-inducing enzyme, caspase-1 under a Schwann cell specific promoter for schwannoma. This therapeutic delivery strategy is novel and being explored for a number of diseases.

Versatility of acyl-acyl carrier protein synthetases

DOE PAGES

Beld, Joris; Finzel, Kara; Burkart, Michael D.

2014-10-09

The acyl carrier protein (ACP) requires posttranslational modification with a 4'-phosphopantetheine arm for activity, and this thiol-terminated modification carries cargo between enzymes in ACP-dependent metabolic pathways. In this paper, we show that acyl-ACP synthetases (AasSs) from different organisms are able to load even, odd, and unnatural fatty acids onto E. coli ACP in vitro. Vibrio harveyi AasS not only shows promiscuity for the acid substrate, but also is active upon various alternate carrier proteins. AasS activity also extends to functional activation in living organisms. We show that exogenously supplied carboxylic acids are loaded onto ACP and extended by the E.more » coli fatty acid synthase, including unnatural fatty acid analogs. These analogs are further integrated into cellular lipids. Finally, in vitro characterization of four different adenylate-forming enzymes allowed us to disambiguate CoA-ligases and AasSs, and further in vivo studies show the potential for functional application in other organisms.« less

A new approach to synthesis of benzyl cinnamate: Optimization by response surface methodology.

PubMed

Zhang, Dong-Hao; Zhang, Jiang-Yan; Che, Wen-Cai; Wang, Yun

2016-09-01

In this work, the new approach to synthesis of benzyl cinnamate by enzymatic esterification of cinnamic acid with benzyl alcohol is optimized by response surface methodology. The effects of various reaction conditions, including temperature, enzyme loading, substrate molar ratio of benzyl alcohol to cinnamic acid, and reaction time, are investigated. A 5-level-4-factor central composite design is employed to search for the optimal yield of benzyl cinnamate. A quadratic polynomial regression model is used to analyze the experimental data at a 95% confidence level (P<0.05). The coefficient of determination of this model is found to be 0.9851. Three sets of optimum reaction conditions are established, and the verified experimental trials are performed for validating the optimum points. Under the optimum conditions (40°C, 31mg/mL enzyme loading, 2.6:1 molar ratio, 27h), the yield reaches 97.7%, which provides an efficient processes for industrial production of benzyl cinnamate. Copyright © 2016 Elsevier Ltd. All rights reserved.

Synthesis of α-linolenic acid-rich triacylglycerol using a newly prepared immobilized lipase.

PubMed

Kim, Heejin; Choi, Nakyung; Oh, Se-Wook; Kim, Yangha; Hee Kim, Byung; Kim, In-Hwan

2017-12-15

An α-linolenic acid (ALA)-rich triacylglycerol (TAG) was synthesized from an ALA-rich fatty acid (FA) from perilla oil and glycerol, using a newly prepared immobilized lipase under vacuum. The ALA-rich FA (purity >90wt%) used as the substrate was prepared by urea complexation from perilla oil FAs. Liquid Lipozyme TL 100L lipase from Thermomyces lanuginosus was used for immobilization. Nine different hydrophilic and hydrophobic carriers for immobilization were tested, and Duolite A568, which is a hydrophilic resin, was selected as the best carrier. This immobilized lipase was used to synthesize TAG by direct esterification under vacuum. The parameters investigated were temperature, enzyme loading, and vacuum level. The optimum reaction conditions were a temperature of 60°C, an enzyme loading of 15% (based on the total weight of the substrate), and a vacuum of 0.7kPa, respectively. The maximum conversion to TAG of ca. 88wt% was obtained in 12h under the optimum conditions. Copyright © 2017 Elsevier Ltd. All rights reserved.

The feasibility study of crude palm oil transesterification at 30 °C operation.

PubMed

Sim, Jia Huey; Kamaruddin, Azlina Harun; Bhatia, Subhash

2010-12-01

The objective of this research is to investigate the potential of transesterification of crude palm oil (CPO) to biodiesel at 30 degrees C. The mass transfer limitations problem crucial at 30 degrees C due to the viscosity of CPO has been addressed. The process parameters that are closely related to mass transfer effects like enzyme loading, agitation speed and reaction time were optimized. An optimum methanol to oil substrate molar ratio at 6.5:1 was observed and maintained throughout the experiments. The optimum operating condition for the transesterification process was found at 6.67 wt% of enzyme loading and at 150 rpm of agitation speed. The corresponding initial reaction and FAME yield obtained at 6 h were 89.29% FAME yield/hr and 85.01%, respectively. The 85% FAME yield obtained at 30 degrees C operation of CPO transesterification shows that the process is potentially feasible for the biodiesel synthesis. 2010 Elsevier Ltd. All rights reserved.

Production of cellulosic ethanol from cotton processing residues after pretreatment with dilute sodium hydroxide and enzymatic hydrolysis.

PubMed

Fockink, Douglas Henrique; Maceno, Marcelo Adriano Corrêa; Ramos, Luiz Pereira

2015-01-01

In this study, production of cellulosic ethanol from two cotton processing residues was investigated after pretreatment with dilute sodium hydroxide. Pretreatment performance was investigated using a 2(2) factorial design and the highest glucan conversion was achieved at the most severe alkaline conditions (0.4g NaOH g(-1) of dry biomass and 120°C), reaching 51.6% and 38.8% for cotton gin waste (CGW) and cotton gin dust (CGD), respectively. The susceptibility of pretreated substrates to enzymatic hydrolysis was also investigated and the best condition was achieved at the lowest total solids (5wt%) and the highest enzyme loading (85mg of Cellic CTec2 g(-1) of dry substrate). However, the highest concentration of fermentable sugars - 47.8 and 42.5gL(-1) for CGD and CGW, respectively - was obtained at 15wt% total solids using this same enzyme loading. Substrate hydrolysates had no inhibitory effects on the fermenting microorganism. Copyright © 2015. Published by Elsevier Ltd.

Suppression of cucumber stachyose synthase gene (CsSTS) inhibits phloem loading and reduces low temperature stress tolerance.

PubMed

Lü, Jianguo; Sui, Xiaolei; Ma, Si; Li, Xin; Liu, Huan; Zhang, Zhenxian

2017-09-01

Stachyose is the main transporting sugar in phloem of Raffinose family oligosaccharides-transporting species. Stachyose synthase (STS) is a key enzyme for stachyose biosynthesis, but the gene encoding STS is poorly characterized in cucumber (Cucumis sativus L.), which is a model plant for studying stachyose metabolism and phloem function. In this research, stachyose synthase gene (CsSTS) from cucumber was isolated and its physiological functions were analyzed. CsSTS expressed mainly in the phloem of the minor veins in mature leaves and localized to companion cells. Reverse genetics with CsSTS RNAi lines revealed obviously reductions in STS activity and stachyose content along with a small amount of starch accumulation in leaves, suggesting that CsSTS is involved in phloem loading of cucumber leaves. After 6 °C low temperature stress, malondialdehyde content and electrical conductivity increased, especially in CsSTS-RNAi plants. But CsSTS expression was up-regulated, STS activity and stachyose level increased, the activities of reactive-oxygen-scavenging enzyme in cucumber seedlings improved significantly and starch accumulation reduced, especially in CsSTS-OE lines. These results demonstrate clearly that CsSTS is involved in phloem loading, carbohydrate distribution and tolerance of cucumber seedlings to low temperature stress.

Effect of poultry litter biochar on Saccharomyces cerevisiae growth and ethanol production from steam-exploded poplar and corn stover

NASA Astrophysics Data System (ADS)

Diallo, Oumou

The use of ethanol produced from lignocellulosic biomass for transportation fuel offers solutions in reducing environmental emission and the use of non-renewable fuels. However, lignocellulosic ethanol production is still hampered by economic and technical obstacles. For instance, the inhibitory effect of toxic compounds produced during biomass pretreatment was reported to inhibit the fermenting microorganisms, hence there was a decrease in ethanol yield and productivity. Thus, there is a need to improve the bioconversion of lignocellulosic biomass to ethanol in order to promote its commercialization. The research reported here investigated the use of poultry litter biochar to improve the ethanol production from steam-exploded poplar and corn stover. The effect of poultry litter biochar was first studied on Saccharomyces cerevisiae ATCC 204508/S288C growth, and second on the enzyme hydrolysis and fermentation of two steam-exploded biomasses: (poplar and corn stover). The third part of the study investigated optimal process parameters (biochar loading, biomass loading, and enzyme loading) on the reducing sugars production, and ethanol yield from steam-exploded corn stover. In this study, it has been shown that poultry litter biochar improved the S. cerevisiae growth and ethanol productivity; therefore poultry litter biochar could potentially be used to improve the ethanol production from steam-exploded lignocellulosic biomass.

13C-Breath Tests for Sucrose Digestion in Congenital Sucrase Isomaltase Deficient and Sacrosidase Supplemented Patients

PubMed Central

Robayo-Torres, Claudia C.; Opekun, Antone R.; Quezada-Calvillo, Roberto; Xavier, Villa; Smith, E. O’Brian; Navarrete, Marilyn; Baker, S. Susan; Nichols, Buford L

2008-01-01

Congenital sucrase-isomaltase deficiency (CSID) is characterized by absence or deficiency of the mucosal sucrase-isomaltase enzyme. Specific diagnosis requires upper gastrointestinal biopsy with evidence of low to absent sucrase enzyme activity and normal histology. The hydrogen breath test (BT) is useful but is not specific for confirmation of CSID. We investigated a more specific 13C-sucrose labeled BT. Objectives were to determine if CSID can be detected with the 13C-sucrose BT without duodenal biopsy sucrase assay and if the 13C-sucrose BT can document restoration of sucrose digestion by CSID patients after oral supplementation with sacrosidase (Sucraid®). Methods Ten CSID patients were diagnosed by low biopsy sucrase activity. Ten controls were children who underwent endoscopy and biopsy because of dyspepsia or chronic diarrhea with normal mucosal enzymes activity and histology. Uniformly-labeled 13C-glucose and 13C-sucrose loads were orally administered. 13CO2 breath enrichments were assayed using an infrared spectrophotometer. In CSID patients the 13C-sucrose load was repeated adding Sucraid®. Sucrose digestion and oxidation were calculated as a mean % coefficient of glucose oxidation (% CGO) averaged between 30 and 90 minutes. Results Classification of patients by 13C-sucrose BT % CGO agreed with biopsy sucrase activity. The breath test also documented the return to normal of sucrose digestion and oxidation after supplementation of CSID patients with Sucraid®. Conclusion 13C-sucrose BT is an accurate and specific non-invasive confirmatory test for CSID and for enzyme replacement management. PMID:19330928

Artificial Intelligence vs. Statistical Modeling and Optimization of Continuous Bead Milling Process for Bacterial Cell Lysis.

PubMed

Haque, Shafiul; Khan, Saif; Wahid, Mohd; Dar, Sajad A; Soni, Nipunjot; Mandal, Raju K; Singh, Vineeta; Tiwari, Dileep; Lohani, Mohtashim; Areeshi, Mohammed Y; Govender, Thavendran; Kruger, Hendrik G; Jawed, Arshad

2016-01-01

For a commercially viable recombinant intracellular protein production process, efficient cell lysis and protein release is a major bottleneck. The recovery of recombinant protein, cholesterol oxidase (COD) was studied in a continuous bead milling process. A full factorial response surface methodology (RSM) design was employed and compared to artificial neural networks coupled with genetic algorithm (ANN-GA). Significant process variables, cell slurry feed rate (A), bead load (B), cell load (C), and run time (D), were investigated and optimized for maximizing COD recovery. RSM predicted an optimum of feed rate of 310.73 mL/h, bead loading of 79.9% (v/v), cell loading OD 600 nm of 74, and run time of 29.9 min with a recovery of ~3.2 g/L. ANN-GA predicted a maximum COD recovery of ~3.5 g/L at an optimum feed rate (mL/h): 258.08, bead loading (%, v/v): 80%, cell loading (OD 600 nm ): 73.99, and run time of 32 min. An overall 3.7-fold increase in productivity is obtained when compared to a batch process. Optimization and comparison of statistical vs. artificial intelligence techniques in continuous bead milling process has been attempted for the very first time in our study. We were able to successfully represent the complex non-linear multivariable dependence of enzyme recovery on bead milling parameters. The quadratic second order response functions are not flexible enough to represent such complex non-linear dependence. ANN being a summation function of multiple layers are capable to represent complex non-linear dependence of variables in this case; enzyme recovery as a function of bead milling parameters. Since GA can even optimize discontinuous functions present study cites a perfect example of using machine learning (ANN) in combination with evolutionary optimization (GA) for representing undefined biological functions which is the case for common industrial processes involving biological moieties.

Artificial Intelligence vs. Statistical Modeling and Optimization of Continuous Bead Milling Process for Bacterial Cell Lysis

PubMed Central

Haque, Shafiul; Khan, Saif; Wahid, Mohd; Dar, Sajad A.; Soni, Nipunjot; Mandal, Raju K.; Singh, Vineeta; Tiwari, Dileep; Lohani, Mohtashim; Areeshi, Mohammed Y.; Govender, Thavendran; Kruger, Hendrik G.; Jawed, Arshad

2016-01-01

For a commercially viable recombinant intracellular protein production process, efficient cell lysis and protein release is a major bottleneck. The recovery of recombinant protein, cholesterol oxidase (COD) was studied in a continuous bead milling process. A full factorial response surface methodology (RSM) design was employed and compared to artificial neural networks coupled with genetic algorithm (ANN-GA). Significant process variables, cell slurry feed rate (A), bead load (B), cell load (C), and run time (D), were investigated and optimized for maximizing COD recovery. RSM predicted an optimum of feed rate of 310.73 mL/h, bead loading of 79.9% (v/v), cell loading OD600 nm of 74, and run time of 29.9 min with a recovery of ~3.2 g/L. ANN-GA predicted a maximum COD recovery of ~3.5 g/L at an optimum feed rate (mL/h): 258.08, bead loading (%, v/v): 80%, cell loading (OD600 nm): 73.99, and run time of 32 min. An overall 3.7-fold increase in productivity is obtained when compared to a batch process. Optimization and comparison of statistical vs. artificial intelligence techniques in continuous bead milling process has been attempted for the very first time in our study. We were able to successfully represent the complex non-linear multivariable dependence of enzyme recovery on bead milling parameters. The quadratic second order response functions are not flexible enough to represent such complex non-linear dependence. ANN being a summation function of multiple layers are capable to represent complex non-linear dependence of variables in this case; enzyme recovery as a function of bead milling parameters. Since GA can even optimize discontinuous functions present study cites a perfect example of using machine learning (ANN) in combination with evolutionary optimization (GA) for representing undefined biological functions which is the case for common industrial processes involving biological moieties. PMID:27920762

Serum ceruloplasmin protein expression and activity increases in iron-deficient rats and is further enhanced by higher dietary copper intake

PubMed Central

Ranganathan, Perungavur N.; Lu, Yan; Jiang, Lingli; Kim, Changae

2011-01-01

Increases in serum and liver copper content are noted during iron deficiency in mammals, suggesting that copper-dependent processes participate during iron deprivation. One point of intersection between the 2 metals is the liver-derived, multicopper ferroxidase ceruloplasmin (Cp) that is important for iron release from certain tissues. The current study sought to explore Cp expression and activity during physiologic states in which hepatic copper loading occurs (eg, iron deficiency). Weanling rats were fed control or low iron diets containing low, normal, or high copper for ∼ 5 weeks, and parameters of iron homeostasis were measured. Liver copper increased in control and iron-deficient rats fed extra copper. Hepatic Cp mRNA levels did not change; however, serum Cp protein was higher during iron deprivation and with higher copper consumption. In-gel and spectrophotometric ferroxidase and amine oxidase assays demonstrated that Cp activity was enhanced when hepatic copper loading occurred. Interestingly, liver copper levels strongly correlated with Cp protein expression and activity. These observations support the possibility that liver copper loading increases metallation of the Cp protein, leading to increased production of the holo enzyme. Moreover, this phenomenon may play an important role in the compensatory response to maintain iron homeostasis during iron deficiency. PMID:21768302

Accessory enzymes influence cellulase hydrolysis of the model substrate and the realistic lignocellulosic biomass.

PubMed

Sun, Fubao Fuebiol; Hong, Jiapeng; Hu, Jinguang; Saddler, Jack N; Fang, Xu; Zhang, Zhenyu; Shen, Song

2015-11-01

The potential of cellulase enzymes in the developing and ongoing "biorefinery" industry has provided a great motivation to develop an efficient cellulase mixture. Recent work has shown how important the role that the so-called accessory enzymes can play in an effective enzymatic hydrolysis. In this study, three newest Novozymes Cellic CTec cellulase preparations (CTec 1/2/3) were compared to hydrolyze steam pretreated lignocellulosic substrates and model substances at an identical FPA loading. These cellulase preparations were found to display significantly different hydrolytic performances irrelevant with the FPA. And this difference was even observed on the filter paper itself when the FPA based assay was revisited. The analysis of specific enzyme activity in cellulase preparations demonstrated that different accessory enzymes were mainly responsible for the discrepancy of enzymatic hydrolysis between diversified substrates and various cellulases. Such the active role of accessory enzymes present in cellulase preparations was finally verified by supplementation with β-glucosidase, xylanase and lytic polysaccharide monooxygenases AA9. This paper provides new insights into the role of accessory enzymes, which can further provide a useful reference for the rational customization of cellulase cocktails in order to realize an efficient conversion of natural lignocellulosic substrates. Copyright © 2015 Elsevier Inc. All rights reserved.

Enhanced cell adhesion on bioinert ceramics mediated by the osteogenic cell membrane enzyme alkaline phosphatase.

PubMed

Aminian, Alieh; Shirzadi, Bahareh; Azizi, Zahra; Maedler, Kathrin; Volkmann, Eike; Hildebrand, Nils; Maas, Michael; Treccani, Laura; Rezwan, Kurosch

2016-12-01

Functional bone and dental implant materials are required to guide cell response, offering cues that provide specific instructions to cells at the implant/tissue interface while maintaining full biocompatibility as well as the desired structural requirements and functions. In this work we investigate the influence of covalently immobilized alkaline phosphatase (ALP), an enzyme involved in bone mineralization, on the first contact and initial cell adhesion. To this end, ALP is covalently immobilized by carbodiimide-mediated chemoligation on two highly bioinert ceramics, alpha-alumina (Al2O3) and yttria-stabilized zirconia (Y-TZP) that are well-established for load-bearing applications. The physicochemical surface properties are evaluated by profilometry, zeta potential and water contact angle measurements. The initial cell adhesion of human osteoblasts (HOBs), human osteoblast-like cells (MG-63) and mesenchymal stromal cells (hMSCs) was investigated. Cell adhesion was assessed at serum free condition via quantification of percentage of adherent cells, adhesion area and staining of the focal adhesion protein vinculin. Our findings show that after ALP immobilization, the Al2O3 and Y-TZP surfaces gained a negative charge and their hydrophilicity was increased. In the presence of surface-immobilized ALP, a higher cell adhesion, more pronounced cell spreading and a higher number of focal contact points were found. Thereby, this work gives evidence that surface functionalization with ALP can be utilized to modify inert materials for biological conversion and faster bone regeneration on inert and potentially load-bearing implant materials. Copyright © 2016 Elsevier B.V. All rights reserved.

Enhanced cellulase recovery without β-glucosidase supplementation for cellulosic ethanol production using an engineered strain and surfactant.

PubMed

Huang, Renliang; Guo, Hong; Su, Rongxin; Qi, Wei; He, Zhimin

2017-03-01

Recycling cellulases by substrate adsorption is a promising strategy for reducing the enzyme cost of cellulosic ethanol production. However, β-glucosidase has no carbohydrate-binding module (CBM). Thus, additional enzymes are required in each cycle to achieve a high ethanol yield. In this study, we report a new method of recycling cellulases without β-glucosidase supplementation using lignocellulosic substrate, an engineered strain expressing β-glucosidase and Tween 80. The cellulases and Tween 80 were added to an aqueous suspension of diluted sulfuric acid/ammonia-treated corncobs in a simultaneous saccharification and fermentation (SSF) process for ethanol production. Subsequently, the addition of fresh pretreated corncobs to the fermentation liquor and remaining solid residue provided substrates with absorbed cellulases for the next SSF cycle. This method provided excellent ethanol production in three successive SSF cycles without requiring the addition of new cellulases. For a 10% (w/v) solid loading, a cellulase dosage of 30 filter paper units (FPU)/g cellulose, 0.5% Tween 80, and 2 g/L of the engineered strain, approximately 90% of the initial ethanol concentration from the first SSF process was obtained in the next two SSF processes, with a total ethanol production of 306.27 g/kg corncobs and an enzyme productivity of 0.044 g/FPU. Tween 80 played an important role in enhancing cellulase recovery. This new enzyme recycling method is more efficient and practical than other reported methods. Biotechnol. Bioeng. 2017;114: 543-551. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Sulfhydryl oxidases: emerging catalysts of protein disulfide bond formation in eukaryotes.

PubMed

Thorpe, Colin; Hoober, Karen L; Raje, Sonali; Glynn, Nicole M; Burnside, Joan; Turi, George K; Coppock, Donald L

2002-09-01

Members of the Quiescin-sulfhydryl oxidase (QSOX) family utilize a thioredoxin domain and a small FAD-binding domain homologous to the yeast ERV1p protein to oxidize sulfhydryl groups to disulfides with the reduction of oxygen to hydrogen peroxide. QSOX enzymes are found in all multicellular organisms for which complete genomes exist and in Trypanosoma brucei, but are not found in yeast. The avian QSOX is the best understood enzymatically: its preferred substrates are peptides and proteins, not monothiols such as glutathione. Mixtures of avian QSOX and protein disulfide isomerase catalyze the rapid insertion of the correct disulfide pairings in reduced RNase. Immunohistochemical studies of human tissues show a marked and highly localized concentration of QSOX in cell types associated with heavy secretory loads. Consistent with this role in the formation of disulfide bonds, QSOX is typically found in the cell in the endoplasmic reticulum and Golgi and outside the cell. In sum, this review suggests that QSOX enzymes play a significant role in oxidative folding of a large variety of proteins in a wide range of multicellular organisms.

Hydrolysis-acidogenesis of food waste in solid-liquid-separating continuous stirred tank reactor (SLS-CSTR) for volatile organic acid production.

PubMed

Karthikeyan, Obulisamy Parthiba; Selvam, Ammaiyappan; Wong, Jonathan W C

2016-01-01

The use of conventional continuous stirred tank reactor (CSTR) can affect the methane (CH4) recovery in a two-stage anaerobic digestion of food waste (FW) due to carbon short circuiting in the hydrolysis-acidogenesis (Hy-Aci) stage. In this research, we have designed and tested a solid-liquid-separating CSTR (SLS-CSTR) for effective Hy-Aci of FW. The working conditions were pH 6 and 9 (SLS-CSTR-1 and -2, respectively); temperature-37°C; agitation-300rpm; and organic loading rate (OLR)-2gVSL(-1)day(-1). The volatile fatty acids (VFA), enzyme activities and bacterial population (by qPCR) were determined as test parameters. Results showed that the Hy-Aci of FW at pH 9 produced ∼35% excess VFA as compared to that at pH 6, with acetic and butyric acids as major precursors, which correlated with the high enzyme activities and low lactic acid bacteria. The design provided efficient solid-liquid separation there by improved the organic acid yields from FW. Copyright © 2015 Elsevier Ltd. All rights reserved.

Single cell oil production by Mortierella isabellina from steam exploded corn stover degraded by three-stage enzymatic hydrolysis in the context of on-site enzyme production.

PubMed

Fang, Hao; Zhao, Chen; Chen, Shaolin

2016-09-01

Single cell oil (SCO), promising as alternative oil source, was produced from steam exploded corn stover (SECS) by Mortierella isabellina. Different bioprocesses from SECS to SCO were compared and the bioprocess C using the three-stage enzymatic hydrolysis was found to be the most efficient one. The bioprocess C used the lowest enzyme input 20FPIU cellulase/g glucan and the shortest time 222h, but produced 44.94g dry cell biomass and 25.77g lipid from 327.63g dry SECS. It had the highest lipid content 57.34%, and its productivities and yields were much higher than those of the bioprocess B and comparable to the bioprocess A, indicating that the three-stage enzymatic hydrolysis could greatly improve the efficiency of the bioprocess from high solid loading SECS to SCO by Mortierella isabellina. This work testified the application value of three-stage enzymatic hydrolysis in lignocellulose-based bioprocesses. Copyright © 2016 Elsevier Ltd. All rights reserved.

Multi-step approach to add value to corncob: Production of biomass-degrading enzymes, lignin and fermentable sugars.

PubMed

Michelin, Michele; Ruiz, Héctor A; Polizeli, Maria de Lourdes T M; Teixeira, José A

2018-01-01

This work presents an integrated and multi-step approach for the recovery and/or application of the lignocellulosic fractions from corncob in the production of high value added compounds as xylo-oligosaccharides, enzymes, fermentable sugars, and lignin in terms of biorefinery concept. For that, liquid hot water followed by enzymatic hydrolysis were used. Liquid hot water was performed using different residence times (10-50min) and holding temperature (180-200°C), corresponding to severities (log(R 0 )) of 3.36-4.64. The most severe conditions showed higher xylo-oligosaccharides extraction (maximum of 93%) into the hydrolysates and higher recovery of cellulose on pretreated solids (maximum of 65%). Subsequently, hydrolysates and solids were used in the production of xylanases and cellulases, respectively, as well as, pretreated solids were also subjected to enzymatic hydrolysis for the recovery of lignin and fermentable sugars from cellulose. Maximum glucose yield (100%) was achieved for solids pretreated at log(R 0 ) of 4.42 and 5% solid loading. Copyright © 2017 Elsevier Ltd. All rights reserved.

Purification of pectinase from mango (Mangifera indica L. cv. Chokanan) waste using an aqueous organic phase system: a potential low cost source of the enzyme.

PubMed

Amid, Mehrnoush; Abdul Manap, Mohd Yazid; Mustafa, Shuhaimi

2013-07-15

As a novel method of purification, an aqueous organic phase system (AOPS) was employed to purify pectinase from mango waste. The effect of different parameters, such as the alcohol concentration (ethanol, 1-propanol, and 2-propanol), the salt type and concentration (ammonium sulfate, potassium phosphate and sodium citrate), the feed stock crude load, the aqueous phase pH and NaCl concentration, were investigated in the recovery of pectinase from mango peel. The partition coefficient (K), selectivity (S), purification factor (PF) and yield (Y, %) were investigated in this study as important parameters for the evaluation of enzyme recovery. The desirable partition efficiency for pectinase purification was achieved in an AOPS of 19% (w/w) ethanol and 22% (w/w) potassium phosphate in the presence of 5% (w/w) NaCl at pH 7.0. Based on the system, the purification factor of pectinase was enhanced 11.7, with a high yield of 97.1%. Copyright © 2013 Elsevier B.V. All rights reserved.

Biodegradation of polycyclic aromatic hydrocarbons (PAHs) by laccase from Trametes versicolor covalently immobilized on amino-functionalized SBA-15.

PubMed

Bautista, Luis Fernando; Morales, Gabriel; Sanz, Raquel

2015-10-01

A covalent immobilization method based on glutaraldehyde and amino-functionalized SBA-15 supports has been successfully applied to covalently and stably immobilize laccase from Trametes versicolor. The resultant biocatalysts displayed high incorporation yields of enzyme and led to excellent biodegradation rates of selected HPAs models, i.e. naphthalene, phenanthrene and anthracene, in water. The nature of the hydrocarbon chain accompanying the amino group has been shown as determinant for the immobilization as well as for the activity and reusability of the materials. Thus, alkyl moieties displayed higher enzyme loadings than phenyl moieties, being more adequate the larger n-butyl tethering residue likely due to its higher mobility. Using the aminobutyl-based laccase-SBA-15, 82%, 73%, and 55% conversion of naphthalene, phenanthrene and anthracene, respectively, were achieved after 48 h, very close to the values obtained with free laccase under the same reaction conditions. On the other hand, aminopropyl-based laccase-SBA-15 biocatalysts displayed the best reusability properties, retaining higher activity after four repeated uses than the corresponding aminobutyl-based materials. Copyright © 2015 Elsevier Ltd. All rights reserved.

Structured Mono- and Diacylglycerols with a High Content of Medium Chain Fatty Acids.

PubMed

Esperón-Rojas, Alaina A; Baeza-Jiménez, R; Cano-Sarmiento, Cynthia; García, Hugo S

2017-09-01

In the present work, direct enzyme-catalysed esterification of medium chain fatty acids (MCFA) from three different sources (Medium chain triacylglycerols, MCT; saponified MCT and a mixture of free MCFA) was evaluated to obtain structured mono- and diacylglycerols. The esterifications were carried out mixing the different sources of MCFA with glycerol at two weight ratios (1:1 and 4:1, w/w), using three immobilized lipases: Novozym 435, Lipozyme RM IM and Lipozyme TL IM; different reaction times (t = 0, 15, 30, 60, 120 min); enzyme loadings (5, 10 or 15% with respect to the total weight of substrates). The extent of esterification was determined by gas chromatography (GC) analysis of the acylglycerols produced. The highest incorporation of free MCFA into glycerol was obtained for a 1:1 (w/w) glycerol to free MCFA ratio, 5% of Novozym 435, at 50°C, 300 rpm, 10% of molecular sieves and a commercial MCFA mixture as starting material. Under these conditions, incorporation of at least 90% of MCFA into glycerol was achieved after 30 min of reaction.

Ethanol/water pulps from sugar cane straw and their biobleaching with xylanase from Bacillus pumilus.

PubMed

Moriya, Regina Y; Gonçalves, Adilson R; Duarte, Marta C T

2007-04-01

The influence of independent variables (temperature and time) on the cooking of sugar cane straw with ethanol/water mixtures was studied to determine operating conditions that obtain pulp with high cellulose contents and a low lignin content. An experimental 2(2) design was applied for temperatures of 185 and 215 degrees C, and time of 1 and 2.5 h with the ethanol/water mixture concentration and constant straw-to-solvent ratio. The system was scaled-up at 200 degrees C cooking temperature for 2 h with 50% ethanol-water concentration, and 1:10 (w/v) straw-to-solvent ratio to obtain a pulp with 3.14 cP viscosity, 58.09 kappa-number, and the chemical composition of the pulps were 3.2% pentosan and 31.5% lignin. Xylanase from Bacillus pumilus was then applied at a loading of 5-150 IU/g dry pulp in the sugar cane straw ethanol/water pulp at 50 degrees C for 2 and 20 h. To ethanol/water pulps, the best enzyme dosage was found to be 20 IU/g dry pulp at 20 h, and a high enzyme dosage of 150 IU/g dry pulp did not decrease the kappa-number of the pulp.

Whole cell immobilization of refractory glucose isomerase using tris(hydroxymethyl)phosphine as crosslinker for preparation of high fructose corn syrup at elevated temperature.

PubMed

Jia, Dong-Xu; Wang, Teng; Liu, Zi-Jian; Jin, Li-Qun; Li, Jia-Jia; Liao, Cheng-Jun; Chen, De-Shui; Zheng, Yu-Guo

2018-04-04

Glucose isomerase (GI) responsible for catalyzing the isomerization from d-glucose to d-fructose, was an important enzyme for producing high fructose corn syrup (HFCS). In a quest to prepare HFCS at elevated temperature and facilitate enzymatic recovery, an effective procedure for whole cell immobilization of refractory Thermus oshimai glucose isomerase (ToGI) onto Celite 545 using tris(hydroxymethyl)phosphine (THP) as crosslinker was established. The immobilized biocatalyst showed an activity of approximate 127.3 U/(g·immobilized product) via optimization in terms of cells loading, crosslinker concentration and crosslinking time. The pH optimum of the immobilized biocatalyst was displaced from pH 8.0 of native enzyme to neutral pH 7.0. Compared with conventional glutaraldehyde (GLU)-immobilized cells, it possessed the enhanced thermostability with 70.1% residual activity retaining after incubation at 90°C for 72 h. Moreover, the THP-immobilized biocatalyst exhibited superior operational stability, in which it retained 85.8% of initial activity after 15 batches of bioconversion at 85°C. This study paved a way for reducing catalysis cost for upscale preparation of HFCS with higher d-fructose concentration. Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Studies on acute toxic effects to keratinocytes induced by hematoporphyrin derivatives and laser light.

PubMed

Artuc, M; Ramshad, M; Kappus, H

1989-01-01

Human epidermal keratinocytes were grown in culture and the uptake of hematoporphyrin derivatives (HPDs) used in photodynamic therapy was estimated. Keratinocytes loaded with HPDs were irradiated with laser light of 632 nm generated by a helium-neon laser and cell toxicity was determined by the trypan blue exclusion test and the measurement of enzyme release. With increasing intracellular concentration of HPDs and with increasing intensity of the laser light, an increasing number of cells took up trypan blue and released the cytosolic enzyme lactate dehydrogenase and the lysosomal enzyme acid phosphatase after 1 h incubation of the irradiated cells at 37 degrees C. Cytotoxicity was less pronounced when the irradiated cells were incubated at 0 degree C indicating the involvement of enzyme reactions in cell death. No lipid peroxidation as measured by malondialdehyde and ethane formation was detectable. Our results suggest that during photodynamic therapy with HPDs and laser light epidermal keratinocytes may be seriously damaged. The data indicate that not lipid peroxidation but rather the activation of lysosomal enzymes is responsible for the cytotoxicity observed.

Immobilization of lipase and keratinase on functionalized SBA-15 nanostructured materials

NASA Astrophysics Data System (ADS)

Le, Hy G.; Vu, Tuan A.; Tran, Hoa T. K.; Dang, Phuong T.

2013-12-01

SBA-15 nanostructured materials were synthesized via hydrothermal treatment and were functionalized with 3- aminopropyltriethoxysilane (APTES). The obtained samples were characterized by different techniques such as XRD, BET, TEM, IR and DTA. After functionalization, it showed that these nanostrucrured materials still maintained the hexagonal pore structure of the parent SBA-15. The model enzyms chosen in this study were lipase and keratinase. Lipase was a biocatalyst for hydrolyzation of long chain triglycerides or methyl esters of long chain alcohols and fatty acids; keratinase is a proteolytic enzyme that catalyzes the cleavage of keratin. The functionalized SBA-15 materials were used to immobilize lipase and keratinase, exhibiting higher activity than that of the unfunctionalized pure silica SBA-15 ones. This might be due to the enhancing of surface hydrophobicity upon functionalization. The surface functionalization of the nanostructured silicas with organic groups can favor the interaction between enzyme and the supports and consequently increasing the operational stability of the immobilized enzymes. The loading of lipase on functionalized SBA-15 materials was higher than that of keratinase. This might be rationalized by the difference in size of enzyms.

Performance comparison of new generation HCV core antigen test versus HCV RNA test in management of hepatitis C virus infection.

PubMed

Çetiner, Salih; Çetin Duran, Alev; Kibar, Filiz; Yaman, Akgün

2017-06-01

The study has evaluated the performance of HCV core antigen (Cag) test by comparing HCV RNA PCR assay which is considered the gold standard for management of HCV infection. Totally, 132 samples sent for HCV RNA (real-time PCR) test were included in the study. Anti-HCV antibody test and HCV Cag test were performed by chemiluminescent enzyme immunoassay (CMEI). Anti-HCV test was positive in all samples. HCV RNA was detected in 112/132 (84.8%) samples, and HCV Cag in 105/132 (79.5%). The most common HCV genotype was genotype 1 (86%). Considering the HCV RNA test as gold standard; the sensitivity, specificity, positive predictive value, negative predictive value and accuracy of Cag test were found to be 93.75%, 100%, 100%, 74.07% and 94.69%, respectively, and paired test results were detected as highly concordant. A high level of correlation was seen between HCV RNA and Cag tests, however, the concordance between the two tests appeared to be disrupted at viral loads lower than 10 3 IU/mL. On the contrary, the correlation reached significance for the values higher than 10 3 IU/mL. Viral loads were in the 17-2500IU/mL range for the negative results for Cag test. Pearson's correlation coefficient revealed a considerably high correlation. The concordance between HCV RNA and Cag tests was disrupted under a viral load lower than 10 3 IU/mL. Therefore, it would be appropriate to consider cost effectiveness, advantages and limitations of the HCV RNA and Cag tests during the decision on which method to use for patient management. Copyright © 2017 Elsevier Ltd. All rights reserved.

Characterisation of bare and tannase-loaded calcium alginate beads by microscopic, thermogravimetric, FTIR and XRD analyses.

PubMed

Larosa, Claudio; Salerno, Marco; de Lima, Juliana Silva; Merijs Meri, Remo; da Silva, Milena Fernandes; de Carvalho, Luiz Bezerra; Converti, Attilio

2018-08-01

Incorporating enzymes into calcium alginate beads is an effective method to immobilise them and to preserve, at the same time, their catalytic activity. Sodium alginate was mixed with Aspergillus ficuum tannase in aqueous solution, and tannase-loaded calcium alginate beads were prepared using a simple droplet-based microfluidic system. Extensive experimental analysis was carried out to characterise the samples. Microscopic imaging revealed morphological differences between the surfaces of bare alginate matrix and tannase-loaded alginate beads. Thermal analysis allowed assessing the hydration contents of alginate and revealed the presence of tannase entrapped in the loaded beads, which was confirmed by vibrational spectroscopy. X-ray diffraction allowed us to conclude that alginate of tannase-loaded beads is not crystalline, which would make them suitable as carriers for possible controlled release. Moreover, they could be used in food applications to improve tea quality or clarify juices. Copyright © 2018 Elsevier B.V. All rights reserved.

Bioelectrochemistry of non-covalent immobilized alcohol dehydrogenase on oxidized diamond nanoparticles.

PubMed

Nicolau, Eduardo; Méndez, Jessica; Fonseca, José J; Griebenow, Kai; Cabrera, Carlos R

2012-06-01

Diamond nanoparticles are considered a biocompatible material mainly due to their non-cytotoxicity and remarkable cellular uptake. Model proteins such as cytochrome c and lysozyme have been physically adsorbed onto diamond nanoparticles, proving it to be a suitable surface for high protein loading. Herein, we explore the non-covalent immobilization of the redox enzyme alcohol dehydrogenase (ADH) from Saccharomyces cerevisiae (E.C.1.1.1.1) onto oxidized diamond nanoparticles for bioelectrochemical applications. Diamond nanoparticles were first oxidized and physically characterized by X-ray diffraction (XRD), FT-IR and TEM. Langmuir isotherms were constructed to investigate the ADH adsorption onto the diamond nanoparticles as a function of pH. It was found that a higher packing density is achieved at the isoelectric point of the enzyme. Moreover, the relative activity of the immobilized enzyme on diamond nanoparticles was addressed under optimum pH conditions able to retain up to 70% of its initial activity. Thereafter, an ethanol bioelectrochemical cell was constructed by employing the immobilized alcohol dehydrogenase onto diamond nanoparticles, this being able to provide a current increment of 72% when compared to the blank solution. The results of this investigation suggest that this technology may be useful for the construction of alcohol biosensors or biofuel cells in the near future. Copyright © 2011 Elsevier B.V. All rights reserved.

Diffusion Coefficients of Endogenous Cytosolic Proteins from Rabbit Skinned Muscle Fibers

PubMed Central

Carlson, Brian E.; Vigoreaux, Jim O.; Maughan, David W.

2014-01-01

Efflux time courses of endogenous cytosolic proteins were obtained from rabbit psoas muscle fibers skinned in oil and transferred to physiological salt solution. Proteins were separated by gel electrophoresis and compared to load-matched standards for quantitative analysis. A radial diffusion model incorporating the dissociation and dissipation of supramolecular complexes accounts for an initial lag and subsequent efflux of glycolytic and glycogenolytic enzymes. The model includes terms representing protein crowding, myofilament lattice hindrance, and binding to the cytomatrix. Optimization algorithms returned estimates of the apparent diffusion coefficients, D(r,t), that were very low at the onset of diffusion (∼10−10 cm2 s−1) but increased with time as cytosolic protein density, which was initially high, decreased. D(r,t) at later times ranged from 2.11 × 10−7 cm2 s−1 (parvalbumin) to 0.20 × 10−7 cm2 s−1 (phosphofructose kinase), values that are 3.6- to 12.3-fold lower than those predicted in bulk water. The low initial values are consistent with the presence of complexes in situ; the higher later values are consistent with molecular sieving and transient binding of dissociated proteins. Channeling of metabolic intermediates via enzyme complexes may enhance production of adenosine triphosphate at rates beyond that possible with randomly and/or sparsely distributed enzymes, thereby matching supply with demand. PMID:24559981

DENITRIFICATION ENZYME ACTIVITY OF FRINGE SALT MARSHES IN NEW ENGLAND (USA)

EPA Science Inventory

Coastal salt marshes are a buffer between the uplands and adjacent coastal waters in New England (USA). With increasing N loads from developed watersheds, salt marshes could play an important role in the water quality maintenance of coastal waters. In this study we examined seaso...

Effects of Macroporous Resin Size on Candida antarctica Lipase B Adsorption, Fraction of Active Molecules, and Catalytic Activity for Polyester Synthesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen,B.; Miller, E.; Miller, L.

2007-01-01

Methyl methacrylate resins with identical average pore diameter (250 {angstrom}) and surface area (500 m{sup 2}/g) but with varied particle size (35 to 560-710 {mu}m) were employed to study how immobilization resin particle size influences Candida antarctica Lipase B (CALB) loading, fraction of active sites, and catalytic properties for polyester synthesis. CALB adsorbed more rapidly on smaller beads. Saturation occurred in less than 30 s and 48 h for beads with diameters 35 and 560-710 {mu}m, respectively. Linearization of adsorption isotherm data by the Scatchard analysis showed for the 35 {mu}m resin that: (1) CALB loading at saturation was wellmore » below that required to form a monolayer and fully cover the support surface and (2) CALB has a high affinity for this resin surface. Infrared microspectroscopy showed that CALB forms protein loading fronts for resins with particle sizes 560-710 and 120 {mu}m. In contrast, CALB appears evenly distributed throughout 35 {mu}m resins. By titration with p-nitrophenyl n-hexyl phosphate (MNPHP), the fraction of active CALB molecules adsorbed onto resins was <50% which was not influenced by particle size. The fraction of active CALB molecules on the 35 {mu}m support increased from 30 to 43% as enzyme loading was increased from 0.9 to 5.7% (w/w) leading to increased activity for {epsilon}-caprolactone ({epsilon}-CL) ring-opening polymerization. At about 5% w/w CALB loading, by decreasing the immobilization support diameter from 560-710 to 120, 75, and 35 {mu}m, conversion of {epsilon}-CL % to polyester increased (20 to 36, 42, and 61%, respectively, at 80 min). Similar trends were observed for condensation polymerizations between 1,8-octanediol and adipic acid.« less

Enzymatic hydrolysis of autohydrolyzed wheat straw followed by refining to produce fermentable sugars.

PubMed

Ertas, Murat; Han, Qiang; Jameel, Hasan; Chang, Hou-min

2014-01-01

Wheat straw was pretreated using an autohydrolysis process with different temperatures (160-200 °C) and times (10-20 min) in order to allow the recovery of hemicellulose in the filtrate and help open up the structure of the biomass for improved accessibility of enzymes during enzymatic hydrolysis. Autohydrolysis at 190 °C for 10 min provided the highest overall sugar (12.2/100g raw wheat straw) in the autohydrolysis filtrate and recovered 62.3% of solid residue. Before enzymatic hydrolysis, the pulps obtained from each pretreatment condition were subjected to a refining post-treatment to improve enzyme accessibility. Enzymatic hydrolysis was performed for all the pretreated solids with and without refining post-treatment at the enzyme loadings of 4 and 10 FPU/g oven dry substrate for 96 h. A total of 30.4 g sugars can be recovered from 100g wheat straw at 180 °C for 20 min with 4 FPU/g enzyme charge. Copyright © 2013 Elsevier Ltd. All rights reserved.

Tissue-specific biomass recalcitrance in corn stover pretreated with liquid hot-water: enzymatic hydrolysis (part 1).

PubMed

Zeng, Meijuan; Ximenes, Eduardo; Ladisch, Michael R; Mosier, Nathan S; Vermerris, Wilfred; Huang, Chia-Ping; Sherman, Debra M

2012-02-01

Lignin content, composition, distribution as well as cell wall thickness, structures, and type of tissue have a measurable effect on enzymatic hydrolysis of cellulose in lignocellulosic feedstocks. The first part of our work combined compositional analysis, pretreatment and enzyme hydrolysis for fractionated pith, rind, and leaf tissues from a hybrid stay-green corn, in order to identify the role of structural characteristics on enzyme hydrolysis of cell walls. The extent of enzyme hydrolysis follows the sequence rind < leaves < pith with 90% conversion of cellulose to glucose in 24 h in the best cases. Physical fractionation of corn stalks or other C(4) grasses into soft and hard tissue types could reduce cost of cellulose conversion by enabling reduced enzyme loadings to hydrolyze soft tissue, and directing the hard tissue to other uses such as thermal processing, combustion, or recycle to the land from which the corn was harvested. Copyright © 2011 Wiley Periodicals, Inc.

Two New Preyssler-Type Polyoxometalate-Based Coordination Polymers and Their Application in Horseradish Peroxidase Immobilization.

PubMed

Du, Jing; Cao, Mei-Da; Feng, Shu-Li; Su, Fang; Sang, Xiao-Jing; Zhang, Lan-Cui; You, Wan-Sheng; Yang, Mei; Zhu, Zai-Ming

2017-10-17

Enzyme immobilization is of increasing importance for biocatalysis, for which good supports are critical. Herein, two new Preyssler-type polyoxometalate (POM)-based coordination polymers, namely, {[Cu(H 2 biim) 2 ][{Cu(H 2 biim) 2 (μ-H 2 O)} 2 Cu(H 2 biim)(H 2 O) 2 ]H[({Cu(H 2 biim)(H 2 O) 2 } 0.5 ) 2 ((μ-C 3 HN 2 Cl 2 ){Cu(H 2 biim)} 2 ){Z(H 2 O)P 5 W 30 O 110 }]⋅x H 2 O} n (1: Z=Na, x=9; 2: Z=Ag, x=10; H 2 biim=2,2'-biimidazole) were designed and synthesized. Compounds 1 and 2 exhibit the same skeletons, which contain multiple Cu II complex fragments and penta-supported {ZP 5 W 30 } (Z=Na, Ag) clusters. They were first employed to immobilize horseradish peroxidase (HRP). Results show that compounds 1 and 2 are good supports for HRP immobilization, and exhibit higher enzyme loading, lower loading times, and excellent reusability. The immobilized HRP (HRP/1 or HRP/2) was further applied to detect H 2 O 2 , and good sensitivity, wide linear range, low detection limit, and fast response were achieved. This work shows that POM-based hybrid materials are a new kind of promising support for enzyme immobilization. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Pretreatment of lignocellulose: Formation of inhibitory by-products and strategies for minimizing their effects.

PubMed

Jönsson, Leif J; Martín, Carlos

2016-01-01

Biochemical conversion of lignocellulosic feedstocks to advanced biofuels and other commodities through a sugar-platform process involves a pretreatment step enhancing the susceptibility of the cellulose to enzymatic hydrolysis. A side effect of pretreatment is formation of lignocellulose-derived by-products that inhibit microbial and enzymatic biocatalysts. This review provides an overview of the formation of inhibitory by-products from lignocellulosic feedstocks as a consequence of using different pretreatment methods and feedstocks as well as an overview of different strategies used to alleviate problems with inhibitors. As technologies for biorefining of lignocellulose become mature and are transferred from laboratory environments to industrial contexts, the importance of management of inhibition problems is envisaged to increase as issues that become increasingly relevant will include the possibility to use recalcitrant feedstocks, obtaining high product yields and high productivity, minimizing the charges of enzymes and microorganisms, and using high solids loadings to obtain high product titers. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Phloem loading--not metaphysical, only complex: towards a unified model of phloem loading.

PubMed

Komor, E; Orlich, G; Weig, A; Köckenberger, W

1996-08-01

Phloem loading comprises the entire pathway of phloem-mobile solutes from their place of generation (or delivery) to the sieve tubes in a sequence of transport steps across or passing by several different cell types. Each of these steps can be classified as symplastic or apoplastic. The detailed anatomical-cytological work in the past ten years made clear that the symplastic continuity from mesophyll to sieve tubes may be very different for different plant species or even in different vein orders. Therefore data from one species are not transferable to another species and a well-rounded picture involving different experimental methods has to be aimed at for each species separately. The information obtained with the Ricinus seedling, where phloem loading and sieve tube sap analysis can be achieved relatively easily, is presented. The analysis of the radioactive labelling of sucrose from the sieve tubes of cotyledons, in which external and intracellular sucrose had been differently labelled, revealed that at sucrose concentrations close to the natural one, 50% of sucrose is loaded directly from the external medium. The other 50% is first taken up by mesophyll and then released for uptake into the sieve tubes. No bundle tissue works as obligate, intermediate sucrose storage. The apoplast therefore definitely serves as a transit reservoir for sucrose destined to be loaded into the sieve tubes. The sieve tube sap contains glycolytic metabolites at concentrations higher than found in the hypocotyl tissue, whereas the corresponding glycolytic enzymes are missing. It is concluded that the enzymes are sequestered in the companion cell or by parietal membrane stacks. Not only the sieve tubes but nearly all cotyledonary cells are equipped with a sucrose-H(+) symporter able to achieve sucrose accumulation and sensitive to inhibition by high salt concentrations or SH reagents. A cDNA clone coding for a sucrose carrier was isolated. It is transcribed at approximately the same level in most organs of the seedling and throughout the germination period. Leaves of adult Ricinus have significantly lower levels of this transcript. Recirculation of excess, phloem-delivered solutes from the sink back to the source is shown not only to be a common feature of long-distance transport, but the only way that an imbalance between supply to and consumption of nutrients in the sink can be adjusted in the source. It is a pathway by which sink activity regulates phloem loading. Non-invasive NMR imaging revealed the flow rates and flow speeds in phloem and xylem in the intact seedling and proved directly the existence of an internal circulating solution flow. A unified model of phloem loading is proposed, based on a pump-and-leak model, where active sucrose carriers (and other carriers) accumulate solutes in the sieve tubes with a concomitant build-up of pressure resulting in mass flow. Plasmodesmata are leaks (as are the transport carriers, too), slowing down the transport rate, but they also serve as diffusion channels for substances which are produced in the neighbouring cell. Therefore, compounds, which are not made in the sieve tubes themselves are translocated together with the bulk solution of sieve tube sap.

Pancreatic enzyme replacement therapy for pancreatic exocrine insufficiency in the 21(st) century.

PubMed

Trang, Tony; Chan, Johanna; Graham, David Y

2014-09-07

Restitution of normal fat absorption in exocrine pancreatic insufficiency remains an elusive goal. Although many patients achieve satisfactory clinical results with enzyme therapy, few experience normalization of fat absorption, and many, if not most, will require individualized therapy. Increasing the quantity of lipase administered rarely eliminates steatorrhea but increases the cost of therapy. Enteric coated enzyme microbead formulations tend to separate from nutrients in the stomach precluding coordinated emptying of enzymes and nutrients. Unprotected enzymes mix well and empty with nutrients but are inactivated at pH 4 or below. We describe approaches for improving the results of enzyme therapy including changing to, or adding, a different product, adding non-enteric coated enzymes, (e.g., giving unprotected enzymes at the start of the meal and acid-protected formulations later), use of antisecretory drugs and/or antacids, and changing the timing of enzyme administration. Because considerable lipid is emptied in the first postprandial hour, it is prudent to start therapy with enteric coated microbead prior to the meal so that some enzymes are available during that first hour. Patients with hyperacidity may benefit from adjuvant antisecretory therapy to reduce the duodenal acid load and possibly also sodium bicarbonate to prevent duodenal acidity. Comparative studies of clinical effectiveness of different formulations as well as the characteristics of dispersion, emptying, and dissolution of enteric-coated microspheres of different diameter and density are needed; many such studies have been completed but not yet made public. We discuss the history of pancreatic enzyme therapy and describe current use of modern preparations, approaches to overcoming unsatisfactory clinical responses, as well as studies needed to be able to provide reliably effective therapy.

Pancreatic enzyme replacement therapy for pancreatic exocrine insufficiency in the 21st century

PubMed Central

Trang, Tony; Chan, Johanna; Graham, David Y

2014-01-01

Restitution of normal fat absorption in exocrine pancreatic insufficiency remains an elusive goal. Although many patients achieve satisfactory clinical results with enzyme therapy, few experience normalization of fat absorption, and many, if not most, will require individualized therapy. Increasing the quantity of lipase administered rarely eliminates steatorrhea but increases the cost of therapy. Enteric coated enzyme microbead formulations tend to separate from nutrients in the stomach precluding coordinated emptying of enzymes and nutrients. Unprotected enzymes mix well and empty with nutrients but are inactivated at pH 4 or below. We describe approaches for improving the results of enzyme therapy including changing to, or adding, a different product, adding non-enteric coated enzymes, (e.g., giving unprotected enzymes at the start of the meal and acid-protected formulations later), use of antisecretory drugs and/or antacids, and changing the timing of enzyme administration. Because considerable lipid is emptied in the first postprandial hour, it is prudent to start therapy with enteric coated microbead prior to the meal so that some enzymes are available during that first hour. Patients with hyperacidity may benefit from adjuvant antisecretory therapy to reduce the duodenal acid load and possibly also sodium bicarbonate to prevent duodenal acidity. Comparative studies of clinical effectiveness of different formulations as well as the characteristics of dispersion, emptying, and dissolution of enteric-coated microspheres of different diameter and density are needed; many such studies have been completed but not yet made public. We discuss the history of pancreatic enzyme therapy and describe current use of modern preparations, approaches to overcoming unsatisfactory clinical responses, as well as studies needed to be able to provide reliably effective therapy. PMID:25206255

A practical workshop for generating simple DNA fingerprints of plants.

PubMed

Rouzière, A-S; Redman, J E

2011-01-01

Gel electrophoresis DNA fingerprints offer a graphical and visually appealing illumination of the similarities and differences between DNA sequences of different species and individuals. A polymerase chain reaction (PCR) and restriction digest protocol was designed to give high-school students the opportunity to generate simple fingerprints of plants thereby illustrating concepts and techniques in genetics and molecular biology. Three combinations of primers/restriction enzyme targeting chloroplast DNA were sufficient to generate patterns that enabled visual discrimination of plant species. The protocol was tested with a range of common fruit, vegetable, and herb plants that could be easily cultivated and handled in the laboratory. Toxic or hazardous materials such as ethidium bromide and liquid nitrogen were avoided. The protocol was validated as a university outreach workshop targeted at a group of up to 10 high-school students. In a teaching laboratory, students sampled plants, setup the PCR reaction and restriction digest using microliter pipettes, and loaded the digested samples on an agarose gel. The workshop was structured as 2 × 2.5-hour sessions on separate days. The main challenges stemmed from the speed and accuracy of pipetting, especially at the gel loading stage. Feedback from students was largely positive, with the majority reporting that they had both enjoyed and learnt from the experience. Copyright © 2010 Wiley Periodicals, Inc.

[A Case of Severe Chronic Active Epstein-Barr Virus Infection with Aplastic Anemia and Hepatitis].

PubMed

Lee, Ja In; Lee, Sung Won; Han, Nam Ik; Ro, Sang Mi; Noh, Yong-Sun; Jang, Jeong Won; Bae, Si Hyun; Choi, Jong Young; Yoon, Seung Kew

2016-01-25

Epstein-Barr virus (EBV) causes various acute and chronic diseases. Chronic active EBV infection (CAEBV) is characterized by infectious mononucleosis-like symptoms that persist for more than 6 months with high viral loads in peripheral blood and/or an unusual pattern of anti-EBV antibodies. Severe CAEBV is associated with poor prognosis with severe symptoms, an extremely high EBV-related antibody titer, and hematologic complications that often include hemophagocytic lymphohistiocytosis. However, CAEBV which led to the development of aplastic anemia (AA) has not been reported yet. A 73-year-old woman was admitted to our hospital with intermittent fever, general weakness and elevated liver enzymes. In the serologic test, EBV-related antibody titer was elevated, and real-time quantitative-PCR in peripheral blood showed viral loads exceeding 10(4) copies/μg DNA. Liver biopsy showed characteristic histopathological changes of EBV hepatitis and in situ hybridization with EBV-encoded RNA-1 was positive for EBV. Pancytopenia was detected in peripheral blood, and the bone marrow aspiration biopsy showed hypocellularity with replacement by adipocytes. AA progressed and the patient was treated with prednisolone but deceased 8 months after the diagnosis due to multiple organ failure and opportunistic infection. Herein, we report a rare case of severe CAEBV in an adult patient accompanied by AA and persistent hepatitis.

A Comparison of Simple Rheological Parameters and Simulation Data for Zymomonas mobilis Fermentation Broths with High Substrate Loading in a 3-L Bioreactor

NASA Astrophysics Data System (ADS)

Um, Byung-Hwan; Hanley, Thomas R.

Traditionally, as much as 80% or more of an ethanol fermentation broth is water that must be removed. This mixture is not only costly to separate but also produces a large aqueous stream that must then be disposed of or recycled. Integrative approaches to water reduction include increasing the biomass concentration during fermentation. In this paper, experimental results are presented for the rheological behavior of high-solids enzymatic cellulose hydrolysis and ethanol fermentation for biomass conversion using Solka Floc as the model feedstock. The experimental determination of the viscosity, shear stress, and shear rate relationships of the 10 to 20% slurry concentrations with constant enzyme concentrations are performed with a variable speed rotational viscometer (2.0 to 200 rpm) at 40 °C. The viscosities of enzymatic suspension observed were in range of 0.0418 to 0.0144, 0.233 to 0.0348, and 0.292 to 0.0447 Pa s for shear rates up to 100 reciprocal seconds at 10, 15, and 20% initial solids (w/v), respectively. Computational fluid dynamics analysis of bioreactor mixing demonstrates the change in bioreactor mixing with increasing biomass concentration. The portion-loading method is shown to be effective for processing highsolids slurries.

Using T-RFLP data on denitrifier community composition to inform understanding of denitrification in stream sediments (Invited)

NASA Astrophysics Data System (ADS)

Wang, S.; Somers, K.; Sudduth, E.; Hassett, B.; Bernhardt, E. S.; Urban, D. L.

2010-12-01

We used terminal restriction fragment length polymorphism (T-RFLP), a molecular fingerprinting method, to characterize denitrifier communities in sediments taken from 48 study streams in North Carolina, USA. In addition to characterizing denitrifier communities, we also used denitrification enzyme activity (DEA) assays to measure potential denitrification rates. Due to differences in watershed land-use, study streams covered a gradient of nitrogen and carbon concentrations, as well as a gradient of contaminant loading from stormwater and sanitary sewers. Nitrogen and carbon (i.e., substrate) concentrations are commonly used to make predictions about denitrification rates in streams. Such models do not take into account denitrifier community composition, which may be an important, independent control of denitrification rates, particularly under stressful conditions (e.g., high contaminant loading) that prevent communities from capitalizing on high substrate availability. Our results indicate that substrate availability by itself was a weak predictor of denitrification rates; the same was also true for denitrifier community composition. However, when both factors were incorporated in a multiple regression model, the percent variation explained increased substantially. These findings suggest that T-RFLP, a relatively cost-effective method, can be used to improve our understanding of controls on denitrification rates in streams with varying watershed land-uses.

Characterization of hemicellulase and cellulase from the extremely thermophilic bacterium Caldicellulosiruptor owensensis and their potential application for bioconversion of lignocellulosic biomass without pretreatment.

PubMed

Peng, Xiaowei; Qiao, Weibo; Mi, Shuofu; Jia, Xiaojing; Su, Hong; Han, Yejun

2015-01-01

Pretreatment is currently the common approach for improving the efficiency of enzymatic hydrolysis on lignocellulose. However, the pretreatment process is expensive and will produce inhibitors such as furan derivatives and phenol derivatives. If the lignocellulosic biomass can efficiently be saccharified by enzymolysis without pretreatment, the bioconversion process would be simplified. The genus Caldicellulosiruptor, an obligatory anaerobic and extreme thermophile can produce a diverse set of glycoside hydrolases (GHs) for deconstruction of lignocellulosic biomass. It gives potential opportunities for improving the efficiency of converting native lignocellulosic biomass to fermentable sugars. Both of the extracellular (extra-) and intracellular (intra-) enzymes of C. owensensis cultivated on corncob xylan or xylose had cellulase (including endoglucanase, cellobiohydrolase and β-glucosidase) and hemicellulase (including xylanase, xylosidase, arabinofuranosidase and acetyl xylan esterase) activities. The enzymes of C. owensensis had high ability for degrading hemicellulose of native corn stover and corncob with the conversion rates of xylan 16.7 % and araban 60.0 %. Moreover, they had remarkable synergetic function with the commercial enzyme cocktail Cellic CTec2 (Novoyzmes). When the native corn stover and corncob were respectively, sequentially hydrolyzed by the extra-enzymes of C. owensensis and CTec2, the glucan conversion rates were 31.2 and 37.9 %,which were 1.7- and 1.9-fold of each control (hydrolyzed by CTec2 alone), whereas the glucan conversion rates of the steam-exploded corn stover and corncob hydrolyzed by CTec2 alone on the same loading rate were 38.2 and 39.6 %, respectively. These results show that hydrolysis by the extra-enzyme of C. owensensis made almost the same contribution as steam-exploded pretreatment on degradation of native lignocellulosic biomass. A new process for saccharification of lignocellulosic biomass by sequential hydrolysis is demonstrated in the present research, namely hyperthermal enzymolysis (70-80 °C) by enzymes of C. owensensis followed with mesothermal enzymolysis (50-55 °C) by commercial cellulase. This process has the advantages of no sugar loss, few inhibitors generation and consolidated with sterilization. The enzymes of C. owensensis demonstrated an enhanced ability to degrade the hemicellulose of native lignocellulose. The pretreatment and detoxification steps may be removed from the bioconversion process of the lignocellulosic biomass by using the enzymes from C. owensensis.

Development of a carbonate absorption-based process for post-combustion CO2 capture: The role of biocatalyst to promote CO2 absorption rate

USGS Publications Warehouse

Lu, Y.; Ye, X.; Zhang, Z.; Khodayari, A.; Djukadi, T.

2011-01-01

An Integrated Vacuum Carbonate Absorption Process (IVCAP) for post-combustion carbon dioxide (CO2) capture is described. IVCAP employs potassium carbonate (PC) as a solvent, uses waste or low quality steam from the power plant for CO2 stripping, and employs a biocatalyst, carbonic anhydrase (CA) enzyme, for promoting the CO2 absorption into PC solution. A series of experiments were performed to evaluate the activity of CA enzyme mixed in PC solutions in a stirred tank reactor system under various temperatures, CA dosages, CO2 loadings, CO2 partial pressures, and the presence of major flue gas contaminants. It was demonstrated that CA enzyme is an effective biocatalyst for CO2 absorption under IVCAP conditions. ?? 2011 Published by Elsevier Ltd.

Desorption of Lipases Immobilized on Octyl-Agarose Beads and Coated with Ionic Polymers after Thermal Inactivation. Stronger Adsorption of Polymers/Unfolded Protein Composites.

PubMed

Virgen-Ortíz, Jose J; Pedrero, Sara G; Fernandez-Lopez, Laura; Lopez-Carrobles, Nerea; Gorines, Beatriz C; Otero, Cristina; Fernandez-Lafuente, Roberto

2017-01-05

Lipases from Candida antarctica (isoform B) and Rhizomucor miehei (CALB and RML) have been immobilized on octyl-agarose (OC) and further coated with polyethylenimine (PEI) and dextran sulfate (DS). The enzymes just immobilized on OC supports could be easily released from the support using 2% SDS at pH 7, both intact or after thermal inactivation (in fact, after inactivation most enzyme molecules were already desorbed). The coating with PEI and DS greatly reduced the enzyme release during thermal inactivation and improved enzyme stability. However, using OC-CALB/RML-PEI-DS, the full release of the immobilized enzyme to reuse the support required more drastic conditions: a pH value of 3, a buffer concentration over 2 M, and temperatures above 45 °C. However, even these conditions were not able to fully release the thermally inactivated enzyme molecules from the support, being necessary to increase the buffer concentration to 4 M sodium phosphate and decrease the pH to 2.5. The formation of unfolded protein/polymers composites seems to be responsible for this strong interaction between the octyl and some anionic groups of OC supports. The support could be reused five cycles using these conditions with similar loading capacity of the support and stability of the immobilized enzyme.

Combining hot-compressed water and ball milling pretreatments to improve the efficiency of the enzymatic hydrolysis of eucalyptus

PubMed Central

Inoue, Hiroyuki; Yano, Shinichi; Endo, Takashi; Sakaki, Tsuyoshi; Sawayama, Shigeki

2008-01-01

Background Lignocellulosic biomass such as wood is an attractive material for fuel ethanol production. Pretreatment technologies that increase the digestibility of cellulose and hemicellulose in the lignocellulosic biomass have a major influence on the cost of the subsequent enzymatic hydrolysis and ethanol fermentation processes. Pretreatments without chemicals such as acids, bases or organic solvents are less effective for an enzymatic hydrolysis process than those with chemicals, but they have a less negative effect on the environment. Results The enzymatic digestibility of eucalyptus was examined following a combined pretreatment without chemicals comprising a ball milling (BM) and hot-compressed water (HCW) treatment. The BM treatment simultaneously improved the digestibility of both glucan and xylan, and was effective in lowering the enzyme loading compared with the HCW treatment. The combination of HCW and BM treatment reduced the BM time. The eucalyptus treated with HCW (160°C, 30 minutes) followed by BM (20 minutes) had an approximately 70% yield of total sugar with a cellulase loading of 4 FPU/g substrate. This yield was comparable to the yields from samples treated with HCW (200°C, 30 minutes) or BM (40 minutes) hydrolyzed with 40 FPU/g substrate. Conclusion The HCW treatment is useful in improving the milling efficiency. The combined HCW-BM treatment can save energy and enzyme loading. PMID:18471309

Time Course and Association of Functional and Biochemical Markers in Severe Semitendinosus Damage Following Intensive Eccentric Leg Curls: Differences between and within Subjects.

PubMed

Carmona, Gerard; Mendiguchía, Jurdan; Alomar, Xavier; Padullés, Josep M; Serrano, David; Nescolarde, Lexa; Rodas, Gil; Cussó, Roser; Balius, Ramón; Cadefau, Joan A

2018-01-01

Purpose: To investigate the extent and evolution of hamstring muscle damage caused by an intensive bout of eccentric leg curls (ELCs) by (1) assessing the time course and association of different indirect markers of muscle damage such as changes in the force-generating capacity (FGC), functional magnetic resonance (fMRI), and serum muscle enzyme levels and (2) analyzing differences in the degree of hamstring muscle damage between and within subjects (limb-to-limb comparison). Methods: Thirteen male participants performed six sets of 10 repetitions of an ELC with each leg. Before and at regular intervals over 7 days after the exercise, FGC was measured with maximal isometric voluntary contraction (MVC). Serum enzyme levels, fMRI transverse relaxation time (T2) and perceived muscle soreness were also assessed and compared against the FGC. Results: Two groups of subjects were identified according to the extent of hamstring muscle damage based on decreased FGC and increased serum enzyme levels: high responders ( n = 10, severe muscle damage) and moderate responders ( n = 3, moderate muscle damage). In the high responders, fMRI T2 analysis revealed that the semitendinosus (ST) muscle suffered severe damage in the three regions measured (proximal, middle, and distal). The biceps femoris short head (BFsh) muscle was also damaged and there were significant differences in the FGC within subjects in the high responders. Conclusion: FGC and serum enzyme levels measured in 10 of the subjects from the sample were consistent with severe muscle damage. However, the results showed a wide range of peak MVC reductions, reflecting different degrees of damage between subjects (high and moderate responders). fMRI analysis confirmed that the ST was the hamstring muscle most damaged by ELCs, with uniform T2 changes across all the measured sections of this muscle. During intensive ELCs, the ST muscle could suffer an anomalous recruitment pattern due to fatigue and damage, placing an excessive load on the BFsh and causing it to perform a synergistic compensation that leads to structural damage. Finally, T2 and MVC values did not correlate for the leg with the smaller FGC decrease in the hamstring muscles, suggesting that long-lasting increases in T2 signals after FGC markers have returned to baseline values might indicate an adaptive process rather than damage.

Time Course and Association of Functional and Biochemical Markers in Severe Semitendinosus Damage Following Intensive Eccentric Leg Curls: Differences between and within Subjects

PubMed Central

Carmona, Gerard; Mendiguchía, Jurdan; Alomar, Xavier; Padullés, Josep M.; Serrano, David; Nescolarde, Lexa; Rodas, Gil; Cussó, Roser; Balius, Ramón; Cadefau, Joan A.

2018-01-01

Purpose: To investigate the extent and evolution of hamstring muscle damage caused by an intensive bout of eccentric leg curls (ELCs) by (1) assessing the time course and association of different indirect markers of muscle damage such as changes in the force-generating capacity (FGC), functional magnetic resonance (fMRI), and serum muscle enzyme levels and (2) analyzing differences in the degree of hamstring muscle damage between and within subjects (limb-to-limb comparison). Methods: Thirteen male participants performed six sets of 10 repetitions of an ELC with each leg. Before and at regular intervals over 7 days after the exercise, FGC was measured with maximal isometric voluntary contraction (MVC). Serum enzyme levels, fMRI transverse relaxation time (T2) and perceived muscle soreness were also assessed and compared against the FGC. Results: Two groups of subjects were identified according to the extent of hamstring muscle damage based on decreased FGC and increased serum enzyme levels: high responders (n = 10, severe muscle damage) and moderate responders (n = 3, moderate muscle damage). In the high responders, fMRI T2 analysis revealed that the semitendinosus (ST) muscle suffered severe damage in the three regions measured (proximal, middle, and distal). The biceps femoris short head (BFsh) muscle was also damaged and there were significant differences in the FGC within subjects in the high responders. Conclusion: FGC and serum enzyme levels measured in 10 of the subjects from the sample were consistent with severe muscle damage. However, the results showed a wide range of peak MVC reductions, reflecting different degrees of damage between subjects (high and moderate responders). fMRI analysis confirmed that the ST was the hamstring muscle most damaged by ELCs, with uniform T2 changes across all the measured sections of this muscle. During intensive ELCs, the ST muscle could suffer an anomalous recruitment pattern due to fatigue and damage, placing an excessive load on the BFsh and causing it to perform a synergistic compensation that leads to structural damage. Finally, T2 and MVC values did not correlate for the leg with the smaller FGC decrease in the hamstring muscles, suggesting that long-lasting increases in T2 signals after FGC markers have returned to baseline values might indicate an adaptive process rather than damage. PMID:29467666

Tuning the Catalytic Activity of Subcellular Nanoreactors.

PubMed

Jakobson, Christopher M; Chen, Yiqun; Slininger, Marilyn F; Valdivia, Elias; Kim, Edward Y; Tullman-Ercek, Danielle

2016-07-31

Bacterial microcompartments are naturally occurring subcellular organelles of bacteria and serve as a promising scaffold for the organization of heterologous biosynthetic pathways. A critical element in the design of custom biosynthetic organelles is quantitative control over the loading of heterologous enzymes to the interior of the organelles. We demonstrate that the loading of heterologous proteins to the 1,2-propanediol utilization microcompartment of Salmonella enterica can be controlled using two strategies: by modulating the transcriptional activation of the microcompartment container and by coordinating the expression of the microcompartment container and the heterologous cargo. These strategies allow general control over the loading of heterologous proteins localized by two different N-terminal targeting peptides and represent an important step toward tuning the catalytic activity of bacterial microcompartments for increased biosynthetic productivity. Copyright © 2016. Published by Elsevier Ltd.

Digital Microfluidics Assisted Sealing of Individual Magnetic Particles in Femtoliter-Sized Reaction Wells for Single-Molecule Detection.

PubMed

Decrop, Deborah; Ruiz, Elena Pérez; Kumar, Phalguni Tewari; Tripodi, Lisa; Kokalj, Tadej; Lammertyn, Jeroen

2017-01-01

Digital microfluidics has emerged in the last years as a promising liquid handling technology for a variety of applications. Here, we describe in detail how to build up an electrowetting-on-dielectric-based digital microfluidic chip with unique advantages for performing single-molecule detection. We illustrate how superparamagnetic particles can be printed with very high loading efficiency (over 98 %) and single-particle resolution in the microwell array patterned in the Teflon-AF ® surface of the grounding plate of the chip. Finally, the potential of the device for its application to single-molecule detection is demonstrated by the ultrasensitive detection of the biotinylated enzyme β-Galactosidase captured on streptavidin-coated particles in the described platform.

High endothelin-converting enzyme-1 expression independently predicts poor survival of patients with esophageal squamous cell carcinoma.

PubMed

Wu, Ching-Fang; Lee, Ching-Tai; Kuo, Yao-Hung; Chen, Tzu-Haw; Chang, Chi-Yang; Chang, I-Wei; Wang, Wen-Lun

2017-09-01

Patients with esophageal squamous cell carcinoma have poor survival and high recurrence rate, thus an effective prognostic biomarker is needed. Endothelin-converting enzyme-1 is responsible for biosynthesis of endothelin-1, which promotes growth and invasion of human cancers. The role of endothelin-converting enzyme-1 in esophageal squamous cell carcinoma is still unknown. Therefore, this study investigated the significance of endothelin-converting enzyme-1 expression in esophageal squamous cell carcinoma clinically. We enrolled patients with esophageal squamous cell carcinoma who provided pretreated tumor tissues. Tumor endothelin-converting enzyme-1 expression was evaluated by immunohistochemistry and was defined as either low or high expression. Then we evaluated whether tumor endothelin-converting enzyme-1 expression had any association with clinicopathological findings or predicted survival of patients with esophageal squamous cell carcinoma. Overall, 54 of 99 patients with esophageal squamous cell carcinoma had high tumor endothelin-converting enzyme-1 expression, which was significantly associated with lymph node metastasis ( p = 0.04). In addition, tumor endothelin-converting enzyme-1 expression independently predicted survival of patients with esophageal squamous cell carcinoma, and the 5-year survival was poorer in patients with high tumor endothelin-converting enzyme-1 expression ( p = 0.016). Among patients with locally advanced and potentially resectable esophageal squamous cell carcinoma (stage II and III), 5-year survival was poorer with high tumor endothelin-converting enzyme-1 expression ( p = 0.003). High tumor endothelin-converting enzyme-1 expression also significantly predicted poorer survival of patients in this population. In patients with esophageal squamous cell carcinoma, high tumor endothelin-converting enzyme-1 expression might indicate high tumor invasive property. Therefore, tumor endothelin-converting enzyme-1 expression could be a good biomarker to identify patients with worse survival and higher risks of recurrence, who might benefit from the treatment by endothelin-converting enzyme-1 inhibitor.

Molecular Cloning and Sequence of Channel Catfish (Ictalurus punctatus, Rafinesque 1818) Cathepsin S gene

USDA-ARS?s Scientific Manuscript database

Cathepsin S is a lysosomal cysteine endopeptidase of the papain family. This enzyme digests the invariant chain molecules so that antigenic peptides are able to load on the class II-associated invariant chain peptide of MHC. The complexes can subsequently be presented to the CD4 cell surface. In ...

Ex vivo permeation of tamoxifen and its 4-OH metabolite through rat intestine from lecithin/chitosan nanoparticles.

PubMed

Barbieri, S; Buttini, F; Rossi, A; Bettini, R; Colombo, P; Ponchel, G; Sonvico, F; Colombo, G

2015-08-01

Tamoxifen citrate is an anticancer drug slightly soluble in water. Administered orally, it shows great intra- and inter-patient variations in bioavailability. We developed a nanoformulation based on phospholipid and chitosan able to efficiently load tamoxifen and showing an enzyme triggered release. In this work the permeation of tamoxifen released from lecithin/chitosan nanoparticles across excised rat intestinal wall mounted in an Ussing chamber was investigated. Compared to tamoxifen citrate suspension, the amount of the drug permeated using the nanoformulation was increased from 1.5 to 90 times, in absence or in presence of pancreatin or lipase, respectively. It was also evidenced the formation of an active metabolite of tamoxifen, 4-hydroxy tamoxifen, however, the amount of metabolite permeated remained roughly constant in all experiments. The effect of enzymes on intestinal permeation of tamoxifen was shown only when tamoxifen-loaded nanoparticles were in intimate contact with the mucosal surface. The encapsulation of tamoxifen in lecithin/chitosan nanoparticles improved the non-metabolized drug passing through the rat intestinal tissue via paracellular transport. Copyright © 2015 Elsevier B.V. All rights reserved.

Stimuli-responsive protamine-based biodegradable nanocapsules for enhanced bioavailability and intracellular delivery of anticancer agents

NASA Astrophysics Data System (ADS)

Radhakrishnan, Krishna; Thomas, Midhun B.; Pulakkat, Sreeranjini; Gnanadhas, Divya P.; Chakravortty, Dipshikha; Raichur, Ashok M.

2015-08-01

Enzyme- and pH-responsive polyelectrolyte nanocapsules having diameters in the range of 200 ± 20 nm were fabricated by means of Layer-by-Layer assembly of biopolymers, protamine, and heparin, and then loaded with anticancer drug doxorubicin. The incorporation of the FDA-approved peptide drug protamine as a wall component rendered the capsules responsive to enzyme stimuli. The stimuli-responsive drug release from these nanocapsules was evaluated, and further modulation of capsule permeability to avoid premature release was demonstrated by crosslinking the wall components. The interaction of the nanocapsules with cancer cells was studied using MCF-7 breast cancer cells. These capsules were readily internalized and disintegrated inside the cells, culminating in the release of the loaded doxorubicin and subsequent cell death as observed by confocal microscopy and MTT Assay. The bioavailability studies performed using BALB/c mice revealed that the encapsulated doxorubicin exhibited enhanced bioavailability compared to free doxorubicin. Our results indicate that this stimuli-responsive system fabricated from clinically used FDA-approved molecules and exhibiting minimal premature release has great potential for drug-delivery applications.

A Sensitive Assay for Virus Discovery in Respiratory Clinical Samples

PubMed Central

de Vries, Michel; Deijs, Martin; Canuti, Marta; van Schaik, Barbera D. C.; Faria, Nuno R.; van de Garde, Martijn D. B.; Jachimowski, Loes C. M.; Jebbink, Maarten F.; Jakobs, Marja; Luyf, Angela C. M.; Coenjaerts, Frank E. J.; Claas, Eric C. J.; Molenkamp, Richard; Koekkoek, Sylvie M.; Lammens, Christine; Leus, Frank; Goossens, Herman; Ieven, Margareta; Baas, Frank; van der Hoek, Lia

2011-01-01

In 5–40% of respiratory infections in children, the diagnostics remain negative, suggesting that the patients might be infected with a yet unknown pathogen. Virus discovery cDNA-AFLP (VIDISCA) is a virus discovery method based on recognition of restriction enzyme cleavage sites, ligation of adaptors and subsequent amplification by PCR. However, direct discovery of unknown pathogens in nasopharyngeal swabs is difficult due to the high concentration of ribosomal RNA (rRNA) that acts as competitor. In the current study we optimized VIDISCA by adjusting the reverse transcription enzymes and decreasing rRNA amplification in the reverse transcription, using hexamer oligonucleotides that do not anneal to rRNA. Residual cDNA synthesis on rRNA templates was further reduced with oligonucleotides that anneal to rRNA but can not be extended due to 3′-dideoxy-C6-modification. With these modifications >90% reduction of rRNA amplification was established. Further improvement of the VIDISCA sensitivity was obtained by high throughput sequencing (VIDISCA-454). Eighteen nasopharyngeal swabs were analysed, all containing known respiratory viruses. We could identify the proper virus in the majority of samples tested (11/18). The median load in the VIDISCA-454 positive samples was 7.2 E5 viral genome copies/ml (ranging from 1.4 E3–7.7 E6). Our results show that optimization of VIDISCA and subsequent high-throughput-sequencing enhances sensitivity drastically and provides the opportunity to perform virus discovery directly in patient material. PMID:21283679

Effect of inhibition of microsomal Ca(2+)-ATPase on cytoplasmic calcium and enzyme secretion in pancreatic acini.

PubMed

Metz, D C; Pradhan, T K; Mrozinski, J E; Jensen, R T; Turner, R J; Patto, R J; Gardner, J D

1994-01-13

We used thapsigargin (TG), 2,5-di-tert-butyl-1,4-benzohydroquinone (BHQ) and cyclopiazonic acid (CPA), each of which inhibits microsomal Ca(2+)-ATPase, to evaluate the effects of this inhibition on cytoplasmic free calcium ([Ca2+]i) and secretagogue-stimulated enzyme secretion in rat pancreatic acini. Using single-cell microspectrofluorimetry of fura-2-loaded acini we found that all three agents caused a sustained increase in [Ca2+]i by mobilizing calcium from inositol-(1,4,5)-trisphosphate-sensitive intracellular calcium stores and by promoting influx of extracellular calcium. Concentrations of all three agents that increased [Ca2+]i potentiated the stimulation of enzyme secretion caused by secretagogues that activate adenylate cyclase but inhibited the stimulation of enzyme secretion caused by secretagogues that activate phospholipase C. With BHQ, potentiation of adenylate cyclase-mediated enzyme secretion occurred immediately whereas inhibition of phospholipase C-mediated enzyme secretion occurred only after several min of incubation. In addition, the effects of BHQ and CPA on both [Ca2+]i and secretagogue-stimulated enzyme secretion were reversed completely by washing whereas the actions of TG could not be reversed by washing. Concentrations of BHQ in excess of those that caused maximal changes in [Ca2+]i inhibited all modes of stimulated enzyme secretion by a mechanism that was apparently unrelated to changes in [Ca2+]i. Finally, in contrast to the findings with TG and BHQ, CPA inhibited bombesin-stimulated enzyme secretion over a range of concentrations that was at least 10-fold lower than the range of concentrations over which CPA potentiated VIP-stimulated enzyme secretion.

Enhanced hydrolysis of cellulose hydrogels by morphological modification.

PubMed

Alfassi, Gilad; Rein, Dmitry M; Cohen, Yachin

2017-11-01

Cellulose is one of the most abundant bio-renewable materials on earth, yet the potential of cellulosic bio-fuels is not fully exploited, primarily due to the high costs of conversion. Hydrogel particles of regenerated cellulose constitute a useful substrate for enzymatic hydrolysis, due to their porous and amorphous structure. This article describes the influence of several structural aspects of the cellulose hydrogel on its hydrolysis. The hydrogel density was shown to be directly proportional to the cellulose concentration in the initial solution, thus affecting its hydrolysis rate. Using high-resolution scanning electron microscopy, we show that the hydrogel particles in aqueous suspension exhibit a dense external surface layer and a more porous internal network. Elimination of the external surface layer accelerated the hydrolysis rate by up to sixfold and rendered the process nearly independent of cellulose concentration. These findings may be of practical relevance to saccharification processing costs, by reducing required solvent quantities and enzyme load.

Advanced treatment of biologically pretreated coal gasification wastewater by a novel integration of catalytic ultrasound oxidation and membrane bioreactor.

PubMed

Jia, Shengyong; Han, Hongjun; Zhuang, Haifeng; Xu, Peng; Hou, Baolin

2015-01-01

Laboratorial scale experiments were conducted to investigate a novel system integrating catalytic ultrasound oxidation (CUO) with membrane bioreactor (CUO-MBR) on advanced treatment of biologically pretreated coal gasification wastewater. Results indicated that CUO with catalyst of FeOx/SBAC (sewage sludge based activated carbon (SBAC) which loaded Fe oxides) represented high efficiencies in eliminating TOC as well as improving the biodegradability. The integrated CUO-MBR system with low energy intensity and high frequency was more effective in eliminating COD, BOD5, TOC and reducing transmembrane pressure than either conventional MBR or ultrasound oxidation integrated MBR. The enhanced hydroxyl radical oxidation, facilitation of substrate diffusion and improvement of cell enzyme secretion were the mechanisms for CUO-MBR performance. Therefore, the integrated CUO-MBR was the promising technology for advanced treatment in engineering applications. Copyright © 2015 Elsevier Ltd. All rights reserved.

Comparison of several ethanol productions using xylanase, inorganic salts, surfactant

NASA Astrophysics Data System (ADS)

Wu, Yan; Lu, Jie; Yang, Rui-feng; Song, Wen-jing; Li, Hai-ming; Wang, Hai-song; Zhou, Jing-hui

2017-03-01

Liquid hot water (LHW) pretreatment is an effective and environmentally friendly method to produce bioethanol with lignocellulosic materials. Corn stover was pretreated with liquid hot water (LHW) and then subjected to semi-simultaneous saccharification and fermentation (S-SSF) to obtain high ethanol concentration and yield. The present study aimed to confirm the effect of several additives on the fermentation digestibility of unwashed WIS of corn stover pretreated with LHW. So we also investigated the process, such as enzyme addition, inorganic salts, surfactant and different loading Triton. Results show that high ethanol concentration is necessary to add xylanase in the stage of saccharification. The ethanol concentration increased mainly with magnesium ion on fermentation. Comparing with Tween 80, Span 80 and Polyethylene glycol, Triton is the best surfactant. In contrast to using xylanase and Triton respectively, optimization can make up the lack of stamina and improve effect of single inorganic salts.

Continuous tank reactors in series: an improved alternative in the removal of phenolic compounds with immobilized peroxidase.

PubMed

Gómez, E; Máximo, M F; Montiel, M C; Gómez, M; Murcia, M D; Ortega, S

2012-01-01

Immobilized derivatives of soybean peroxidase, covalently bound to a glass support, were used in a continuous stirred tank reactor in series, in order to study the removal of two phenolic compounds: phenol and 4-chlorophenol. The use of two reactors in series, rather than one continuous tank, improved the removal efficiencies of phenol and 4-chlorophenol. The distribution of different amounts of enzyme between the two tanks showed that the relative distributions influenced the removal efficiency reached and the degree of the enzyme deactivation. The highest removal percentages were reached at the outlet of the second tank for a distribution of 50% of the enzyme in each tank. However, with a distribution of 75% in the first tank and 25% in the second, the elimination percentage in the second tank was slightly lower than in the previous case, and the effects of deactivation of the enzyme in the first tank were less pronounced. In all the distributions assayed it was observed that the first tank acts as a filter for the second one, which receives a feed with a smaller load of phenolic compounds, thus diminishing enzyme deactivation in the second tank.

Detection of phenolic compounds in flow systems based on tyrosinase-modified reticulated vitreous carbon electrodes.

PubMed

Peña, N; Reviejo, A J; Pingarrón, J M

2001-08-03

The fabrication and performance of a reticulated vitreous carbon (RVC)-based tyrosinase flow-through electrode, in which the enzyme was covalently immobilized, is reported. The bioelectrode was tested as an amperometric detector for phenolic compounds. Variables affecting the construction of the enzyme flow-through electrode such as the RVC chemical pretreatment procedure, the enzyme immobilization method in the RVC matrix, the enzyme loading and the pH value of the buffer solution used, were optimized by flow-injection with amperometric detection. A good immobilization of the enzyme in the RVC matrix, in spite of the hydrodynamic conditions, was found. The same tyrosinase-RVC electrode could be used with no significant loss of the amperometric response for around 20 days, and reproducible responses could be achieved with different electrodes constructed in the same manner. Moreover, the operational stability of the bioelectrode was tested under continuous monitorization conditions. Calibration plots by flow injection with amperometric detection at -0.20 V were obtained for phenol, 2,4-dimethylphenol; 3-chlorophenol; 4-chlorophenol; 4-chloro-3-methylphenol and 2-aminophenol, with detection limits ranging from 2 mug l(-1) (4-chloro-3-methylphenol) to 2 mg l(-1).

Red blood cells serve as intravascular carriers of myeloperoxidase.

PubMed

Adam, Matti; Gajdova, Silvie; Kolarova, Hana; Kubala, Lukas; Lau, Denise; Geisler, Anne; Ravekes, Thorben; Rudolph, Volker; Tsao, Philip S; Blankenberg, Stefan; Baldus, Stephan; Klinke, Anna

2014-09-01

Myeloperoxidase (MPO) is a heme enzyme abundantly expressed in polymorphonuclear neutrophils. MPO is enzymatically capable of catalyzing the generation of reactive oxygen species (ROS) and the consumption of nitric oxide (NO). Thus MPO has both potent microbicidal and, upon binding to the vessel wall, pro-inflammatory properties. Interestingly, MPO - a highly cationic protein - has been shown to bind to both endothelial cells and leukocyte membranes. Given the anionic surface charge of red blood cells, we investigated binding of MPO to erythrocytes. Red blood cells (RBCs) derived from patients with elevated MPO plasma levels showed significantly higher amounts of MPO by flow cytometry and ELISA than healthy controls. Heparin-induced MPO-release from patient-derived RBCs was significantly increased compared to controls. Ex vivo experiments revealed dose and time dependency for MPO-RBC binding, and immunofluorescence staining as well as confocal microscopy localized MPO-RBC interaction to the erythrocyte plasma membrane. NO-consumption by RBC-membrane fragments (erythrocyte "ghosts") increased with incrementally greater concentrations of MPO during incubation, indicating preserved catalytic MPO activity. In vivo infusion of MPO-loaded RBCs into C57BL/6J mice increased local MPO tissue concentrations in liver, spleen, lung, and heart tissue as well as within the cardiac vasculature. Further, NO-dependent relaxation of aortic rings was altered by RBC bound-MPO and systemic vascular resistance significantly increased after infusion of MPO-loaded RBCs into mice. In summary, we find that MPO binds to RBC membranes in vitro and in vivo, is transported by RBCs to remote sites in mice, and affects endothelial function as well as systemic vascular resistance. RBCs may avidly bind circulating MPO, and act as carriers of this leukocyte-derived enzyme. Copyright © 2014 Elsevier Ltd. All rights reserved.

Production of Pectate Lyase by Penicillium viridicatum RFC3 in Solid-State and Submerged Fermentation.

PubMed

Ferreira, Viviani; da Silva, Roberto; Silva, Dênis; Gomes, Eleni

2010-01-01

Pectate lyase (PL) was produced by the filamentous fungus Penicillium viridicatum RFC3 in solid-state cultures of a mixture of orange bagasse and wheat bran (1 : 1 w/w), or orange bagasse, wheat bran and sugarcane bagasse (1 : 1 : 0.5 w/w), and in a submerged liquid culture with orange bagasse and wheat bran (3%) as the carbon source. PL production was highest (1,500 U mL(-1) or 300 Ug(-1) of substrate) in solid-state fermentation (SSF) on wheat bran and orange bagasse at 96 hours. PL production in submerged fermentation (SmF) was influenced by the initial pH of the medium. With the initial pH adjusted to 4.5, 5.0, and 5.5, the peak activity was observed after 72, 48, and 24 hours of fermentation, respectively, when the pH of the medium reached the value 5.0. PL from SSF and SmF were loaded on Sephadex-G75 columns and six activity peaks were obtained from crude enzyme from SSF and designated PL I, II, III, IV, V, and VI, while five peaks were obtained from crude enzyme from SmF and labeled PL I', II', III', IV', and VII'. Crude enzyme and fraction III from each fermentative process were tested further. The optimum pH for crude PL from either process was 5.5, while that for PL III was 8.0. The maximum activity of enzymes from SSF was observed at 35 degrees C, but crude enzyme was more thermotolerant than PL III, maintaining its maximum activity up to 45 degrees C. Crude enzyme from SmF and PL III' showed thermophilic profiles of activity, with maximum activity at 60 and 55 degrees C, respectively. In the absence of substrate, the crude enzyme from SSF was stable over the pH range 3.0-10.0 and PL III was most stable in the pH range 4.0-7.0. Crude enzyme from SmF retained 70%-80% of its maximum activity in the acid-neutral pH range (4.0-7.0), but PIII showed high stability at alkaline pH (7.5-9.5). PL from SSF was more thermolabile than that from SmF. The latter maintained 60% of its initial activity after 1 h at 55 degrees C. The differing behavior of the enzymes with respect to pH and temperature suggests that they are different isozymes.

Bioactive Polymeric Nanoparticles for Periodontal Therapy

PubMed Central

Alfonso-Rodríguez, Camilo Andrés; Medina-Castillo, Antonio L.; Alaminos, Miguel; Toledano, Manuel

2016-01-01

Aims to design calcium and zinc-loaded bioactive and cytocompatible nanoparticles for the treatment of periodontal disease. Methods PolymP-nActive nanoparticles were zinc or calcium loaded. Biomimetic calcium phosphate precipitation on polymeric particles was assessed after 7 days immersion in simulated body fluid, by scanning electron microscopy attached to an energy dispersive analysis system. Amorphous mineral deposition was probed by X-ray diffraction. Cell viability analysis was performed using oral mucosa fibroblasts by: 1) quantifying the liberated deoxyribonucleic acid from dead cells, 2) detecting the amount of lactate dehydrogenase enzyme released by cells with damaged membranes, and 3) by examining the cytoplasmic esterase function and cell membranes integrity with a fluorescence-based method using the Live/Dead commercial kit. Data were analyzed by Kruskal-Wallis and Mann-Whitney tests. Results Precipitation of calcium and phosphate on the nanoparticles surfaces was observed in calcium-loaded nanoparticles. Non-loaded nanoparticles were found to be non-toxic in all the assays, calcium and zinc-loaded particles presented a dose dependent but very low cytotoxic effect. Conclusions The ability of calcium-loaded nanoparticles to promote precipitation of calcium phosphate deposits, together with their observed non-toxicity may offer new strategies for periodontal disease treatment. PMID:27820866

Effects of Porous Polystyrene Resin Parameters on Candida antarctica Lipase B Adsorption, Distribution, and Polyester Synthesis Activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen,B.; Miller, M.; Gross, R.

2007-01-01

Polystyrene resins with varied particle sizes (35 to 350-600 {mu}m) and pore diameters (300-1000 {angstrom}) were employed to study the effects of immobilization resin particle size and pore diameter on Candida antarctica Lipase B (CALB) loading, distribution within resins, fraction of active sites, and catalytic properties for polyester synthesis. CALB adsorbed rapidly (saturation time {<=}4 min) for particle sizes 120 {mu}m (pore size = 300 {angstrom}). Infrared microspectroscopy showed that CALB forms protein loading fronts regardless of resin particle size at similar enzyme loadings ({approx}8%). From the IR images, the fractions of total surface area available to the enzyme aremore » 21, 33, 35, 37, and 88% for particle sizes 350-600, 120, 75, 35 {mu}m (pore size 300 {angstrom}), and 35 {mu}m (pore size 1000 {angstrom}), respectively. Titration with methyl p-nitrophenyl n-hexylphosphate (MNPHP) showed that the fraction of active CALB molecules adsorbed onto resins was {approx}60%. The fraction of active CALB molecules was invariable as a function of resin particle and pore size. At {approx}8% (w/w) CALB loading, by increasing the immobilization support pore diameter from 300 to 1000 {angstrom}, the turnover frequency (TOF) of {var_epsilon}-caprolactone ({var_epsilon}-CL) to polyester increased from 12.4 to 28.2 s{sup -1}. However, the {var_epsilon}-CL conversion rate was not influenced by changes in resin particle size. Similar trends were observed for condensation polymerizations between 1,8-octanediol and adipic acid. The results herein are compared to those obtained with a similar series of methyl methacrylate resins, where variations in particle size largely affected CALB distribution within resins and catalyst activity for polyester synthesis.« less

Modification and Utilization of Nanoporous Gold for Loading and Release of Drugs

NASA Astrophysics Data System (ADS)

Al-badri, Ibtisam

Nanoporous gold (np-Au) is a sponge-like structure of gold, which can be created by removing the less noble element from the precursor alloy, most typically silver or copper, using different chemical or electrochemical methods. It consists of interconnected ligaments and gaps between the ligaments, whose width can range from a few nanometers to a few hundreds of nanometers, creating a high surface area-to-volume ratio. Due to its many important properties (e.g., conductivity, high surface area-to-volume ratio, plasmonic response, biocompatibility, chemically inertness, and physically robustness), np-Au is suitable for different types of applications, including as a transducer for biosensors, in catalysis, for biomolecule separation, as a substrate for enzyme immobilization, and in drug delivery. The widths of the ligaments and gaps of np-Au can be easily tuned by varying conditions during the pre- or post-production process, for example, time kept in an acid bath and post-annealing (e.g. thermal, chemical, and electrochemical), depending on the requirement of the study. Thermal annealing is a commonly used process for tuning the ligaments and pore size of np-Au. However, the effects of thermal annealing on modification of ligaments and gaps sizes are not completely understood and more research needs to be done. Herein, we have explored the effect of annealing time and thickness of the np-Au sample on modification of ligaments and gaps. Furthermore, we used the electroless plating method to cover the pores or gaps partially on the surface without modifying the interior of np-Au. As-prepared np-Au was then studied as a platform for molecular loading and releasing kinetics for the possible use in drug delivery. We have found that simply applying the electroless deposition for 1 to 5 min can drastically decrease the rate of release of the molecules, and flow cell-based loading is the preferred way to load the molecules inside np-Au compared to the static method. The structure of the np-Au monoliths before and after the modification was characterized using Energy-Dispersive X-ray Spectroscopy (EDS) and scanning electron microscopy (SEM), whereas the molecular loading and releasing studies were performed using UV-Vis spectrophotometer.

Regulation of gene expression in intervertebral disc cells by low and high hydrostatic pressure.

PubMed

Neidlinger-Wilke, Cornelia; Würtz, Karin; Urban, Jill P G; Börm, Wolfgang; Arand, Markus; Ignatius, Anita; Wilke, Hans-Joachim; Claes, Lutz E

2006-08-01

Intervertebral disc structures are exposed to wide ranges of intradiscal hydrostatic pressure during different loading exercises and are at their minimum during lying or relaxed sitting and at maximum during lifting weights with a round back. We hypothesize that these different loading magnitudes influence the intervertebral disc (IVD) by alteration of disc matrix turnover depending on their magnitudes. Therefore the aim of this study was to assess changes in gene expression of human nucleus cells after the application of low hydrostatic pressure (0.25 MPa) and high hydrostatic pressure (2.5 MPa). IVD cells isolated from the nucleus of human (n = 18) and bovine (n = 24 from four animals) disc biopsies were seeded into three-dimensional collagen type-I matrices and exposed to the different loading magnitudes by specially developed pressure chambers. The lower pressure range (0.25 MPa, 30 min, 0.1 Hz) was applied with a recently published device by using an external compression cylinder. For the application of higher loads (2.5 MPa, 30 min, 0.1 Hz) the cell-loaded collagen gels were sealed into sterile bags with culture medium and stimulated in a newly developed water-filled compression cylinder by using a loading frame. These methods allowed the comparison of loading regimes in a wide physiological range under an equal three-dimensional culture conditions. Cells were harvested 24 h after the end of stimulation and changes in the expression of genes known to influence IVD matrix turnover (collagen-I, collagen-II, aggrecan, MMP1, MMP2, MMP3, MMP13) were analyzed by real-time RT-PCR. A Wilcoxon signed-rank test(1) and a Wilcoxon 2-sample test(2) were performed to detect differences between the stimulated and control samples(1) and differences between low and high hydrostatic pressure(2). Multiple testing was considered by adjusting the p value appropriately. Both regimes of hydrostatic pressure influenced gene expression in nucleus cells with opposite tendencies for the matrix forming proteins aggrecan and collagen type-I in response to the two different pressure magnitudes: Low hydrostatic-pressure (0.25 MPa) tended to increase collagen-I and aggrecan expression of human nucleus cells (P < 0.05) but only to a small degree. High hydrostatic pressure (2.5 MPa) tended to decrease gene expression of all anabolic proteins with significant effects on aggrecan expression of nucleus cells (P = 0.004). Low hydrostatic pressure had no influence on the expression of matrix metalloproteinases (MMP1, MMP2, MMP3 and MMP13). In contrast, high hydrostatic pressure tended to increase the expression of MMP1, MMP3 and MMP13 of human nucleus cells with high individual-individual variations. The decreased expression of aggrecan (P = 0.008) and collagen type II (P = 0.023) and the increased MMP3 expression (P = 0.008) in response to high hydrostatic pressure could be confirmed in additional experiments with bovine nucleus cells. These results suggest that hydrostatic pressure as one of the physiological stimuli of the IVD may influence matrix turnover in a magnitude dependent way. Low hydrostatic pressure (0.25 MPa) has quite small influences with a tendency to anabolic effects, whereas high hydrostatic pressure (2.5 MPa) tends to decrease the matrix protein expression with a tendency to increase some matrix-turnover enzymes. Therefore, hydrostatic pressure may regulate disc matrix turnover in a dose-dependent way.

A comparative study of ethanol production using dilute acid, ionic liquid and AFEX™ pretreated corn stover

PubMed Central

2014-01-01

Background In a biorefinery producing cellulosic biofuels, biomass pretreatment will significantly influence the efficacy of enzymatic hydrolysis and microbial fermentation. Comparison of different biomass pretreatment techniques by studying the impact of pretreatment on downstream operations at industrially relevant conditions and performing comprehensive mass balances will help focus attention on necessary process improvements, and thereby help reduce the cost of biofuel production. Results An on-going collaboration between the three US Department of Energy (DOE) funded bioenergy research centers (Great Lakes Bioenergy Research Center (GLBRC), Joint BioEnergy Institute (JBEI) and BioEnergy Science Center (BESC)) has given us a unique opportunity to compare the performance of three pretreatment processes, notably dilute acid (DA), ionic liquid (IL) and ammonia fiber expansion (AFEXTM), using the same source of corn stover. Separate hydrolysis and fermentation (SHF) was carried out using various combinations of commercially available enzymes and engineered yeast (Saccharomyces cerevisiae 424A) strain. The optimal commercial enzyme combination (Ctec2: Htec2: Multifect Pectinase, percentage total protein loading basis) was evaluated for each pretreatment with a microplate-based assay using milled pretreated solids at 0.2% glucan loading and 15 mg total protein loading/g of glucan. The best enzyme combinations were 67:33:0 for DA, 39:33:28 for IL and 67:17:17 for AFEX. The amounts of sugar (kg) (glucose: xylose: total gluco- and xylo-oligomers) per 100 kg of untreated corn stover produced after 72 hours of 6% glucan loading enzymatic hydrolysis were: DA (25:2:2), IL (31:15:2) and AFEX (26:13:7). Additionally, the amounts of ethanol (kg) produced per 100 kg of untreated corn stover and the respective ethanol metabolic yield (%) achieved with exogenous nutrient supplemented fermentations were: DA (14.0, 92.0%), IL (21.2, 93.0%) and AFEX (20.5, 95.0%), respectively. The reason for lower ethanol yield for DA is because most of the xylose produced during the pretreatment was removed and not converted to ethanol during fermentation. Conclusions Compositional analysis of the pretreated biomass solids showed no significant change in composition for AFEX treated corn stover, while about 85% of hemicellulose was solubilized after DA pretreatment, and about 90% of lignin was removed after IL pretreatment. As expected, the optimal commercial enzyme combination was different for the solids prepared by different pretreatment technologies. Due to loss of nutrients during the pretreatment and washing steps, DA and IL pretreated hydrolysates required exogenous nutrient supplementation to ferment glucose and xylose efficiently, while AFEX pretreated hydrolysate did not require nutrient supplementation. PMID:24917886

Systematic optimization of fed-batch simultaneous saccharification and fermentation at high-solid loading based on enzymatic hydrolysis and dynamic metabolic modeling of Saccharomyces cerevisiae.

PubMed

Unrean, Pornkamol; Khajeeram, Sutamat; Laoteng, Kobkul

2016-03-01

An integrative simultaneous saccharification and fermentation (SSF) modeling is a useful guiding tool for rapid process optimization to meet the techno-economic requirement of industrial-scale lignocellulosic ethanol production. In this work, we have developed the SSF model composing of a metabolic network of a Saccharomyces cerevisiae cell associated with fermentation kinetics and enzyme hydrolysis model to quantitatively capture dynamic responses of yeast cell growth and fermentation during SSF. By using model-based design of feeding profiles for substrate and yeast cell in the fed-batch SSF process, an efficient ethanol production with high titer of up to 65 g/L and high yield of 85 % of theoretical yield was accomplished. The ethanol titer and productivity was increased by 47 and 41 %, correspondingly, in optimized fed-batch SSF as compared to batch process. The developed integrative SSF model is, therefore, considered as a promising approach for systematic design of economical and sustainable SSF bioprocessing of lignocellulose.

Rapid identification of a novel complex I MT-ND3 m.10134C>A mutation in a Leigh syndrome patient.

PubMed

Miller, David K; Menezes, Minal J; Simons, Cas; Riley, Lisa G; Cooper, Sandra T; Grimmond, Sean M; Thorburn, David R; Christodoulou, John; Taft, Ryan J

2014-01-01

Leigh syndrome (LS) is a rare progressive multi-system neurodegenerative disorder, the genetics of which is frequently difficult to resolve. Rapid determination of the genetic etiology of LS in a 5-year-old girl facilitated inclusion in Edison Pharmaceutical's phase 2B clinical trial of EPI-743. SNP-arrays and high-coverage whole exome sequencing were performed on the proband, both parents and three unaffected siblings. Subsequent multi-tissue targeted high-depth mitochondrial sequencing was performed using custom long-range PCR amplicons. Tissue-specific mutant load was also assessed by qPCR. Complex I was interrogated by spectrophotometric enzyme assays and Western Blot. No putatively causal mutations were identified in nuclear-encoded genes. Analysis of low-coverage off-target mitochondrial reads revealed a previously unreported mitochondrial mutation in the proband in MT-ND3 (m.10134C>A, p.Q26K), a Complex I mitochondrial gene previously associated with LS. Targeted investigations demonstrated that this mutation was 1% heteroplasmic in the mother's blood and homoplasmic in the proband's blood, fibroblasts, liver and muscle. Enzyme assays revealed decreased Complex I activity. The identification of this novel LS MT-ND3 variant, the genomics of which was accomplished in less than 3.5 weeks, indicates that rapid genomic approaches may prove useful in time-sensitive cases with an unresolved genetic diagnosis.

A novel step towards immobilization of biocatalyst using agro waste and its application for ester synthesis.

PubMed

Tomke, Prerana D; Rathod, Virendra K

2018-05-04

This work explains the utilization of agro-waste (coconut and peanut shell) to produce mesoporous activated carbon which further utilized as a support material for lipase immobilization (Candida antarctica B, CALB). Various parameters affecting the binding of enzyme to activated carbon with high surface area (1603 m 2  g -1 ) were optimized. Maximum 200 μg g -1 CALB has been loaded at 40 °C and pH 6.8 in 12 h by using glutaraldehyde as a cross-linker. The operational parameters such as pH (5.8-8.8) and temperature (30-70 °C) were optimized for free and immobilized form of lipase. In thermal stability (50-70 °C) study, immobilization of enzyme showed 2.35 folds increased half-life with respect to free enzyme. The samples, before and after immobilization, were characterized by specific surface area, FT-IR, SEM, XRD. This immobilized lipase was successfully used for the synthesis of cinnamyl acetate by transesterification reaction producing 94% conversion in 60 min. Catalytic efficiency (58 ± 1.08) was seen to be retained for more than five consecutive cycles of chemical reaction for repeated applications. Sequential results towards activity retention were obtained upto 30 days of storability study. In the context, this process constitutes a clean route for the development of sustainable biocatalysts from agro waste, capable of applications in various area. Copyright © 2017. Published by Elsevier B.V.

Enzyme-mediated bacterial biodegradation of an azo dye (C.I. Acid blue 113): reuse of treated dye wastewater in post-tanning operations.

PubMed

Senthilvelan, T; Kanagaraj, J; Panda, R C

2014-11-01

"Dyeing" is a common practice used to color the hides during the post-tanning operations in leather processing generating plenty of wastewater. The waste stream containing dye as pollutant is severely harmful to living beings. An azo dye (C.I. Acid Blue 113) has been biodegraded effectively by bacterial culture mediated with azoreductase enzyme to reduce the pollution load in the present investigation. The maximum rate of dye degradation was found to be 96 ± 4 and 92 ± 4 % for the initial concentrations of 100 and 200 mg/l, respectively. The enzyme activity was measured using NADH as a substrate. Fourier transform infrared spectroscopy (FT-IR) analysis was confirmed that the transformation of azo linkage could be transformed into N2 or NH3 or incorporated into complete biomass. Breaking down of dye molecules to various metabolites (such as aniline, naphthalene-1,4-diamine, 3-aminobenzenesulfonic acid, naphthalene-1-sulfonic acid, 8-aminonaphthalene-1-sulfonic acid, 5,8-diaminonaphthalene-1-sulfonic acid) was confirmed by gas chromatography and mass spectra (GC-MS) and mass (electrospray ionization (ESI)) spectra analysis. The treated wastewater could be reused for dyeing operation in the leather processing, and the properties of produced leather were evaluated by conventional methods that revealed to have improved dye penetration into the grain layer of experimental leather sample and resulted in high levelness of dyeing, which helps to obtain the desired smoothness and soft leather properties.

Engineering yeasts for raw starch conversion.

PubMed

van Zyl, W H; Bloom, M; Viktor, M J

2012-09-01

Next to cellulose, starch is the most abundant hexose polymer in plants, an import food and feed source and a preferred substrate for the production of many industrial products. Efficient starch hydrolysis requires the activities of both α-1,4 and α-1,6-debranching hydrolases, such as endo-amylases, exo-amylases, debranching enzymes, and transferases. Although amylases are widely distributed in nature, only about 10 % of amylolytic enzymes are able to hydrolyse raw or unmodified starch, with a combination of α-amylases and glucoamylases as minimum requirement for the complete hydrolysis of raw starch. The cost-effective conversion of raw starch for the production of biofuels and other important by-products requires the expression of starch-hydrolysing enzymes in a fermenting yeast strain to achieve liquefaction, hydrolysis, and fermentation (Consolidated Bioprocessing, CBP) by a single organism. The status of engineering amylolytic activities into Saccharomyces cerevisiae as fermentative host is highlighted and progress as well as challenges towards a true CBP organism for raw starch is discussed. Conversion of raw starch by yeast secreting or displaying α-amylases and glucoamylases on their surface has been demonstrated, although not at high starch loading or conversion rates that will be economically viable on industrial scale. Once efficient conversion of raw starch can be demonstrated at commercial level, engineering of yeast to utilize alternative substrates and produce alternative chemicals as part of a sustainable biorefinery can be pursued to ensure the rightful place of starch converting yeasts in the envisaged bio-economy of the future.

Effects of agitation on particle-size distribution and enzymatic hydrolysis of pretreated spruce and giant reed.

PubMed

Kadić, Adnan; Palmqvist, Benny; Lidén, Gunnar

2014-01-01

Mixing is an energy demanding process which has been previously shown to affect enzymatic hydrolysis. Concentrated biomass slurries are associated with high and non-Newtonian viscosities and mixing in these systems is a complex task. Poor mixing can lead to mass and/or heat transfer problems as well as inhomogeneous enzyme distribution, both of which can cause possible yield reduction. Furthermore the stirring energy dissipation may impact the particle size which in turn may affect the enzymatic hydrolysis. The objective of the current work was to specifically quantify the effects of mixing on particle-size distribution (PSD) and relate this to changes in the enzymatic hydrolysis. Two rather different materials were investigated, namely pretreated Norway spruce and giant reed. Changes in glucan hydrolysis and PSD were measured as a function of agitation during enzymatic hydrolysis at fiber loadings of 7 or 13% water-insoluble solids (WIS). Enzymatic conversion of pretreated spruce was strongly affected by agitation rates at the higher WIS content. However, at low WIS content the agitation had almost no effect on hydrolysis. There was some effect of agitation on the hydrolysis of giant reed at high WIS loading, but it was smaller than that for spruce, and there was no measurable effect at low WIS loading. In the case of spruce, intense agitation clearly affected the PSD and resulted in a reduced mean particle size, whereas for giant reed the decrease in particle size was mainly driven by enzymatic action. However, the rate of enzymatic hydrolysis was not increased after size reduction by agitation. The impact of agitation on the enzymatic hydrolysis clearly depends not only on feedstock but also on the solids loading. Agitation was found to affect the PSD differently for the examined pretreated materials spruce and giant reed. The fact that the reduced mean particle diameter could not explain the enhanced hydrolysis rates found for spruce at an elevated agitation suggests that mass transfer at sustained high viscosities plays an important role in determining the rate of enzymatic hydrolysis.

Influence of Butyrate Loaded Clinoptilolite Dietary Supplementation on Growth Performance, Development of Intestine and Antioxidant Capacity in Broiler Chickens

PubMed Central

Wu, Yanan; Zhou, Yanmin; Lu, Changhui; Ahmad, Hussain; Zhang, Hao; He, Jintian; Zhang, Lili; Wang, Tian

2016-01-01

The study was conducted to evaluate the effects of dietary butyrate loaded clinoptilolite (CLI-B) on growth performance, pancreatic digestive enzymes, intestinal development and histomorphology, as well as antioxidant capacity of serum and intestinal mucosal in chickens. Two hundred forty 1-day-old commercial Arbor Acres broilers were randomly assigned to 4 groups: CON group (fed basal diets), SB group (fed basal diet with 0.05% sodium butyrate), CLI group (fed basal diet with 1% clinoptilolite), and CLI-B group (fed basal diet with 1% CLI-B). The results showed that supplementation of CLI-B significantly decreased (P < 0.05) feed conservation ratio at both 21 and 42 days of age, improved the pancreatic digestive enzymes activities (P < 0.05), increased the villus length and villus/crypt ratio (P < 0.05), and decreased the crypt depth of intestine (P < 0.05) as compared to the other experimental groups. Furthermore, the CLI-B environment improved the antioxidant capacity by increasing the antioxidant enzyme activities (P < 0.05) in intestine mucosal, and decreasing the NO content and iNOS activity (P < 0.05) in serum. In addition, CLI-B supplementation had improved the development of intestine and antioxidant capacity of broilers than supplementation with either clinoptilolite or butyrate sodium alone. In conclusion, 1% CLI-B supplementation improved the health status, intestine development and antioxidant capacity in broiler chickens, thus appearing as an important feed additive for the poultry industry. PMID:27104860

Simultaneously saccharification and fermentation approach as a tool for enhanced fossil fuels biodesulfurization.

PubMed

Paixão, Susana M; Arez, Bruno F; Roseiro, José C; Alves, Luís

2016-11-01

Biodesulfurization can be a complementary technology to the hydrodesulfurization, the commonly physical-chemical process used for sulfur removal from crude oil. The desulfurizing bacterium Gordonia alkanivorans strain 1B as a fructophilic microorganism requires fructose as C-source. In this context, the main goal of this work was the optimization of a simultaneous saccharification and fermentation (SSF) approach using the Zygosaccharomyces bailii strain Talf1 crude enzymes with invertase activity and sucrose as a cheaper fructose-rich commercial C-source (50% fructose) towards dibenzothiophene (DBT) desulfurization by strain 1B. The determination of optimal conditions, for both sucrose hydrolysis and DBT desulfurization was carried out through two sequential experimental uniform designs according to the Doehlert distribution for two factors: pH (5.5-7.5) and temperature (28-38 °C), with the enzyme load of 1.16 U/g/L; and enzyme load (0-4 U/g/L) and temperature (28-38 °C), with pH at 7.5. Based on 2-hydroxybiphenyl production, the analysis of the response surfaces obtained pointed out for pH 7.5, 32 °C and 1.8 U/g/L as optimal conditions. Further optimized SSF of sucrose during the DBT desulfurization process permitted to attain a 4-fold enhanced biodesulfurization. This study opens a new focus of research through the exploitation of sustainable low cost sucrose-rich feedstocks towards a more economical viable bioprocess scale-up. Copyright © 2016 Elsevier Ltd. All rights reserved.

Mouse mammary tumor virus chromatin in human breast cancer cells is constitutively hypersensitive and exhibits steroid hormone-independent loading of transcription factors in vivo.

PubMed Central

Mymryk, J S; Berard, D; Hager, G L; Archer, T K

1995-01-01

We have stably introduced a reporter gene under the control of the mouse mammary tumor virus (MMTV) long terminal repeat (LTR) into human T47D breast cancer cells to study the action of the progesterone receptor (PR) on transcription from a chromatin template. Unexpectedly, the chromatin organization of the MMTV LTR in these human breast cancer cells differed markedly from what we have observed previously. The region adjacent to the transcription start site (-221 to -75) was found to be constitutively hypersensitive to restriction enzyme cleavage in the absence of hormone. This region is normally encompassed within the second nucleosome of a phased array of six nucleosomes that is assembled when the MMTV LTR is stably maintained in mouse cells. Characteristically, in these rodent cells, the identical DNA sequences show increased restriction enzyme cleavage only in the presence of glucocorticoid. The increased access of restriction enzymes observed in the human PR+ cells was not observed in adjacent nucleosomes and was unaffected by treatment with the progesterone antagonist RU486. In addition, exonuclease III-dependent stops corresponding to the binding sites for nuclear factor 1 and the PR were observed before and after hormone treatment. These results indicate that MMTV chromatin replicated in these cells is organized into a constitutively open architecture and that this open chromatin state is accompanied by hormone-independent loading of a transcription factor complex that is normally excluded from uninduced chromatin. PMID:7799933

Mouse mammary tumor virus chromatin in human breast cancer cells is constitutively hypersensitive and exhibits steroid hormone-independent loading of transcription factors in vivo.

PubMed

Mymryk, J S; Berard, D; Hager, G L; Archer, T K

1995-01-01

We have stably introduced a reporter gene under the control of the mouse mammary tumor virus (MMTV) long terminal repeat (LTR) into human T47D breast cancer cells to study the action of the progesterone receptor (PR) on transcription from a chromatin template. Unexpectedly, the chromatin organization of the MMTV LTR in these human breast cancer cells differed markedly from what we have observed previously. The region adjacent to the transcription start site (-221 to -75) was found to be constitutively hypersensitive to restriction enzyme cleavage in the absence of hormone. This region is normally encompassed within the second nucleosome of a phased array of six nucleosomes that is assembled when the MMTV LTR is stably maintained in mouse cells. Characteristically, in these rodent cells, the identical DNA sequences show increased restriction enzyme cleavage only in the presence of glucocorticoid. The increased access of restriction enzymes observed in the human PR+ cells was not observed in adjacent nucleosomes and was unaffected by treatment with the progesterone antagonist RU486. In addition, exonuclease III-dependent stops corresponding to the binding sites for nuclear factor 1 and the PR were observed before and after hormone treatment. These results indicate that MMTV chromatin replicated in these cells is organized into a constitutively open architecture and that this open chromatin state is accompanied by hormone-independent loading of a transcription factor complex that is normally excluded from uninduced chromatin.

Effect of xylanase supplementation of cellulase on digestion of corn stover solids prepared by leading pretreatment technologies.

PubMed

Kumar, Rajeev; Wyman, Charles E

2009-09-01

Solids resulting from pretreatment of corn stover by ammonia fiber expansion (AFEX), ammonia recycled percolation (ARP), controlled pH, dilute acid, lime, and sulfur dioxide (SO(2)) technologies were hydrolyzed by enzyme cocktails based on cellulase supplemented with beta-glucosidase at an activity ratio of 1:2, respectively, and augmented with up to 11.0 g xylanase protein/g cellulase protein for combined cellulase and beta-glucosidase mass loadings of 14.5 and 29.0 mg protein (about 7.5 and 15 FPU, respectively)/g of original potential glucose. It was found that glucose release increased nearly linearly with residual xylose removal by enzymes for all pretreatments despite substantial differences in their relative yields. The ratio of the fraction of glucan removed by enzymes to that for xylose was defined as leverage and correlated statistically at two combined cellulase and beta-glucosidase mass loadings with pretreatment type. However, no direct relationship was found between leverage and solid features following different pretreatments such as residual xylan or acetyl content. However, acetyl content not only affected how xylanase impacted cellulase action but also enhanced accessibility of cellulose and/or cellulase effectiveness, as determined by hydrolysis with purified CBHI (Cel7A). Statistical modeling showed that cellulose crystallinity, among the main substrate features, played a vital role in cellulase-xylanase interactions, and a mechanism is suggested to explain the incremental increase in glucose release with xylanase supplementation.

Beta-glucuronidase and hexosaminidase are marker enzymes for different compartments of the endo-lysosomal system in mussel digestive cells.

PubMed

Izagirre, U; Angulo, E; Wade, S C; ap Gwynn, I; Marigómez, I

2009-02-01

In environmental toxicology, the most commonly used techniques used to visualise lysosomes in order to determine their responses to pollutants (LSC test: lysosomal structural changes test; LMS test: lysosomal membrane stability test) are based on the histochemical application of lysosomal marker enzymes. In mussel digestive cells, the marker enzymes used are beta-glucuronidase (beta-Gus) and hexosaminidase (Hex). The present work has been aimed at determining the distribution of these lysosomal marker enzymes in the various compartments of the endo-lysosomal system (ELS) of mussel digestive cells and at exploring whether intercellular transfer of lysosomal enzymes occurs between digestive and basophilic cells. Immunogold cytochemistry has allowed us to conclude that beta-Gus is present in every compartment of the digestive cell ELS, whereas Hex is not so widely distributed. Moreover, Hex is intimately linked to the lysosomal membrane, whereas beta-Gus appears to be not necessarily membrane-bound. Therefore, two populations of heterolysosomes with different enzyme load and membrane stability have been distinguished in the digestive cell. In addition, heterolysosomes of different electron density have been commonly observed merging together by contact; we suggest that some might act as storage granules for lysosomal enzymes. On the other hand, beta-Gus seems to be released to the digestive alveolar lumen in secretory lysosomes produced by basophilic cells and endocytosed by digestive cells. Regarding the implications of the present study on the interpretation of lysosomal biomarkers, we conclude that beta-Gus, but not Hex, histochemistry provides an appropriate marker for the LSC test and that, although both lysosomal marker enzymes can be employed in the LMS test, different values would be obtained depending on the marker enzyme employed.

Effects of motor patterns on water-soluble and membrane proteins and cholinesterase activity in subcellular fractions of rat brain tissue

NASA Technical Reports Server (NTRS)

Pevzner, L. Z.; Venkov, L.; Cheresharov, L.

1980-01-01

Albino rats were kept for a year under conditions of daily motor load or constant hypokinesia. An increase in motor activity results in a rise in the acetylcholinesterase activity determined in the synaptosomal and purified mitochondrial fractions while hypokinesia induces a pronounced decrease in this enzyme activity. The butyrylcholinesterase activity somewhat decreases in the synaptosomal fraction after hypokinesia but does not change under the motor load pattern. Motor load causes an increase in the amount of synaptosomal water-soluble proteins possessing an intermediate electrophoretic mobility and seem to correspond to the brain-specific protein 14-3-2. In the synaptosomal fraction the amount of membrane proteins with a low electrophoretic mobility and with the cholinesterase activity rises. Hypokinesia, on the contrary, decreases the amount of these membrane proteins.

On-site cellulase production and efficient saccharification of corn stover employing cbh2 overexpressing Trichoderma reesei with novel induction system.

PubMed

Li, Yonghao; Zhang, Xiaoyue; Xiong, Liang; Mehmood, Muhammad Aamer; Zhao, Xinqing; Bai, Fengwu

2017-08-01

Although on-site cellulase production offers cost-effective saccharification of lignocellulosic biomass, low enzyme titer is still a barrier for achieving robustness. In the present study, a strain of T. reesei was developed for enhanced production of cellulase via overexpression of Cellobiohydrolase II. Furthermore, optimum enzyme production was achieved using a novel inducer mixture containing synthesized glucose-sophorose (MGD) and alkali pre-treated corn stover (APCS). Within 60h, a remarkably higher cellulase productivity and activity were achieved in the fed-batch fermentation using the optimized ratio of MGD and APCS in the inducer mixture, compared to those reported using cellulosic biomass as the sole inducer. After the enzyme production, APCS was added directly into the fermentation broth at 20% solid loading, which produced 122.5g/L glucose and 40.21g/L xylose, leading to the highest yield reported so far. The improved enzyme titers during on-site cellulase production would benefit cost-competitive saccharification of lignocellulosic biomass. Copyright © 2017 Elsevier Ltd. All rights reserved.

Enhanced therapeutic efficacy of budesonide in experimental colitis with enzyme/pH dual-sensitive polymeric nanoparticles.

PubMed

Naeem, Muhammad; Cao, Jiafu; Choi, Moonjeong; Kim, Woo Seong; Moon, Hyung Ryong; Lee, Bok Luel; Kim, Min-Soo; Jung, Yunjin; Yoo, Jin-Wook

2015-01-01

Current colon-targeted drug-delivery approaches for colitis therapy often utilize single pH-triggered systems, which are less reliable due to the variation of gut pH in individuals and in disease conditions. Herein, we prepared budesonide-loaded dual-sensitive nanoparticles using enzyme-sensitive azo-polyurethane and pH-sensitive methacrylate copolymer for the treatment of colitis. The therapeutic potential of the enzyme/pH dual-sensitive nanoparticles was evaluated using a rat colitis model and compared to single pH-triggered nanoparticles. Clinical activity scores, colon/body weight ratios, myeloperoxidase activity, and proinflammatory cytokine levels were markedly decreased by dual-sensitive nanoparticles compared to single pH-triggered nanoparticles and budesonide solution. Moreover, dual-sensitive nanoparticles accumulated selectively in inflamed segments of the colon. In addition, dual-sensitive nanoparticle plasma concentrations were lower than single pH-triggered nanoparticles, and no noticeable in vitro or in vivo toxicity was observed. Our results demonstrate that enzyme/pH dual-sensitive nanoparticles are an effective and safe colon-targeted delivery system for colitis therapy.

Enhanced therapeutic efficacy of budesonide in experimental colitis with enzyme/pH dual-sensitive polymeric nanoparticles

PubMed Central

Naeem, Muhammad; Cao, Jiafu; Choi, Moonjeong; Kim, Woo Seong; Moon, Hyung Ryong; Lee, Bok Luel; Kim, Min-Soo; Jung, Yunjin; Yoo, Jin-Wook

2015-01-01

Current colon-targeted drug-delivery approaches for colitis therapy often utilize single pH-triggered systems, which are less reliable due to the variation of gut pH in individuals and in disease conditions. Herein, we prepared budesonide-loaded dual-sensitive nanoparticles using enzyme-sensitive azo-polyurethane and pH-sensitive methacrylate copolymer for the treatment of colitis. The therapeutic potential of the enzyme/pH dual-sensitive nanoparticles was evaluated using a rat colitis model and compared to single pH-triggered nanoparticles. Clinical activity scores, colon/body weight ratios, myeloperoxidase activity, and proinflammatory cytokine levels were markedly decreased by dual-sensitive nanoparticles compared to single pH-triggered nanoparticles and budesonide solution. Moreover, dual-sensitive nanoparticles accumulated selectively in inflamed segments of the colon. In addition, dual-sensitive nanoparticle plasma concentrations were lower than single pH-triggered nanoparticles, and no noticeable in vitro or in vivo toxicity was observed. Our results demonstrate that enzyme/pH dual-sensitive nanoparticles are an effective and safe colon-targeted delivery system for colitis therapy. PMID:26213469

Response surface optimization of corn stover pretreatment using dilute phosphoric acid for enzymatic hydrolysis and ethanol production

USDA-ARS?s Scientific Manuscript database

Dilute H3PO4 (0.0 - 2.0%, v/v) was used to pretreat corn stover (10%, w/w) for conversion to ethanol. Pretreatment conditions were optimized for temperature, acid loading, and time using a central composite design. Optimal pretreatment conditions were chosen to promote sugar yields following enzym...

Monitoring of the lactonase activity of paraoxonase-1 enzyme in HIV-1-infection.

PubMed

Dias, Clara; Marinho, Aline; Morello, Judit; Almeida, Gabriela; Caixas, Umbelina; Soto, Karina; Monteiro, Emilia; Pereira, Sofia

2014-01-01

Paraoxonase-1 (PON1) is a high-density lipoprotein (HDL)-associated enzyme known as a free radical scavenging system (1). PON-1 has three main activities, responsible for its antioxidant and anti-inflammatory potential: paraoxonase, arylesterase and lactonase (LACase), the latest to be discovered and pointed out to be its native activity (2). Among other physiological roles, the LACase might minimize the deleterious effects of hyperhomocysteinaemia in infection, by detoxifying the highly reactive metabolite homocysteine-thiolactone (HcyTL) (3),4. In the present work, we have developed and applied a method to quantify LACase activity and to explore the role of this enzyme in HIV-infection and virological response. The LACase activity was monitored in a cohort of HIV-1-infected patients, through the titration of 3-(o-hydroxyphenyl) propionic acid, formed upon the LACase-mediated hydrolysis of the substrate dihydrocoumarin. The study protocol was approved by the Ethics Committee of Centro Hospitalar de Lisboa Central and Hospital Prof. Doutor Fernando Fonseca. All patients gave their written informed consent and were adults with documented HIV-1-infection, regardless of combined antiretroviral therapy (cART) use. Naïve patients and patients who had received continuous antiretroviral treatment for more than one month were included. A total of 179 HIV-1-infected patients were included on this study (51% Men, 39% non-Caucasian, 45±13 years old). Patients with non-suppressed viraemia, either from the non-cART (n=89, 12±4 kU/L, p<0.01) or from the cART with detectable viral load (n=11, 10±5 kU/L, p<0.05) groups, had lower activity than the cART with suppressed viraemia (n=79, 15±7 kU/L) (Kruskal-Wallis test). Among naïve patients, higher viral load (> 31,500 cps/mL, Spearman r=-0.535, p=0.003) and lower CD4+ T-cells count (< 500 cell/mm(3), Pearson r=0.326, p=0.024) were associated with the LACase activity. The present study suggests that lower LACase activity is associated with uncontrolled HIV-1-infection, particularly with non-suppressed viraemia, despite of cART. This data seems to point to LACase role in HIV-infection, probably reflecting an increased formation of HcyTL deleterious species. A better knowledge of the LACase and its role in HcyTL pathophysiology might identify new therapeutic targets in HIV-1-infected patients. Acknowledgements: EXPL/DTP-FTO/0204/2012; EXPL/DTP-PIC/1758/2013.

Characterization of a dual specificity aryl acid adenylation enzyme with dual function in nikkomycin biosynthesis.

PubMed

Moon, Mary; Van Lanen, Steven G

2010-09-01

Nikkomycin Z is a dipeptide antifungal antibiotic characterized by two nonproteinogenic amino acids, nikkomycin C(Z) and 4-(4'-hydroxy-2'-pyridinyl)-homothreonine (HPHT). The HPHT scaffold is assembled by an aldol reaction between 2-oxobutyrate and picolinaldehyde, the latter of which is derived from picolinic acid that is activated and loaded to coenzyme A by the aryl-activating adenylation enzyme, NikE. We now provide evidence that NikE is also involved in the activation and loading of the alpha-keto acid precursor, 4-(2'-pyridinyl)-2-oxo-4-hydroxyisovalerate (POHIV), to a phosphopantetheinyl group of an acyl carrier protein domain of NikT. POHIV was synthesized using Escherichia coli 2-dehydro-3-deoxy-phosphogluconate aldolase, and phenylalanine dehydrogenase from Bacillus sp. NRRL B-14911 was used to prepare the alpha-amino acid, 4-(2'-pyridinyl)-homothreonine (PHT). Using the carboxylic acid-dependent, ATP-[(32)P]PP(i) exchange assay, NikE is shown to activate both picolinic acid and POHIV but not PHT. Furthermore, NikE loads POHIV to holo-NikT to generate a new thioester-linked intermediate, which was not observed using a NikT(S33A) mutant. Thus, NikE activates two distinct carboxylic acids to form two new thioester intermediates, one of which is subsequently reduced to the aldehyde and the other that likely serves as a substrate for the aminotransferase domain of NikT prior to condensation with nikkomycin C(Z) to yield the dipeptide. Copyright 2010 Wiley Periodicals, Inc.

Brain glucose metabolism in an animal model of depression.

PubMed

Detka, J; Kurek, A; Kucharczyk, M; Głombik, K; Basta-Kaim, A; Kubera, M; Lasoń, W; Budziszewska, B

2015-06-04

An increasing number of data support the involvement of disturbances in glucose metabolism in the pathogenesis of depression. We previously reported that glucose and glycogen concentrations in brain structures important for depression are higher in a prenatal stress model of depression when compared with control animals. A marked rise in the concentrations of these carbohydrates and glucose transporters were evident in prenatally stressed animals subjected to acute stress and glucose loading in adulthood. To determine whether elevated levels of brain glucose are associated with a change in its metabolism in this model, we assessed key glycolytic enzymes (hexokinase, phosphofructokinase and pyruvate kinase), products of glycolysis, i.e., pyruvate and lactate, and two selected enzymes of the tricarboxylic acid cycle (pyruvate dehydrogenase and α-ketoglutarate dehydrogenase) in the hippocampus and frontal cortex. Additionally, we assessed glucose-6-phosphate dehydrogenase activity, a key enzyme in the pentose phosphate pathway (PPP). Prenatal stress increased the levels of phosphofructokinase, an important glycolytic enzyme, in the hippocampus and frontal cortex. However, prenatal stress had no effect on hexokinase or pyruvate kinase levels. The lactate concentration was elevated in prenatally stressed rats in the frontal cortex, and pyruvate levels remained unchanged. Among the tricarboxylic acid cycle enzymes, prenatal stress decreased the level of pyruvate dehydrogenase in the hippocampus, but it had no effect on α-ketoglutarate dehydrogenase. Like in the case of glucose and its transporters, also in the present study, differences in markers of glucose metabolism between control animals and those subjected to prenatal stress were not observed under basal conditions but in rats subjected to acute stress and glucose load in adulthood. Glucose-6-phosphate dehydrogenase activity was not reduced by prenatal stress but was found to be even higher in animals exposed to all experimental conditions, i.e., prenatal stress, acute stress, and glucose administration. Our data indicate that glycolysis is increased and the Krebs cycle is decreased in the brain of a prenatal stress animal model of depression. Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

Continuous D-tagatose production by immobilized thermostable L-arabinose isomerase in a packed-bed bioreactor.

PubMed

Ryu, Se-Ah; Kim, Chang Sup; Kim, Hye-Jung; Baek, Dae Heoun; Oh, Deok-Kun

2003-01-01

D-Tagatose was continuously produced using thermostable L-arabinose isomerase immobilized in alginate with D-galactose solution in a packed-bed bioreactor. Bead size, L/D (length/diameter) of reactor, dilution rate, total loaded enzyme amount, and substrate concentration were found to be optimal at 0.8 mm, 520/7 mm, 0.375 h(-1), 5.65 units, and 300 g/L, respectively. Under these conditions, the bioreactor produced about 145 g/L tagatose with an average productivity of 54 g tagatose/L x h and an average conversion yield of 48% (w/w). Operational stability of the immobilized enzyme was demonstrated, with a tagatose production half-life of 24 days.

Mediator-free interaction of glucose oxidase, as model enzyme for immobilization, with Al-doped and undoped ZnO thin films laser-deposited on polycarbonate supports.

PubMed

V T K P, Fidal; Inguva, Saikumar; Krishnamurthy, Satheesh; Marsili, Enrico; Mosnier, Jean-Paul; T S, Chandra

2017-01-01

Al doped and undoped ZnO thin films were deposited by pulsed-laser deposition on polycarbonate sheets. The films were characterized by optical transmission, Hall effect measurement, XRD and SEM. Optical transmission and surface reflectometry studies showed good transparency with thicknesses ∼100nm and surface roughness of 10nm. Hall effect measurements showed that the sheet carrier concentration was -1.44×10 15 cm -2 for AZO and -6×10 14 cm -2 for ZnO. The films were then modified by drop-casting glucose oxidase (GOx) without the use of any mediators. Higher protein concentration was observed on ZnO as compared to AZO with higher specific activity for ZnO (0.042Umg -1 ) compared to AZO (0.032Umg -1 ), and was in agreement with cyclic voltemmetry (CV). X-ray photoelectron spectroscopy (XPS) suggested that the protein was bound by dipole interactions between AZO lattice oxygen and the amino group of the enzyme. Chronoamperometry showed sensitivity of 5.5μAmM -1 cm -2 towards glucose for GOx/AZO and 2.2μAmM -1 cm -2 for GOx/ZnO. The limit of detection (LoD) was 167μM of glucose for GOx/AZO, as compared to 360μM for GOx/ZnO. The linearity was 0.28-28mM for GOx/AZO whereas it was 0.6-28mM for GOx/ZnO with a response time of 10s. Possibly due to higher enzyme loading, the decrease of impedance in presence of glucose was larger for GOx/ZnO as compared to GOx/AZO in electrochemical impedance spectroscopy (EIS). Analyses with clinical blood serum samples showed that the systems had good reproducibility and accuracy. The characteristics of novel ZnO and AZO thin films with GOx as a model enzyme, should prove useful for the future fabrication of inexpensive, highly sensitive, disposable electrochemical biosensors for high throughput diagnostics. Copyright © 2016 Elsevier Inc. All rights reserved.

Simultaneous saccharification and co-fermentation of paper sludge to ethanol by Saccharomyces cerevisiae RWB222--Part I: kinetic modeling and parameters.

PubMed

Zhang, Jiayi; Shao, Xiongjun; Townsend, Oliver V; Lynd, Lee R

2009-12-01

A kinetic model was developed to predict batch simultaneous saccharification and co-fermentation (SSCF) of paper sludge by the xylose-utilizing yeast Saccharomyces cerevisiae RWB222 and the commercial cellulase preparation Spezyme CP. The model accounts for cellulose and xylan enzymatic hydrolysis and competitive uptake of glucose and xylose. Experimental results show that glucan and xylan enzymatic hydrolysis are highly correlated, and that the low concentrations of xylose encountered during SSCF do not have a significant inhibitory effect on enzymatic hydrolysis. Ethanol is found to not only inhibit the specific growth rate, but also to accelerate cell death. Glucose and xylose uptake rates were found to be competitively inhibitory, but this did not have a large impact during SSCF because the sugar concentrations are low. The model was used to evaluate which constants had the greatest impact on ethanol titer for a fixed substrate loading, enzyme loading, and fermentation time. The cellulose adsorption capacity and cellulose hydrolysis rate constants were found to have the greatest impact among enzymatic hydrolysis related constants, and ethanol yield and maximum ethanol tolerance had the greatest impact among fermentation related constants.

Analysis of enzyme production by submerged culture of Aspergillus oryzae using whole barley.

PubMed

Masuda, Susumu; Kikuchi, Kaori; Matsumoto, Yuko; Sugimoto, Toshikazu; Shoji, Hiroshi; Tanabe, Masayuki

2009-10-01

We have reported on high enzyme production by submerged culture of Aspergillus kawachii using barley with the husk (whole barley). To elucidate the mechanism underlying this high enzyme production, we performed a detailed analysis. Aspergillus oryzae RIB40 was submerged-cultured using whole barley and milled whole barley. Enzyme production was analyzed in terms of changes in medium components and gene expression levels. When whole barley was used, high production of glucoamylase and alpha-amylase and high gene expression levels of these enzymes were observed. Low ammonium concentrations were maintained with nitrate ion uptake continuing into the late stage using whole barley. These findings suggest that the sustainability of nitrogen metabolism is related to high enzyme production, and that a mechanism other than that associated with the conventional amylase expression system is involved in this relationship.

Comparative evaluation of acid and alkaline sulfite pretreatments for enzymatic saccharification of bagasses from three different sugarcane hybrids.

PubMed

Monte, Joseana R; Laurito-Friend, Debora F; Ferraz, André; Milagres, Adriane M F

2018-04-26

Sugarcane bagasses from three experimental sugarcane hybrids and a mill-reference sample were used to compare the efficiency and mode of action of acid and alkaline sulfite pretreatment processes. Varied chemical loads and reaction temperatures were used to prepare samples with distinguished characteristics regarding xylan and lignin removals, as well as sulfonation levels of residual lignins. The pretreatment with low sulfite loads (5%) under acidic conditions (pH 2) provided maximum glucose yield of 70% during enzymatic hydrolysis with cellulases (10 FPU/g) and β-glucosidases (20 UI/g bagasse). In this case, glucan enzymatic conversion from pretreated materials was mostly associated with extensive xylan removal (70-100%) and partial delignification occurred during the pretreatment. The use of low sulfite loads under acidic conditions required pretreatment temperatures of 160°C. In contrast, at a lower pretreatment temperature (120°C), alkaline sulfite process achieved similar glucan digestibility, but required a higher sulfite load (7.5%). Residual xylans from acid pretreated materials were almost completely hydrolysed by commercial enzymes, contrasting with relatively lower xylan to xylose conversions observed in alkaline pretreated samples. Efficient xylan removal during acid sulfite pretreatment and also during enzymatic digestion can be useful to enhance glucan accessibility and digestibility by cellulases. Alkaline sulfite process also provided substrates with high glucan digestibility, mainly associated with delignification and sulfonation of residual lignins. The results demonstrate that temperature, pH and sulfite can be combined for reducing lignocellulose recalcitrance and achieve similar glucan conversion rates in the alkaline and acid sulfite pretreated bagasses. This article is protected by copyright. All rights reserved. © 2018 American Institute of Chemical Engineers.

Engineering of GlcNAc-1-Phosphotransferase for Production of Highly Phosphorylated Lysosomal Enzymes for Enzyme Replacement Therapy.

PubMed

Liu, Lin; Lee, Wang-Sik; Doray, Balraj; Kornfeld, Stuart

2017-06-16

Several lysosomal enzymes currently used for enzyme replacement therapy in patients with lysosomal storage diseases contain very low levels of mannose 6-phosphate, limiting their uptake via mannose 6-phosphate receptors on the surface of the deficient cells. These enzymes are produced at high levels by mammalian cells and depend on endogenous GlcNAc-1-phosphotransferase α/β precursor to phosphorylate the mannose residues on their glycan chains. We show that co-expression of an engineered truncated GlcNAc-1-phosphotransferase α/β precursor and the lysosomal enzyme of interest in the producing cells resulted in markedly increased phosphorylation and cellular uptake of the secreted lysosomal enzyme. This method also results in the production of highly phosphorylated acid β-glucocerebrosidase, a lysosomal enzyme that normally has just trace amounts of this modification.

Digital microfluidics-enabled single-molecule detection by printing and sealing single magnetic beads in femtoliter droplets.

PubMed

Witters, Daan; Knez, Karel; Ceyssens, Frederik; Puers, Robert; Lammertyn, Jeroen

2013-06-07

Digital microfluidics is introduced as a novel platform with unique advantages for performing single-molecule detection. We demonstrate how superparamagnetic beads, used for capturing single protein molecules, can be printed with unprecedentedly high loading efficiency and single bead resolution on an electrowetting-on-dielectric-based digital microfluidic chip by micropatterning the Teflon-AF surface of the device. By transporting droplets containing suspended superparamagnetic beads over a hydrophilic-in-hydrophobic micropatterned Teflon-AF surface, single beads are trapped inside the hydrophilic microwells due to their selective wettability and tailored dimensions. Digital microfluidics presents the following advantages for printing and sealing magnetic beads for single-molecule detection: (i) droplets containing suspended beads can be transported back and forth over the array of hydrophilic microwells to obtain high loading efficiencies of microwells with single beads, (ii) the use of hydrophilic-in-hydrophobic patterns permits the use of a magnet to speed up the bead transfer process to the wells, while the receding droplet meniscus removes excess beads off the chip surface and thereby shortens the bead patterning time, and (iii) reagents can be transported over the printed beads multiple times, while capillary forces and a magnet hold the printed beads in place. High loading efficiencies (98% with a CV of 0.9%) of single beads in microwells were obtained by transporting droplets of suspended beads over the array 10 times in less than 1 min, which is much higher than previously reported methods (40-60%), while the total surface area needed for performing single-molecule detection can be decreased. The performance of the device was demonstrated by fluorescent detection of the presence of the biotinylated enzyme β-galactosidase on streptavidin-coated beads with a linear dynamic range of 4 orders of magnitude ranging from 10 aM to 90 fM.

Methotrexate-F127 conjugated mesoporous zinc hydroxyapatite as an efficient drug delivery system for overcoming chemotherapy resistance in osteosarcoma cells.

PubMed

Meshkini, Azadeh; Oveisi, Hamid

2017-10-01

The resistance of cancer cells to chemotherapeutic agents and the poor selectivity of drugs toward tumor cells are regarded as the main impediments in successful cancer therapy. Currently, the design and fabrication of stimulus-responsive drug delivery systems with high specificity toward cancer cells are gaining increasing attention and they show a promising potential for clinical applications. In this study, mesoporous zinc-substituted hydroxyapatite has been synthesized and served as a drug delivery vehicle owing to its biocompatibility and high drug loading capacity. The mesoporous nanoparticles were decorated with F127 and subsequently conjugated with methotrexate (MTX) through a stable amide linkage. Since folate receptors are overexpressed on many tumor cell surfaces, MTX on the nanocarrier system plays a dual role as a targeting molecule and a chemotherapeutic drug. The evaluation of the drug release profile revealed that MTX was cleaved from the nanoparticles by a certain type of enzyme under low pH conditions that are meant to simulate the intracellular conditions in the lysosome. Cell viability studies on primary osteosarcoma cells (Saos-2) and MTX-resistance cell lines (RSaos-2/MTX) revealed that the drug-loaded nanoparticles possess high antitumor efficacy on both of the cell lines relative to free MTX. It was also found that the inhibition of P-glycoproteins by F127 and the release of Zn 2+ ions from the nanoparticles in an acidic environment effectively potentiate the antitumor efficacy of the drug-loaded nanoparticles, leading to caspase-mediated cell death. Based on these data, MTX-F127@ZnHAP nanoparticles are pH-responsive nanocarriers with precise controlled drug release and targeting effect. Therefore, they are considered to be promising candidates capable of overcoming resistance in osteosarcoma cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Ligand-conjugated mesoporous silica nanorattles based on enzyme targeted prodrug delivery system for effective lung cancer therapy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sundarraj, Shenbagamoorthy, E-mail: sundarrajbu09@gmail.com; Thangam, Ramar; Department of Virology, King Institute of Preventive Medicine and Research, Guindy, Chennai 600 032, TN

2014-03-15

Epidermal growth factor receptor antibody (EGFRAb) conjugated silica nanorattles (SNs) were synthesized and used to develop receptor mediated endocytosis for targeted drug delivery strategies for cancer therapy. The present study determined that the rate of internalization of silica nanorattles was found to be high in lung cancer cells when compared with the normal lung cells. EGFRAb can specifically bind to EGFR, a receptor that is highly expressed in lung cancer cells, but is expressed at low levels in other normal cells. Furthermore, in vitro studies clearly substantiated that the cPLA{sub 2}α activity, arachidonic acid release and cell proliferation were considerablymore » reduced by pyrrolidine-2 loaded EGFRAb-SN in H460 cells. The cytotoxicity, cell cycle arrest and apoptosis were significantly induced by the treatment of pyrrolidine-2 loaded EGFRAb-SN when compared with free pyrrolidine-2 and pyrrolidine-2 loaded SNs in human non-small cell lung cancer cells. An in vivo toxicity assessment showed that silica nanorattles and EGFRAb-SN-pyrrolidine-2 exhibited low systemic toxicity in healthy Balb/c mice. The EGFRAb-SN-pyrrolidine-2 showed a much better antitumor activity (38%) with enhanced tumor inhibition rate than the pyrrolidine-2 on the non-small cell lung carcinoma subcutaneous model. Thus, the present findings validated the low toxicity and high therapeutic potentials of EGFRAb-SN-pyrrolidine-2, which may provide a convincing evidence of the silica nanorattles as new potential carriers for targeted drug delivery systems. - Highlights: • EGFRAb-SN developed for receptor-mediated Drug delivery system (DDS). • EGFRAb-SN-pyrrolidine-2 targeted DDS for cPLA2α inhibition in NSLC. • Study indicates EGFRAb-SN-pyrrolidine-2 as an efficient in target dug delivery carrier. • Study explains entire efficiency of EGFRAb-SN-pyrrolidine-2 in vitro and in vivo models.« less

Molecular architectures and functions of radical enzymes and their (re)activating proteins.

PubMed

Shibata, Naoki; Toraya, Tetsuo

2015-10-01

Certain proteins utilize the high reactivity of radicals for catalysing chemically challenging reactions. These proteins contain or form a radical and therefore named 'radical enzymes'. Radicals are introduced by enzymes themselves or by (re)activating proteins called (re)activases. The X-ray structures of radical enzymes and their (re)activases revealed some structural features of these molecular apparatuses which solved common enigmas of radical enzymes—i.e. how the enzymes form or introduce radicals at the active sites, how they use the high reactivity of radicals for catalysis, how they suppress undesired side reactions of highly reactive radicals and how they are (re)activated when inactivated by extinction of radicals. This review highlights molecular architectures of radical B12 enzymes, radical SAM enzymes, tyrosyl radical enzymes, glycyl radical enzymes and their (re)activating proteins that support their functions. For generalization, comparisons of the recently reported structures of radical enzymes with those of canonical radical enzymes are summarized here. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Soil nitrogen mineralization and enzymatic activities in fire and fire surrogate treatments in California

Treesearch

J. R. Miesel; R. E. J. Boerner; C. N. Skinner

2011-01-01

Forest thinning and prescribed fire are management strategies used to reduce hazardous fuel loads and catastrophic wildfires in western mixed-conifer forests. We evaluated effects of thinning (Thin) and prescribed fire (Burn), alone and in combination (Thin+Burn), on N transformations and microbial enzyme activities relative to an untreated control (Control) at 1 and 3...

Synthesis of novel laccase-biotitania biocatalysts for malachite green decolorization.

PubMed

Zhang, Xinying; Wang, Meiyin; Lin, Linlin; Xiao, Gao; Tang, Zhenping; Zhu, Xuefeng

2018-07-01

Biomimetic mineralization has emerged as a novel tool for generating excellent supports for enzyme stabilization. In this work, protamine was used to induce titanium (IV) bis(ammonium lactato) dihydroxide (Ti-BALDH) into titania nanoparticles. This biomimetic titanification process was adopted for laccase immobilization. Laccase-biotitania biocatalyst was prepared and the effect of different parameters (buffer solution, titania precursor concentration, protamine concentration, and enzyme loading) on the encapsulation efficiency and recovery of laccase were evaluated. Compared with free laccase, the thermal and pH stability of immobilized laccase were improved significantly. In addition, laccase loaded on titania was effective at enhancing its storage stability. After seven consecutive cycles, the immobilized laccase still retained 51% of its original activity. Finally, laccase-biotitania biocatalysts showed good performance on decolorization of malachite green (MG), which can be attributed to an adsorption and degradation effect. The intermediates of the MG degradation were identified by gas chromatography-mass spectrometry (GC-MS) analysis, and the most probable degradation pathway was proposed. This study provides deeper understanding of the laccase-biotitania particles as a fast biocatalyst for MG decolorization. Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Modeling and optimization of phospholipase A₁-catalyzed hydrolysis of phosphatidylcholine using response surface methodology for lysophosphatidylcholine production.

PubMed

Lim, Chang Wan; Kim, Byung Hee; Kim, In-Hwan; Lee, Moon-Won

2015-01-01

Modeling the phospholipase A1 (PLA1 )-catalyzed partial hydrolysis of soy phosphatidylcholine (PC) in hexane for the production of lysophosphatidylcholine (LPC) and optimizing the reaction conditions using response surface methodology were described. The reaction was performed with 4 g of PC in a stirred batch reactor using a commercial PLA1 (Lecitase Ultra) as the biocatalyst. The effects of temperature, reaction time, water content, and enzyme loading on LPC and glycerylphosphorylcholine (GPC) content in the reaction products were elucidated using the models established. Optimal reaction conditions for maximizing the LPC content while suppressing acyl migration, which causes GPC formation, were as follows: temperature, 60°C; reaction time, 3 h; water content, 10% of PC; and enzyme loading, 1% of PC. When the reaction was conducted with 40 g of PC under these conditions, the reaction products contained 83.7 mol % LPC and were free of GPC. LPC had a higher total unsaturated fatty acid content than original PC had and was mainly composed of linoleic acid (78.0 mol % of the total fatty acids). © 2014 American Institute of Chemical Engineers.

Enzymatic hydrolysis of pretreated Alfa fibers (Stipa tenacissima) using β-d-glucosidase and xylanase of Talaromyces thermophilus from solid-state fermentation.

PubMed

Mallek-Fakhfakh, Hanen; Fakhfakh, Jawhar; Walha, Kamel; Hassairi, Hajer; Gargouri, Ali; Belghith, Hafedh

2017-10-01

This work aims at realizing an optimal hydrolysis of pretreated Alfa fibers (Stipa tenacissima) through the use of enzymes produced from Talaromyces thermophilus AX4, namely β-d-glucosidase and xylanase, by a solid state fermentation process of an agro-industrial waste (wheat bran supplemented with lactose). The carbon source was firstly selected and the optimal values of three other parameters were determined: substrate loading (10g), moisture content (85%) and production time (10days); which led to an optimized enzymatic juice. The outcome was then supplemented with cellulases of T. reesei and used to optimize the enzymatic saccharification of alkali-pretreated Alfa fibers (PAF). The maximum saccharification yield of 83.23% was achieved under optimized conditions (substrate concentration 3.7% (w/v), time 144h and enzyme loading of 0.8 FPU, 15U CMCase, 60U β-d-glucosidase and 125U xylanase).The structural modification of PAF due to enzymatic saccharification was supported by the changes of morphologic and chemical composition observed through macroscopic representation, FTIR and X-Ray analysis. Copyright © 2017 Elsevier B.V. All rights reserved.

Synthesis of structured lipids by transesterification of trilinolein catalyzed by Lipozyme IM60.

PubMed

Sellappan, S; Akoh, C C

2001-04-01

Structured lipids (SL) containing caprylic, stearic, and linoleic acids were synthesized by enzymatic transesterification using Lipozyme IM60. Pure trilinolein and free fatty acids were used as substrates. Incorporation of stearic acid was higher than that of caprylic acid in all parameters. Highest incorporations of both acids were achieved at 32 h, mole ratio of 1:4:4 (trilinolein/caprylic/stearic acids), water content of 1% (wt %), temperature of 55 degrees C, and 10% (wt %) enzyme load. The maximal incorporations of caprylic and stearic acids were 23.73 and 62.46 mol %, respectively. Reaction time, water content, and enzyme load had major influences on the reaction, whereas substrate mole ratio and temperature showed less influence. Lipozyme showed good stability over six reuses. Differential scanning calorimetric analysis of SL gave a melting profile with a very low melting peak of 0-3.3 degrees C and a solid fat content of 25.21% at 0 degrees C. The melting profile and solid fat content of SL were compared with those of fats extracted from commercially available solid and liquid margarine products. The data suggest that enzymatically produced SL could be used in liquid margarine products.

Enzyme immobilization in novel alginate-chitosan core-shell microcapsules.

PubMed

Taqieddin, Ehab; Amiji, Mansoor

2004-05-01

Alginate-chitosan core-shell microcapsules were prepared in order to develop a biocompatible matrix for enzyme immobilization, where the protein is retained either in a liquid or solid core and the shell allows permeability control over substrates and products. The permeability coefficients of different molecular weight compounds (vitamin B2, vitamin B12, and myoglobin) were determined through sodium tripolyphosphate (Na-TPP)-crosslinked chitosan membrane. The microcapsule core was formed by crosslinking sodium alginate with either calcium or barium ions. The crosslinked alginate core was uniformly coated with a chitosan layer and crosslinked with Na-TPP. In the case of calcium alginate, the phosphate ions of Na-TPP were able to extract the calcium ions from alginate and liquefy the core. A model enzyme, beta-galactosidase, was immobilized in the alginate core and the catalytic activity was measured with o-nitrophenyl-beta-D-galactopyranoside (ONPG). Change in the activity of free and immobilized enzyme was determined at three different temperatures. Na-TPP crosslinked chitosan membranes were found to be permeable to solutes of up to 17,000Da molecular weight. The enzyme loading efficiency was higher in the barium alginate core (100%) as compared to the calcium alginate core (60%). The rate of ONPG conversion to o-nitrophenol was faster in the case of calcium alginate-chitosan microcapsules as compared to barium alginate-chitosan microcapsules. Barium alginate-chitosan microcapsules, however, did improve the stability of the enzyme at 37 degrees C relative to calcium alginate-chitosan microcapsules or free enzyme. This study illustrates a new method of enzyme immobilization for biotechnology applications using liquid or solid core and shell microcapsule technology.

Impact of heavy metal on activity of some microbial enzymes in the riverbed sediments: Ecotoxicological implications in the Ganga River (India).

PubMed

Jaiswal, Deepa; Pandey, Jitendra

2018-04-15

We studied the extracellular enzyme activity (EEA) in the riverbed sediment along a 518km gradient of the Ganga River receiving carbon and nutrient load from varied human sources. Also, we tested, together with substrate-driven stimulation, if the heavy metal accumulated in the sediment inhibits enzyme activities. Because pristine values are not available, we considered Dev Prayag, a least polluted site located 624km upstream to main study stretch, as a reference site. There were distinct increases in enzyme activities in the sediment along the study gradient from Dev Prayag, however, between-site differences were in concordance with sediment carbon(C), nitrogen (N) and phosphorus (P). Fluorescein diacetate hydrolysis (FDAase), β-glucosidase (Glu) and protease activities showed positive correlation with C, N and P while alkaline phosphatase was found negatively correlated with P. Enzyme activities were found negatively correlated with heavy metal, although ecological risk index (E R i ) varied with site and metal species. Dynamic fit curves showed significant positive correlation between heavy metal and microbial metabolic quotient (qCO 2 ) indicating a decrease in microbial activity in response to increasing heavy metal concentrations. This study forms the first report linking microbial enzyme activities to regional scale sediment heavy metal accumulation in the Ganga River, suggests that the microbial enzyme activities in the riverbed sediment were well associated with the proportion of C, N and P and appeared to be a sensitive indicator of C, N and P accumulation in the river. Heavy metal accumulated in the sediment inhibits enzyme activities, although C rich sediment showed relatively low toxicity due probably to reduced bioavailability of the metal. The study has relevance from ecotoxicological as well as from biomonitoring perspectives. Copyright © 2017 Elsevier Inc. All rights reserved.

Abeta-degrading enzymes in Alzheimer's disease.

PubMed

Miners, James Scott; Baig, Shabnam; Palmer, Jennifer; Palmer, Laura E; Kehoe, Patrick G; Love, Seth

2008-04-01

In Alzheimer's disease (AD) Abeta accumulates because of imbalance between the production of Abeta and its removal from the brain. There is increasing evidence that in most sporadic forms of AD, the accumulation of Abeta is partly, if not in some cases solely, because of defects in its removal--mediated through a combination of diffusion along perivascular extracellular matrix, transport across vessel walls into the blood stream and enzymatic degradation. Multiple enzymes within the central nervous system (CNS) are capable of degrading Abeta. Most are produced by neurons or glia, but some are expressed in the cerebral vasculature, where reduced Abeta-degrading activity may contribute to the development of cerebral amyloid angiopathy (CAA). Neprilysin and insulin-degrading enzyme (IDE), which have been most extensively studied, are expressed both neuronally and within the vasculature. The levels of both of these enzymes are reduced in AD although the correlation with enzyme activity is still not entirely clear. Other enzymes shown capable of degrading Abetain vitro or in animal studies include plasmin; endothelin-converting enzymes ECE-1 and -2; matrix metalloproteinases MMP-2, -3 and -9; and angiotensin-converting enzyme (ACE). The levels of plasmin and plasminogen activators (uPA and tPA) and ECE-2 are reported to be reduced in AD. Reductions in neprilysin, IDE and plasmin in AD have been associated with possession of APOEepsilon4. We found no change in the level or activity of MMP-2, -3 or -9 in AD. The level and activity of ACE are increased, the level being directly related to Abeta plaque load. Up-regulation of some Abeta-degrading enzymes may initially compensate for declining activity of others, but as age, genetic factors and diseases such as hypertension and diabetes diminish the effectiveness of other Abeta-clearance pathways, reductions in the activity of particular Abeta-degrading enzymes may become critical, leading to the development of AD and CAA.

Performance comparison between multienzymes loaded single and dual electrodes for the simultaneous electrochemical detection of adenosine and metabolites in cancerous cells.

PubMed

Hussain, Khalil K; Akhtar, Mahmood H; Kim, Moo-Hyun; Jung, Dong-Keun; Shim, Yoon-Bo

2018-06-30

The analytical performance of the multi enzymes loaded single electrode sensor (SES) and dual electrode sensor (DES) was compared for the detection of adenosine and metabolites. The SES was fabricated by covalent binding of tri-enzymes, adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP), and xanthine oxidase (XO) along with hydrazine (Hyd) onto a functionalized conducting polymer [2,2:5,2-terthiophene-3-(p-benzoic acid)] (pTTBA). The enzyme reaction electrode in DES was fabricated by covalent binding of ADA and PNP onto pTTBA coated on Au nanoparticles. The detection electrode in DES was constructed by covalent binding of XO and Hyd onto pTTBA coated on porous Au. Due to the higher amount (3.5 folds) of the immobilized enzymes and Hyd onto the DES than SES, and the lower Michaelis constant (Km) value for DES (28.7 µM) compared to SES (36.1 µM), the sensitivity was significantly enhanced for the DES (8.2 folds). The dynamic range obtained using DES was from 0.5 nM to 120.0 µM with a detection limit of 1.43 nM ± 0.02, 0.76 nM ± 0.02, and 0.48 nM ± 0.01, for adenosine (AD), inosine (IN), and hypoxanthine (Hypo) respectively. Further, the DES was coupled with an electrochemical potential modulated microchannel for the separation and simultaneous detection of AD, IN, and Hypo in an extracellular matrix of cancerous (A549) and non-cancerous (Vero) cells. The sensor probe confirms a higher basal level of extracellular AD and its metabolites in cancer cells compared to normal cells. In addition, the effect of dipyridamole on released adenosine in A549 cells was investigated. Copyright © 2018 Elsevier B.V. All rights reserved.

Microtubule Depolymerization as a Driver for Chromosome Motion

NASA Astrophysics Data System (ADS)

McIntosh, Richard

2014-03-01

Microtubules (MTs) are rigid polymers of the protein, tubulin, which function as intracellular struts. They are also tracks along which motor enzymes can run, carrying cargo to specific cellular locations. Most MTs are dynamic; they assemble and disassemble rapidly, particularly during cell division when the cell forms the ``mitotic spindle,'' a machine that organizes the duplicated chromosomes into a planar disk, then pulls the duplicate copies apart, moving them to opposite ends of the cell. This process is necessary for the daughter cells to have a full complement of DNA. The mitotic spindle is a labile framework that exerts several kinds of forces on the chromosomes to move them in well organized ways. It contains many motor enzymes that contribute to spindle formation, but genetic evidence shows that the motors that attach to chromosomes and might contribute to chromosome motion are dispensable for normal mitosis. Apparently MT dynamics can also serve as a motor and is an important source of force for chromosome motion. We have studied this process and find that MTs can be coupled to a load by specific spindle proteins so that MT depolymerization can exert substantial force. With the yeast protein, Dam1, a single MT can generate 30 pN, about 5-fold more than is generated by a motor enzyme like kinesin or myosin. The resulting motions are processive, so a depolymerizing MT can carry its load for many micrometers. However, Dam1 is found only in fungi. We have therefore sought other proteins that can serve as analogous couplers. Several MT-dependent motor enzymes can do the job in ways that do not require ATP, their normal source of energy. Some non-motor MT-associated proteins will also work, e.g., the kinetochore proteins NDC80 and CENP-F. Data will be presented that show the strengths and weaknesses of each coupler, allowing some generalization about how the mitotic machinery works. Supported by NIH GM033787.

Reducing sugar production of sweet sorghum bagasse kraft pulp

NASA Astrophysics Data System (ADS)

Solihat, Nissa Nurfajrin; Fajriutami, Triyani; Adi, Deddy Triyono Nugroho; Fatriasari, Widya; Hermiati, Euis

2017-01-01

Kraft pulping of sweet sorghum bagasse (SSB) has been used for effective delignification method for cellulose production. This study was conducted to evaluate the performance pulp kraft of SSB for reducing sugar production. The study intended to investigate the effect of active alkali and sulfidity loading variation of SSB pulp kraft on reducing sugar yield per biomass. The SSB pulp was prepared after pulping using three variations of active alkali (17, 19, and 22%) and sulfidity loading (20, 22, and 24%) at 170°C for 4 h with liquor to wood ratio of 10. A total of 9 pulps were obtained from these pretreatments. Delignification pretreatment has been succesfully removed lignin and hemicellulose more than 90% and 50%, respectively. Increasing active alkali and sulfidity loading has significantly increased lignin removal caused by disruption of the cell wall structure for releasing lignin into black liquor in the cellulose extraction. The enzymatic hydrolysis of pulp was carried out with cellulase loading of 40 FPU per g substrate in the shaking incubator at 50°C and 150 rpm for 78 h. For each 24 h, the reducing sugar yield (DNS assay) has been observed. Even though the lignin and hemicellulose loss occurred along with higher active alkali loading, this condition tends to decrease its yield. The reducing sugar concentration varied between 7-8 g/L. Increasing active alkali and sulfidity was significantly decreased the reducing sugar per biomass. Pulp delignified by 17% active alkali and 20% sulfidity has demonstrated the maximum reducing sugar yield per biomass of 45.57% resulted after 72 h enzymatic hydrolysis. These results indicated that kraft pulping was success to degrade more lignin and hemicellulose content to facilitate the enzyme for breaking down the cellulose into its sugar monomer. A high loss of lignin and hemicellulose are not single factor to improve digestibility of SSB. This sugar has potential for yeast fermented into bioethanol.

Diagnosis and management of trimethylaminuria (FMO3 deficiency) in children.

PubMed

Chalmers, R A; Bain, M D; Michelakakis, H; Zschocke, J; Iles, R A

2006-02-01

Persistent trimethylaminuria in children is caused by autosomal recessively inherited impairment of hepatic trimethylamine (TMA) oxidation due to deficiency of flavin monooxygenase 3 (FMO3) secondary to mutations in the FMO3 gene. Trimethylaminuria or 'fish odour syndrome' is due to excessive excretion into body fluids and breath of TMA derived from the enterobacterial metabolism of dietary precursors. The disorder is present from birth but becomes apparent as foods containing high amounts of choline or of trimethylamine N-oxide (TMAO) from marine (sea or saltwater) fish are introduced into the diet. In our experience, trimethylaminuria (FMO3 deficiency) in children is rare. We have compared the dynamics and diagnostic efficacy of choline loading with marine fish meals in six children with trimethylaminuria. Loading with a marine fish meal provides a simple and acceptable method for confirmation of diagnosis of suspected trimethylaminuria in children, with the effects being cleared more quickly than with a choline load test. However, oral loading with choline bitartrate allows estimation of residual oxidative capacity in vivo and is a useful adjunct to molecular studies. Patients homozygous for the 'common' P153L mutation in the FMO3 gene showed virtual complete lack of residual TMA N-oxidative capacity, consistent with a nonfunctional or absent FMO3 enzyme, whereas a patient with the M82T mutation showed some residual oxidative capacity. A patient compound heterozygous for two novel mutations, G193E and R483T, showed considerable residual N-oxidative capacity. A further patient, heterozygous for two novel sequence variations in the FMO3 gene, consistently showed malodour and elevated urinary TMA/TMAO ratios under basal conditions but a negative response to both choline and marine fish meal loading. Comparison of the effects of administration of antibiotics (metronidazole, amoxicillin, neomycin) on gut bacterial production of trimethylamine from choline showed they all reduced TMA production to a limited extent, with neomycin being most effective. 'Best-practice' diagnostic and treatment guidelines are summarized.

Pancreatic and Pancreatic-Like Microbial Proteases Accelerate Gut Maturation in Neonatal Rats

PubMed Central

Prykhodko, Olena; Pierzynowski, Stefan G.; Nikpey, Elham; Arevalo Sureda, Ester; Fedkiv, Olexandr; Weström, Björn R.

2015-01-01

Objectives Postnatal gut maturation in neonatal mammals, either at natural weaning or after precocious inducement, is coinciding with enhanced enzymes production by exocrine pancreas. Since the involvement of enzymes in gut functional maturation was overlooked, the present study aimed to investigate the role of enzymes in gut functional maturation using neonatal rats. Methods Suckling rats (Rattus norvegicus) were instagastrically gavaged with porcine pancreatic enzymes (Creon), microbial-derived amylase, protease, lipase and mixture thereof, while controls received α-lactalbumin or water once per day during 14–16 d of age. At 17 d of age the animals were euthanized and visceral organs were dissected, weighed and analyzed for structural and functional properties. For some of the rats, gavage with the macromolecular markers such as bovine serum albumin and bovine IgG was performed 3 hours prior to blood collection to assess the intestinal permeability. Results Gavage with the pancreatic or pancreatic-like enzymes resulted in stimulated gut growth, increased gastric acid secretion and switched intestinal disaccharidases, with decreased lactase and increased maltase and sucrase activities. The fetal-type vacuolated enterocytes were replaced by the adult-type in the distal intestine, and macromolecular transfer to the blood was declined. Enzyme exposure also promoted pancreas growth with increased amylase and trypsin production. These effects were confined to the proteases in a dose-dependent manner. Conclusion Feeding exogenous enzymes, containing proteases, induced precocious gut maturation in suckling rats. This suggests that luminal exposure to proteases by oral loading or, possibly, via enhanced pancreatic secretion involves in the gut maturation of young mammals. PMID:25658606

Pancreatic and pancreatic-like microbial proteases accelerate gut maturation in neonatal rats.

PubMed

Prykhodko, Olena; Pierzynowski, Stefan G; Nikpey, Elham; Arevalo Sureda, Ester; Fedkiv, Olexandr; Weström, Björn R

2015-01-01

Postnatal gut maturation in neonatal mammals, either at natural weaning or after precocious inducement, is coinciding with enhanced enzymes production by exocrine pancreas. Since the involvement of enzymes in gut functional maturation was overlooked, the present study aimed to investigate the role of enzymes in gut functional maturation using neonatal rats. Suckling rats (Rattus norvegicus) were instagastrically gavaged with porcine pancreatic enzymes (Creon), microbial-derived amylase, protease, lipase and mixture thereof, while controls received α-lactalbumin or water once per day during 14-16 d of age. At 17 d of age the animals were euthanized and visceral organs were dissected, weighed and analyzed for structural and functional properties. For some of the rats, gavage with the macromolecular markers such as bovine serum albumin and bovine IgG was performed 3 hours prior to blood collection to assess the intestinal permeability. Gavage with the pancreatic or pancreatic-like enzymes resulted in stimulated gut growth, increased gastric acid secretion and switched intestinal disaccharidases, with decreased lactase and increased maltase and sucrase activities. The fetal-type vacuolated enterocytes were replaced by the adult-type in the distal intestine, and macromolecular transfer to the blood was declined. Enzyme exposure also promoted pancreas growth with increased amylase and trypsin production. These effects were confined to the proteases in a dose-dependent manner. Feeding exogenous enzymes, containing proteases, induced precocious gut maturation in suckling rats. This suggests that luminal exposure to proteases by oral loading or, possibly, via enhanced pancreatic secretion involves in the gut maturation of young mammals.

Production of Pectate Lyase by Penicillium viridicatum RFC3 in Solid-State and Submerged Fermentation

PubMed Central

Ferreira, Viviani; da Silva, Roberto; Silva, Dênis; Gomes, Eleni

2010-01-01

Pectate lyase (PL) was produced by the filamentous fungus Penicillium viridicatum RFC3 in solid-state cultures of a mixture of orange bagasse and wheat bran (1 : 1 w/w), or orange bagasse, wheat bran and sugarcane bagasse (1 : 1 : 0.5 w/w), and in a submerged liquid culture with orange bagasse and wheat bran (3%) as the carbon source. PL production was highest (1,500 U  mL−1 or 300 Ug−1 of substrate) in solid-state fermentation (SSF) on wheat bran and orange bagasse at 96 hours. PL production in submerged fermentation (SmF) was influenced by the initial pH of the medium. With the initial pH adjusted to 4.5, 5.0, and 5.5, the peak activity was observed after 72, 48, and 24 hours of fermentation, respectively, when the pH of the medium reached the value 5.0. PL from SSF and SmF were loaded on Sephadex-G75 columns and six activity peaks were obtained from crude enzyme from SSF and designated PL I, II, III, IV, V, and VI, while five peaks were obtained from crude enzyme from SmF and labeled PL  I′, II′, III′, IV′, and VII′. Crude enzyme and fraction III from each fermentative process were tested further. The optimum pH for crude PL from either process was 5.5, while that for PL III was 8.0. The maximum activity of enzymes from SSF was observed at 35°C, but crude enzyme was more thermotolerant than PL III, maintaining its maximum activity up to 45°C. Crude enzyme from SmF and PL III′ showed thermophilic profiles of activity, with maximum activity at 60 and 55°C, respectively. In the absence of substrate, the crude enzyme from SSF was stable over the pH range 3.0–10.0 and PL III was most stable in the pH range 4.0–7.0. Crude enzyme from SmF retained 70%–80% of its maximum activity in the acid-neutral pH range (4.0–7.0), but PIII showed high stability at alkaline pH (7.5–9.5). PL from SSF was more thermolabile than that from SmF. The latter maintained 60% of its initial activity after 1 h at 55°C. The differing behavior of the enzymes with respect to pH and temperature suggests that they are different isozymes. PMID:20689719

Reverse-Bumpy-Ball-Type-Nanoreactor-Loaded Nylon Membranes as Peroxidase-Mimic Membrane Reactors for a Colorimetric Assay for H₂O₂.

PubMed

Tong, Ying; Jiao, Xiangyu; Yang, Hankun; Wen, Yongqiang; Su, Lei; Zhang, Xueji

2016-04-01

Herein we report for the first time fabrication of reverse bumpy ball (RBB)-type-nanoreactor-based flexible peroxidase-mimic membrane reactors (MRs). The RBB-type nanoreactors with gold nanoparticles embedded in the inner walls of carbon shells were loaded on nylon membranes through a facile filtration approach. The as-prepared flexible catalytic membrane was studied as a peroxidase-mimic MR. It was found that the obtained peroxidase-mimic MR could exhibit several advantages over natural enzymes, such as facile and good recyclability, long-term stability and easy storage. Moreover, the RBB NS-modified nylon MRs as a peroxidase mimic provide a useful colorimetric assay for H₂O₂.

EFICAz2.5: application of a high-precision enzyme function predictor to 396 proteomes.

PubMed

Kumar, Narendra; Skolnick, Jeffrey

2012-10-15

High-quality enzyme function annotation is essential for understanding the biochemistry, metabolism and disease processes of organisms. Previously, we developed a multi-component high-precision enzyme function predictor, EFICAz(2) (enzyme function inference by a combined approach). Here, we present an updated improved version, EFICAz(2.5), that is trained on a significantly larger data set of enzyme sequences and PROSITE patterns. We also present the results of the application of EFICAz(2.5) to the enzyme reannotation of 396 genomes cataloged in the ENSEMBL database. The EFICAz(2.5) server and database is freely available with a use-friendly interface at http://cssb.biology.gatech.edu/EFICAz2.5.

Towards improved artificial lungs through biocatalysis.

PubMed

Kaar, Joel L; Oh, Heung-Il; Russell, Alan J; Federspiel, William J

2007-07-01

Inefficient CO(2) removal due to limited diffusion represents a significant barrier in the development of artificial lungs and respiratory assist devices, which use hollow fiber membranes (HFMs) as the blood-gas interface and can require large blood-contacting membrane area. To offset the underlying diffusional challenge, "bioactive" HFMs that facilitate CO(2) diffusion were prepared via covalent immobilization of carbonic anhydrase (CA), an enzyme which catalyzes the conversion of bicarbonate in blood to CO(2), onto the surface of plasma-modified conventional HFMs. This study examines the impact of enzyme attachment on the diffusional properties and the rate of CO(2) removal of the bioactive membranes. Plasma deposition of surface reactive hydroxyls, to which CA could be attached, did not change gas permeance of the HFMs or generate membrane defects, as determined by scanning electron microscopy, when low plasma discharge power and short exposure times were employed. Cyanogen bromide activation of the surface hydroxyls and subsequent modification with CA resulted in near monolayer enzyme coverage (88%) on the membrane. The effect of increased plasma discharge power and exposure time on enzyme loading was negligible while gas permeance studies showed enzyme attachment did not impede CO(2) or O(2) diffusion. Furthermore, when employed in a model respiratory assist device, the bioactive membranes improved CO(2) removal rates by as much as 75% from physiological bicarbonate solutions with no enzyme leaching. These results demonstrate the potential of bioactive HFMs with immobilized CA to enhance CO(2) exchange in respiratory devices.

L: (+)-Lactic acid production from non-food carbohydrates by thermotolerant Bacillus coagulans.

PubMed

Ou, Mark S; Ingram, Lonnie O; Shanmugam, K T

2011-05-01

Lactic acid is used as an additive in foods, pharmaceuticals, and cosmetics, and is also an industrial chemical. Optically pure lactic acid is increasingly used as a renewable bio-based product to replace petroleum-based plastics. However, current production of lactic acid depends on carbohydrate feedstocks that have alternate uses as foods. The use of non-food feedstocks by current commercial biocatalysts is limited by inefficient pathways for pentose utilization. B. coagulans strain 36D1 is a thermotolerant bacterium that can grow and efficiently ferment pentoses using the pentose-phosphate pathway and all other sugar constituents of lignocellulosic biomass at 50°C and pH 5.0, conditions that also favor simultaneous enzymatic saccharification and fermentation (SSF) of cellulose. Using this bacterial biocatalyst, high levels (150-180 g l(-1)) of lactic acid were produced from xylose and glucose with minimal by-products in mineral salts medium. In a fed-batch SSF of crystalline cellulose with fungal enzymes and B. coagulans, lactic acid titer was 80 g l(-1) and the yield was close to 80%. These results demonstrate that B. coagulans can effectively ferment non-food carbohydrates from lignocellulose to L: (+)-lactic acid at sufficient concentrations for commercial application. The high temperature fermentation of pentoses and hexoses to lactic acid by B. coagulans has these additional advantages: reduction in cellulase loading in SSF of cellulose with a decrease in enzyme cost in the process and a reduction in contamination of large-scale fermentations.

Metabolic characterization of anaerobic fungi provides a path forward for bioprocessing of crude lignocellulose

DOE Office of Scientific and Technical Information (OSTI.GOV)

Henske, John K.; Wilken, St. Elmo; Solomon, Kevin V.

The conversion of lignocellulose-rich biomass to bio-based chemicals and higher order fuels remains a grand challenge, as single-microbe approaches often cannot drive both deconstruction and chemical production steps. In contrast, consortia based bioprocessing leverages the strengths of different microbes to distribute metabolic loads and achieve process synergy, product diversity, and bolster yields. Here, we describe a biphasic fermentation scheme that combines the lignocellulolytic action of anaerobic fungi isolated from large herbivores with domesticated microbes for bioproduction. When grown in batch culture, anaerobic fungi release excess sugars from both cellulose and crude biomass due to a wealth of highly expressed carbohydratemore » active enzymes (CAZymes), converting as much as 49% of cellulose to free glucose. This sugar-rich hydrolysate readily supports growth of Saccharomyces cerevisiae, which can be engineered to produce a range of value-added chemicals. Further, construction of metabolic pathways from transcriptomic data reveals that anaerobic fungi do not catabolize all sugars that their enzymes hydrolyze from biomass, leaving other carbohydrates such as galactose, arabinose, and mannose available as nutritional links to other microbes in their consortium. Although basal expression of CAZymes in anaerobic fungi is high, it is drastically amplified by cellobiose breakout products encountered during biomass hydrolysis. Overall, these results suggest that anaerobic fungi provide a nutritional benefit to the rumen microbiome, which can be harnessed to design synthetic microbial communities that compartmentalize biomass degradation and bioproduct formation.« less

Niosome-loaded cold-set whey protein hydrogels.

PubMed

Abaee, Arash; Madadlou, Ashkan

2016-04-01

The α-tocopherol-carrying niosomes with mean diameter of 5.7 μm were fabricated and charged into a transglutaminase-cross-linked whey protein solution that was subsequently gelled with glucono delta-lactone. Encapsulation efficiency of α-tocopherol within niosomes was ≈80% and encapsulation did not influence the radical scavenging activity of α-tocopherol. Fourier transform infrared (FTIR) spectroscopy suggested formation of ε-(γ-glutamyl) lysine cross-linkages by transglutaminase and that enzymatic cross-linking increased proteins hydrophobicity. FTIR also proposed hydrogen bonding between niosomes and proteins. Dynamic rheometry indicated that transglutaminase cross-linking and niosomes charging of the protein solution enhanced the gelation process. However, charging the cross-linked protein solution with niosomal suspension resulted in lower elastic modulus (G') of the subsequently formed gel compared with both non-cross-linked niosome-loaded and cross-linked niosome-free counterparts. Electron microscopy indicated a discontinuous network for the niosome-loaded cross-linked sample. Niosome loading into the protein gel matrix increased its swelling extent in the enzyme-free simulated gastric fluid. Copyright © 2015 Elsevier Ltd. All rights reserved.

Release Profile and Inhibition Test of The Nanoparticles A. Paniculata Extract as Inhibitor of α-Glucosidase in The Process of Carbohydrates Breakdown Into Glucose Diabetes Mellitus

NASA Astrophysics Data System (ADS)

Imansari, Farisa; Sahlan, Muhammad; Arbianti, Rita

2017-07-01

Andrographis paniculata (A.paniculata) contain the main active substances Andrographolide which helps lower glucose levels in diabetics by inhibiting the enzyme α-glucosidase. The ability of the extract A.paniculata in lowering glucose levels will increase with the technique encapsulation with a coating of composition Chitosan-STPP as a drug delivery to the target organ. This study aimed to get an overview of A.paniculata release profile of nanoparticles in a synthetic fluid media with various concentrations of coating and inhibition testing nasty shard extract in inhibiting the enzyme α-glucosidase. This research resulted in nanoparticles by coating efficiency and loading capacity of chitosan greatest variation of 2% and 1% STPP 60% and 46.29%. chitosan greatest variation of 2% and 1% STPP 60% and 46.29%. The ability of A.paniculata extracts as α-glucosidase enzyme inhibitors has been demonstrated in this study, the percent inhibition of 33.17%.

Multistage process for the production of bioethanol from almond shell.

PubMed

Kacem, Imen; Koubaa, Mohamed; Maktouf, Sameh; Chaari, Fatma; Najar, Taha; Chaabouni, Moncef; Ettis, Nadia; Ellouz Chaabouni, Semia

2016-07-01

This work describes the feasibility of using almond shell as feedstock for bioethanol production. A pre-treatment step was carried out using 4% NaOH for 60min at 121°C followed by 1% sulfuric acid for 60min at 121°C. Enzymatic saccharification of the pre-treated almond shell was performed using Penicillium occitanis enzymes. The process was optimized using a hybrid design with four parameters including the incubation time, temperature, enzyme loads, and polyethylene glycol (PEG) concentration. The optimum hydrolysis conditions led to a sugar yield of 13.5%. A detoxification step of the enzymatic hydrolysate was carried out at pH 5 using 1U/ml of laccase enzyme produced by Polyporus ciliatus. Fermenting efficiency of the hydrolysates was greatly improved by laccase treatment, increasing the ethanol yield from 30% to 84%. These results demonstrated the efficiency of using almond shell as a promising source for bioethanol production. Copyright © 2016 Elsevier Ltd. All rights reserved.

A Urea Biosensor from Stacked Sol-Gel Films with Immobilized Nile Blue Chromoionophore and Urease Enzyme.

PubMed

Alqasaimeh, Muawia Salameh; Heng, Lee Yook; Ahmad, Musa

2007-10-11

An optical urea biosensor was fabricated by stacking several layers of sol-gelfilms. The stacking of the sol-gel films allowed the immobilization of a Nile Bluechromoionophore (ETH 5294) and urease enzyme separately without the need of anychemical attachment procedure. The absorbance response of the biosensor was monitoredat 550 nm, i.e. the deprotonation of the chromoionophore. This multi-layer sol-gel filmformat enabled higher enzyme loading in the biosensor to be achieved. The urea opticalbiosensor constructed from three layers of sol-gel films that contained urease demonstrateda much wider linear response range of up to 100 mM urea when compared with biosensorsthat constructed from 1-2 layers of films. Analysis of urea in urine samples with thisoptical urea biosensor yielded results similar to that determined by a spectrophotometricmethod using the reagent p-dimethylaminobenzaldehyde (R² = 0.982, n = 6). The averagerecovery of urea from urine samples using this urea biosensor is approximately 103%.

A Urea Biosensor from Stacked Sol-Gel Films with Immobilized Nile Blue Chromoionophore and Urease Enzyme

PubMed Central

Alqasaimeh, Muawia Salameh; Heng, Lee Yook; Ahmad, Musa

2007-01-01

An optical urea biosensor was fabricated by stacking several layers of sol-gel films. The stacking of the sol-gel films allowed the immobilization of a Nile Blue chromoionophore (ETH 5294) and urease enzyme separately without the need of any chemical attachment procedure. The absorbance response of the biosensor was monitored at 550 nm, i.e. the deprotonation of the chromoionophore. This multi-layer sol-gel film format enabled higher enzyme loading in the biosensor to be achieved. The urea optical biosensor constructed from three layers of sol-gel films that contained urease demonstrated a much wider linear response range of up to 100 mM urea when compared with biosensors that constructed from 1-2 layers of films. Analysis of urea in urine samples with this optical urea biosensor yielded results similar to that determined by a spectrophotometric method using the reagent p-dimethylaminobenzaldehyde (R2 = 0.982, n = 6). The average recovery of urea from urine samples using this urea biosensor is approximately 103%.

Imparting functionality to biocatalysts via embedding enzymes into nanoporous materials by a de novo approach: size-selective sheltering of catalase in metal-organic framework microcrystals.

PubMed

Shieh, Fa-Kuen; Wang, Shao-Chun; Yen, Chia-I; Wu, Chang-Cheng; Dutta, Saikat; Chou, Lien-Yang; Morabito, Joseph V; Hu, Pan; Hsu, Ming-Hua; Wu, Kevin C-W; Tsung, Chia-Kuang

2015-04-08

We develop a new concept to impart new functions to biocatalysts by combining enzymes and metal-organic frameworks (MOFs). The proof-of-concept design is demonstrated by embedding catalase molecules into uniformly sized ZIF-90 crystals via a de novo approach. We have carried out electron microscopy, X-ray diffraction, nitrogen sorption, electrophoresis, thermogravimetric analysis, and confocal microscopy to confirm that the ~10 nm catalase molecules are embedded in 2 μm single-crystalline ZIF-90 crystals with ~5 wt % loading. Because catalase is immobilized and sheltered by the ZIF-90 crystals, the composites show activity in hydrogen peroxide degradation even in the presence of protease proteinase K.

Statistical optimization and operational stability of Rhizomucor miehei lipase supported on magnetic chitosan/chitin nanoparticles for synthesis of pentyl valerate.

PubMed

Rahman, Ida Nurhazwani Abdul; Attan, Nursyafreena; Mahat, Naji Arafat; Jamalis, Joazaizulfazli; Abdul Keyon, Aemi S; Kurniawan, Cepi; Wahab, Roswanira Abdul

2018-04-24

The chemical-catalyzed transesterification process to produce biofuels i.e. pentyl valerate (PeVa) is environmentally unfriendly, energy-intensive with tedious downstream treatment. The present work reports the use of Rhizomucor miehei lipase (RML) crosslinked onto magnetic chitosan/chitin nanoparticles (RML-CS/CH/MNPs). The approach used to immobilize RML onto the CS/CH/MNPs yielded RML-CS/CH/MNPs with an immobilized protein loading and specific activity of 7.6 mg/g and 5.0 U·g -1 , respectively. This was confirmed by assessing data of field emission scanning electron microscopy, X-ray diffraction, thermal gravimetric analysis and Fourier transform infrared spectroscopy. A three-level-four-factor Box-Behnken design (incubation time, temperature, substrate molar ratio, and enzyme loading) was used to optimize the RML-CS/CH/MNP-catalyzed esterification synthesis of PeVa. Under optimum condition, the maximum yield of PeVa (97.8%) can be achieved in 5 h at 50 °C using molar ratio valeric acid:pentanol (1:2) and an enzyme load of 2 mg/mL. Consequently, operational stability experiments showed that the protocol adopted to prepare the CS/CH/MNP nanoparticles had increased the durability of RML. The RML-CS/CH/MNP could catalyze up to eight successive esterification cycles to produce PeVa. The study also demonstrated the functionality of CS/CH/MNP nanoparticles as an eco-friendly support matrix for improving enzymatic activity and operational stability of RML to produce PeVa. Copyright © 2018. Published by Elsevier B.V.

Confocal Raman Microscopy for the Determination of Protein and Quaternary Ammonium Ion Loadings in Biocatalytic Membranes for Electrochemical Energy Conversion and Storage

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cai, Rong; Abdellaoui, Sofiene; Kitt, Jay P.

Here, the need to immobilize active enzyme, while ensuring high rates of substrate turnover and electronic charge transfer with an electrode, is a centrally important challenge in the field of bioelectrocatalysis. In this work, we demonstrate the use of confocal Raman microscopy as a tool for quantitation and molecular-scale structural characterization of ionomers and proteins within biocatalytic membranes to aid in the development of energy efficient biofuel cells. A set of recently available short side chain Aquivion ionomers spanning a range of equivalent weight (EW) suitable for enzyme immobilization was investigated. Aquivion ionomers (790 EW, 830 EW and 980 EW)more » received in the proton-exchanged (SO 3H) form were treated with tetra-n-butylammonium bromide (TBAB) to neutralize the ionomer and expand the size of ionic domains for enzyme incorporation. Through the use of confocal Raman microscopy, membrane TBA+ ion content was predicted in calibration studies to within a few percent of the conventional titrimetric method across the full range of TBA + : SO 3 - ratios of practical interest (0.1 to 1.7). Protein incorporation into membranes was quantified at the levels expected in biofuel cell electrodes. Furthermore, features associated with the catalytically active, enzyme-coordinated copper center were evident between 400 cm -1 - 500 cm -1 in spectra of laccase catalytic membranes, demonstrating the potential to interrogate mechanistic chemistry at the enzyme active site of biocathodes under fuel cell reaction conditions. When benchmarked against the 1100 EW Nafion ionomer in glucose/air enzymatic fuel cells (EFCs), EFCs with laccase air-breathing cathodes prepared from TBA + modified Aquivion ionomers were able to reach maximum power densities (P max) up to 1.5 times higher than EFCs constructed with cathodes prepared from TBA + modified Nafion. The improved performance of EFCs containing the short side chain Aquivion ionomers relative to Nafion is traced to effects of ionomer ion-exchange capacity (IEC, where IEC = EW -1), where the greater density of SO 3 - moieties in the Aquivion materials produces an environment more favourable to mass transport and higher TBA + concentrations.« less

Confocal Raman Microscopy for the Determination of Protein and Quaternary Ammonium Ion Loadings in Biocatalytic Membranes for Electrochemical Energy Conversion and Storage

DOE PAGES

Cai, Rong; Abdellaoui, Sofiene; Kitt, Jay P.; ...

2017-11-14

Here, the need to immobilize active enzyme, while ensuring high rates of substrate turnover and electronic charge transfer with an electrode, is a centrally important challenge in the field of bioelectrocatalysis. In this work, we demonstrate the use of confocal Raman microscopy as a tool for quantitation and molecular-scale structural characterization of ionomers and proteins within biocatalytic membranes to aid in the development of energy efficient biofuel cells. A set of recently available short side chain Aquivion ionomers spanning a range of equivalent weight (EW) suitable for enzyme immobilization was investigated. Aquivion ionomers (790 EW, 830 EW and 980 EW)more » received in the proton-exchanged (SO 3H) form were treated with tetra-n-butylammonium bromide (TBAB) to neutralize the ionomer and expand the size of ionic domains for enzyme incorporation. Through the use of confocal Raman microscopy, membrane TBA+ ion content was predicted in calibration studies to within a few percent of the conventional titrimetric method across the full range of TBA + : SO 3 - ratios of practical interest (0.1 to 1.7). Protein incorporation into membranes was quantified at the levels expected in biofuel cell electrodes. Furthermore, features associated with the catalytically active, enzyme-coordinated copper center were evident between 400 cm -1 - 500 cm -1 in spectra of laccase catalytic membranes, demonstrating the potential to interrogate mechanistic chemistry at the enzyme active site of biocathodes under fuel cell reaction conditions. When benchmarked against the 1100 EW Nafion ionomer in glucose/air enzymatic fuel cells (EFCs), EFCs with laccase air-breathing cathodes prepared from TBA + modified Aquivion ionomers were able to reach maximum power densities (P max) up to 1.5 times higher than EFCs constructed with cathodes prepared from TBA + modified Nafion. The improved performance of EFCs containing the short side chain Aquivion ionomers relative to Nafion is traced to effects of ionomer ion-exchange capacity (IEC, where IEC = EW -1), where the greater density of SO 3 - moieties in the Aquivion materials produces an environment more favourable to mass transport and higher TBA + concentrations.« less

Immune and biochemical responses in skin differ between bovine hosts genetically susceptible and resistant to the cattle tick Rhipicephalus microplus.

PubMed

Franzin, Alessandra Mara; Maruyama, Sandra Regina; Garcia, Gustavo Rocha; Oliveira, Rosane Pereira; Ribeiro, José Marcos Chaves; Bishop, Richard; Maia, Antônio Augusto Mendes; Moré, Daniela Dantas; Ferreira, Beatriz Rossetti; Santos, Isabel Kinney Ferreira de Miranda

2017-01-31

Ticks attach to and penetrate their hosts' skin and inactivate multiple components of host responses in order to acquire a blood meal. Infestation loads with the cattle tick, Rhipicephalus microplus, are heritable: some breeds carry high loads of reproductively successful ticks, whereas in others, few ticks feed and reproduce efficiently. In order to elucidate the mechanisms that result in the different outcomes of infestations with cattle ticks, we examined global gene expression and inflammation induced by tick bites in skins from one resistant and one susceptible breed of cattle that underwent primary infestations with larvae and nymphs of R. microplus. We also examined the expression profiles of genes encoding secreted tick proteins that mediate parasitism in larvae and nymphs feeding on these breeds. Functional analyses of differentially expressed genes in the skin suggest that allergic contact-like dermatitis develops with ensuing production of IL-6, CXCL-8 and CCL-2 and is sustained by HMGB1, ISG15 and PKR, leading to expression of pro-inflammatory chemokines and cytokines that recruit granulocytes and T lymphocytes. Importantly, this response is delayed in susceptible hosts. Histopathological analyses of infested skins showed inflammatory reactions surrounding tick cement cones that enable attachment in both breeds, but in genetically tick-resistant bovines they destabilized the cone. The transcription data provided insights into tick-mediated activation of basophils, which have previously been shown to be a key to host resistance in model systems. Skin from tick-susceptible bovines expressed more transcripts encoding enzymes that detoxify tissues. Interestingly, these enzymes also produce volatile odoriferous compounds and, accordingly, skin rubbings from tick-susceptible bovines attracted significantly more tick larvae than rubbings from resistant hosts. Moreover, transcripts encoding secreted modulatory molecules by the tick were significantly more abundant in larval and in nymphal salivary glands from ticks feeding on susceptible bovines. Compared with tick-susceptible hosts, genes encoding enzymes producing volatile compounds exhibit significantly lower expression in resistant hosts, which may render them less attractive to larvae; resistant hosts expose ticks to an earlier inflammatory response, which in ticks is associated with significantly lower expression of genes encoding salivary proteins that suppress host immunity, inflammation and coagulation.

Ammonium carboxylate production from sugarcane trash using long-term air-lime pretreatment followed by mixed-culture fermentation.

PubMed

Nachiappan, Balasubramaniyan; Fu, Zhihong; Holtzapple, Mark T

2011-03-01

Sugarcane trash (ST) was converted to ammonium carboxylates using a novel bioprocessing strategy known as long-term air-lime pretreatment/mixed-culture fermentation. At mild conditions (50°C, 5 weeks, 1-atm air, and excess lime loading of 0.4 g Ca(OH)(2)/(g dry biomass)), air-lime pretreatment of ST had moderate delignification (64.4%) with little loss in polysaccharides. Without employing detoxification, sterility, expensive nutrients, or costly enzymes, the feedstock (80% treated ST/20% chicken manure) was fermented to primarily ammonium acetate (>75%) and butyrate by a mixed culture of marine microorganisms at 55°C. In the best four-stage countercurrent fermentation, the product yield was 0.36 g total acids/(g VS fed) and the substrate conversion was 64%. Model predictions indicate both high acid concentrations (>47.5 g/L) and high substrate conversions (>70%) are possible at industrial scale. Copyright © 2010 Elsevier Ltd. All rights reserved.

Effect of pretreatment severity in continuous steam explosion on enzymatic conversion of wheat straw: Evidence from kinetic analysis of hydrolysis time courses.

PubMed

Monschein, Mareike; Nidetzky, Bernd

2016-01-01

Focusing on continuous steam explosion, the influence of pretreatment severity due to varied acid loading on hydrolysis of wheat straw by Trichoderma reesei cellulases was investigated based on kinetic evaluation of the saccharification of each pretreated substrate. Using semi-empirical descriptors of the hydrolysis time course, key characteristics of saccharification efficiency were captured in a quantifiable fashion. Not only hydrolysis rates per se, but also the transition point of their bi-phasic decline was crucial for high saccharification degree. After 48h the highest saccharification was achieved for substrate pretreated at relatively low severity (1.2% acid). Higher severity increased enzyme binding to wheat straw, but reduced the specific hydrolysis rates. Higher affinity of the lignocellulosic material for cellulases does not necessarily result in increased saccharification, probably because of lignin modifications occurring at high pretreatment severities. At comparable severity, continuous pretreatment produced a substrate more susceptible to enzymatic hydrolysis than the batch process. Copyright © 2015 Elsevier Ltd. All rights reserved.

Advanced glycation end-products: modifiable environmental factors profoundly mediate insulin resistance

PubMed Central

Ottum, Mona S.; Mistry, Anahita M.

2015-01-01

Advanced glycation end-products are toxic by-products of metabolism and are also acquired from high-temperature processed foods. They promote oxidative damage to proteins, lipids and nucleotides. Aging and chronic diseases are strongly associated with markers for oxidative stress, especially advanced glycation end-products, and resistance to peripheral insulin-mediated glucose uptake. Modifiable environmental factors including high levels of refined and simple carbohydrate diets, hypercaloric diets and sedentary lifestyles drive endogenous formation of advanced glycation end-products via accumulation of highly reactive glycolysis intermediates and activation of the polyol/aldose reductase pathway producing high intracellular fructose. High advanced glycation end-products overwhelm innate defenses of enzymes and receptor-mediated endocytosis and promote cell damage via the pro-inflammatory and pro-oxidant receptor for advanced glycation end-products. Oxidative stress disturbs cell signal transduction, especially insulin-mediated metabolic responses. Here we review emerging evidence that restriction of dietary advanced glycation end-products significantly reduces total systemic load and insulin resistance in animals and humans in diabetes, polycystic ovary syndrome, healthy populations and dementia. Of clinical importance, this insulin sensitizing effect is independent of physical activity, caloric intake and adiposity level. PMID:26236094

Effect of glutamine supplementation on neutrophil function in male judoists.

PubMed

Sasaki, Eiji; Umeda, Takashi; Takahashi, Ippei; Arata, Kojima; Yamamoto, Yousuke; Tanabe, Masaru; Oyamada, Kazuyuki; Hashizume, Erika; Nakaji, Shigeyuki

2013-01-01

Glutamine is an important amino acid for immune function. Though high intensity and prolonged exercise decreases plasma glutamine concentration and causes immune suppression, the relationship between neutrophil functions and glutamine has not yet been found. The purpose of this study was to investigate the impacts of glutamine supplementation on neutrophil function. Twenty-six male university judoists were recruited. Subjects were classified into glutamine and control groups. The glutamine group ingested 3000 mg of glutamine per day and the control group ingested placebo for 2 weeks. Examinations were performed at the start of preunified loading exercise (pre-ULE), then 1 and 2 weeks after ULE (post-ULE). Reactive oxygen species (ROS) production, phagocytic activity, serum opsonic activity and serum myogenic enzymes were measured. Differences between the levels obtained in pre-ULE and post-ULE for the two groups were compared. In the glutamine group, ROS production activity increased 1 week after ULE, whereas it was not observed in the control group (P < 0.001). Though myogenic enzymes increased significantly after ULE (P < 0.001), the glutamine group remained unchanged by supplementation during ULE. Glutamine supplementation has prevented excessive muscle damage and suppression of neutrophil function, especially in ROS production activity, even during an intensive training period. Copyright © 2013 John Wiley & Sons, Ltd.

Androgens and estrogens in skeletal sexual dimorphism

PubMed Central

Laurent, Michaël; Antonio, Leen; Sinnesael, Mieke; Dubois, Vanessa; Gielen, Evelien; Classens, Frank; Vanderschueren, Dirk

2014-01-01

Bone is an endocrine tissue expressing androgen and estrogen receptors as well as steroid metabolizing enzymes. The bioactivity of circulating sex steroids is modulated by sex hormone-binding globulin and local conversion in bone tissue, for example, from testosterone (T) to estradiol (E2) by aromatase, or to dihydrotestosterone by 5α-reductase enzymes. Our understanding of the structural basis for gender differences in bone strength has advanced considerably over recent years due to increasing use of (high resolution) peripheral computed tomography. These microarchitectural insights form the basis to understand sex steroid influences on male peak bone mass and turnover in cortical vs trabecular bone. Recent studies using Cre/LoxP technology have further refined our mechanistic insights from global knockout mice into the direct contributions of sex steroids and their respective nuclear receptors in osteoblasts, osteoclasts, osteocytes, and other cells to male osteoporosis. At the same time, these studies have reinforced the notion that androgen and estrogen deficiency have both direct and pleiotropic effects via interaction with, for example, insulin-like growth factor 1, inflammation, oxidative stress, central nervous system control of bone metabolism, adaptation to mechanical loading, etc., This review will summarize recent advances on these issues in the field of sex steroid actions in male bone homeostasis. PMID:24385015

A covalently cross-linked gel derived from the epidermis of the pilot whale Globicephala melas.

PubMed

Baum, C; Fleischer, L-G; Roessner, D; Meyer, W; Siebers, D

2002-01-01

The rheological properties of the stratum corneum of the pilot whale (Globicephala melas) were investigated with emphasis on their significance to the self-cleaning abilities of the skin surface smoothed by a jelly material enriched with various hydrolytic enzymes. The gel formation of the collected fluid was monitored by applying periodic-harmonic oscillating loads using a stress-controlled rheometer. In the mechanical spectrum of the gel, the plateau region of the storage modulus G' (<1200 Pa) and the loss modulus G" (>120 Pa) were independent of frequency (omega = 43.98 to 0.13 rad x s(-1), tau = 15 Pa, T = 20 degrees C), indicating high elastic performance of a covalently cross-linked viscoelastic solid. In addition, multi-angle laser light scattering experiments (MALLS) were performed to analyse the potential time-dependent changes in the weight-average molar mass of the samples. The observed increase showed that the gel formation is based on the assembly of covalently cross-linked aggregates. The viscoelastic properties and the shear resistance of the gel assure that the enzyme-containing jelly material smoothing the skin surface is not removed from the stratum corneum by shear regimes during dolphin jumping. The even skin surface is considered to be most important for the self-cleaning abilities of the dolphin skin against biofouling.

Predicted Structures of the Proton-Bound Membrane-Embedded Rotor Rings of the Saccharomyces cerevisiae and Escherichia coli ATP Synthases.

PubMed

Zhou, Wenchang; Leone, Vanessa; Krah, Alexander; Faraldo-Gómez, José D

2017-04-20

Recent years have witnessed a renewed interest in the ATP synthase as a drug target against human pathogens. Indeed, clinical, biochemical, and structural data indicate that hydrophobic inhibitors targeting the membrane-embedded proton-binding sites of the c-subunit ring could serve as last-resort antibiotics against multidrug resistant strains. However, because inhibition of the mitochondrial ATP synthase in humans is lethal, it is essential that these inhibitors be not only potent but also highly selective for the bacterial enzyme. To this end, a detailed understanding of the structure of this protein target is arguably instrumental. Here, we use computational methods to predict the atomic structures of the proton-binding sites in two prototypical c-rings: that of the ATP synthase from Saccharomyces cerevisiae, which is a model system for mitochondrial enzymes, and that from Escherichia coli, which can be pathogenic for humans. Our study reveals the structure of these binding sites loaded with protons and in the context of the membrane, that is, in the state that would mediate the recognition of a potential inhibitor. Both structures reflect a mode of proton coordination unlike those previously observed in other c-ring structures, whether experimental or modeled.

Effectiveness of Losartan-Loaded Hyaluronic Acid (HA) Micelles for the Reduction of Advanced Hepatic Fibrosis in C3H/HeN Mice Model

PubMed Central

Thomas, Reju George; Moon, Myeong Ju; Kim, Jo Heon; Lee, Jae Hyuk; Jeong, Yong Yeon

2015-01-01

Advanced hepatic fibrosis therapy using drug-delivering nanoparticles is a relatively unexplored area. Angiotensin type 1 (AT1) receptor blockers such as losartan can be delivered to hepatic stellate cells (HSC), blocking their activation and thereby reducing fibrosis progression in the liver. In our study, we analyzed the possibility of utilizing drug-loaded vehicles such as hyaluronic acid (HA) micelles carrying losartan to attenuate HSC activation. Losartan, which exhibits inherent lipophilicity, was loaded into the hydrophobic core of HA micelles with a 19.5% drug loading efficiency. An advanced liver fibrosis model was developed using C3H/HeN mice subjected to 20 weeks of prolonged TAA/ethanol weight-adapted treatment. The cytocompatibility and cell uptake profile of losartan-HA micelles were studied in murine fibroblast cells (NIH3T3), human hepatic stellate cells (hHSC) and FL83B cells (hepatocyte cell line). The ability of these nanoparticles to attenuate HSC activation was studied in activated HSC cells based on alpha smooth muscle actin (α-sma) expression. Mice treated with oral losartan or losartan-HA micelles were analyzed for serum enzyme levels (ALT/AST, CK and LDH) and collagen deposition (hydroxyproline levels) in the liver. The accumulation of HA micelles was observed in fibrotic livers, which suggests increased delivery of losartan compared to normal livers and specific uptake by HSC. Active reduction of α-sma was observed in hHSC and the liver sections of losartan-HA micelle-treated mice. The serum enzyme levels and collagen deposition of losartan-HA micelle-treated mice was reduced significantly compared to the oral losartan group. Losartan-HA micelles demonstrated significant attenuation of hepatic fibrosis via an HSC-targeting mechanism in our in vitro and in vivo studies. These nanoparticles can be considered as an alternative therapy for liver fibrosis. PMID:26714035

Effectiveness of Losartan-Loaded Hyaluronic Acid (HA) Micelles for the Reduction of Advanced Hepatic Fibrosis in C3H/HeN Mice Model.

PubMed

Thomas, Reju George; Moon, Myeong Ju; Kim, Jo Heon; Lee, Jae Hyuk; Jeong, Yong Yeon

2015-01-01

Advanced hepatic fibrosis therapy using drug-delivering nanoparticles is a relatively unexplored area. Angiotensin type 1 (AT1) receptor blockers such as losartan can be delivered to hepatic stellate cells (HSC), blocking their activation and thereby reducing fibrosis progression in the liver. In our study, we analyzed the possibility of utilizing drug-loaded vehicles such as hyaluronic acid (HA) micelles carrying losartan to attenuate HSC activation. Losartan, which exhibits inherent lipophilicity, was loaded into the hydrophobic core of HA micelles with a 19.5% drug loading efficiency. An advanced liver fibrosis model was developed using C3H/HeN mice subjected to 20 weeks of prolonged TAA/ethanol weight-adapted treatment. The cytocompatibility and cell uptake profile of losartan-HA micelles were studied in murine fibroblast cells (NIH3T3), human hepatic stellate cells (hHSC) and FL83B cells (hepatocyte cell line). The ability of these nanoparticles to attenuate HSC activation was studied in activated HSC cells based on alpha smooth muscle actin (α-sma) expression. Mice treated with oral losartan or losartan-HA micelles were analyzed for serum enzyme levels (ALT/AST, CK and LDH) and collagen deposition (hydroxyproline levels) in the liver. The accumulation of HA micelles was observed in fibrotic livers, which suggests increased delivery of losartan compared to normal livers and specific uptake by HSC. Active reduction of α-sma was observed in hHSC and the liver sections of losartan-HA micelle-treated mice. The serum enzyme levels and collagen deposition of losartan-HA micelle-treated mice was reduced significantly compared to the oral losartan group. Losartan-HA micelles demonstrated significant attenuation of hepatic fibrosis via an HSC-targeting mechanism in our in vitro and in vivo studies. These nanoparticles can be considered as an alternative therapy for liver fibrosis.

A novel stimuli-synchronized alloy-treated matrix for space-defined gastrointestinal delivery of mesalamine in the Large White pig model.

PubMed

Bawa, Priya; Choonara, Yahya E; du Toit, Lisa C; Kumar, Pradeep; Ndesendo, Valence M K; Meyer, Leith C R; Pillay, Viness

2013-03-28

The study focussed on designing a Stimuli-Synchronized Matrix (SSM) for space-defined colonic delivery of the anti-inflammatory drug mesalamine. The configured matrix provided time-independent delivery and stimuli targeting. Formulations were optimized according to a Box-Behnken experimental design that constituted mesalamine-loaded BaSO4-crosslinked chitosan dispersed within a pectin, carboxymethylcellulose and xanthan gum complex. The complex was compressed into matrices and subsequently alloy-treated with pectin and ethylcellulose. In vitro drug release was determined in the presence and absence of colonic enzymes and the mean dissolution time was used for formulation optimization. To mechanistically elucidate the synchronous catalytic action of the enzymes pectinase and glucosidase on the matrix, computer-aided 3D modelling of active fractions of the enzyme-substrate complexes was generated to predict the orientation of residues affecting the substrate domain. Drug release profiles revealed distinct colonic enzyme responsiveness with fractions of 0.402 and 0.152 of mesalamine released in the presence and absence of enzymes, respectively after 24h. The commercial comparator product showed irreproducible release profiles over the same period (SD=0.550) compared to the SSM formulation (SD=0.037). FTIR spectra of alloy-treated matrices showed no peaks from 1589 to 1512cm(-1) after colonic enzyme exposure. With increasing enzyme exposure there were also no peaks between 1646 and 1132cm(-1). This indicated polymeric enzyme cleavage for controlled and space-defined release of mesalamine. Plasma concentration profiles in the Large White pig model produced a Cmax of 3.77±1.375μg/mL compared to 10.604±2.846μg/mL for the comparator formulation. The SSM formulation proved superior over the comparator product by providing superiorly controlled enzyme-responsive colonic drug delivery. Copyright © 2012 Elsevier B.V. All rights reserved.

Strategies to achieve high-solids enzymatic hydrolysis of dilute-acid pretreated corn stover.

PubMed

Geng, Wenhui; Jin, Yongcan; Jameel, Hasan; Park, Sunkyu

2015-01-01

Three strategies were presented to achieve high solids loading while maximizing carbohydrate conversion, which are fed-batch, splitting/thickening, and clarifier processes. Enzymatic hydrolysis was performed at water insoluble solids (WIS) of 15% using washed dilute-acid pretreated corn stover. The carbohydrate concentration increased from 31.8 to 99.3g/L when the insoluble solids content increased from 5% to 15% WIS, while the final carbohydrate conversion was decreased from 78.4% to 73.2%. For the fed-batch process, a carbohydrate conversion efficiency of 76.8% was achieved when solid was split into 60:20:20 ratio, with all enzymes added first. For the splitting/thickening process, a carbohydrate conversion of 76.5% was realized when the filtrate was recycled to simulate a steady-state process. Lastly, the clarifier process was evaluated and the highest carbohydrate conversion of 81.4% was achieved. All of these results suggests the possibility of enzymatic hydrolysis at high solids to make the overall conversion cost-competitive. Copyright © 2015 Elsevier Ltd. All rights reserved.

Intracellular implantation of enzymes in hollow silica nanospheres for protein therapy: cascade system of superoxide dismutase and catalase.

PubMed

Chang, Feng-Peng; Chen, Yi-Ping; Mou, Chung-Yuan

2014-11-01

An approach for enzyme therapeutics is elaborated with cell-implanted nanoreactors that are based on multiple enzymes encapsulated in hollow silica nanospheres (HSNs). The synthesis of HSNs is carried out by silica sol-gel templating of water-in-oil microemulsions so that polyethyleneimine (PEI) modified enzymes in aqueous phase are encapsulated inside the HSNs. PEI-grafted superoxide dismutase (PEI-SOD) and catalase (PEI-CAT) encapsulated in HSNs are prepared with quantitative control of the enzyme loadings. Excellent activities of superoxide dismutation by PEI-SOD@HSN are found and transformation of H2 O2 to water by PEI-CAT@HSN. When PEI-SOD and PEI-CAT are co-encapsulated, cascade transformation of superoxide through hydrogen peroxide to water was facile. Substantial fractions of HSNs exhibit endosome escape to cytosol after their delivery to cells. The production of downstream reactive oxygen species (ROS) and COX-2/p-p38 expression show that co-encapsulated SOD/CAT inside the HSNs renders the highest cell protection against the toxicant N,N'-dimethyl-4,4'-bipyridinium dichloride (paraquat). The rapid cell uptake and strong detoxification effect on superoxide radicals by the SOD/CAT-encapsulated hollow mesoporous silica nanoparticles demonstrate the general concept of implanting catalytic nanoreactors in biological cells with designed functions. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Modeling the Effect of pH and Temperature for Cellulases Immobilized on Enzymogel Nanoparticles.

PubMed

Samaratunga, Ashani; Kudina, Olena; Nahar, Nurun; Zakharchenko, Andrey; Minko, Sergiy; Voronov, Andriy; Pryor, Scott W

2015-06-01

Production costs of cellulosic biofuels can be lowered if cellulases are recovered and reused using particulate carriers that can be extracted after biomass hydrolysis. Such enzyme recovery was recently demonstrated using enzymogel nanoparticles with grafted polymer brushes loaded with cellulases. In this work, cellulase (NS50013) and β-glucosidase (Novozyme 188) were immobilized on enzymogels made of poly(acrylic acid) polymer brushes grafted to the surface of silica nanoparticles. Response surface methodology was used to model effects of pH and temperature on hydrolysis and recovery of free and attached enzymes. Hydrolysis yields using both enzymogels and free cellulase and β-glucosidase were highest at the maximum temperature tested, 50 °C. The optimal pH for cellulase enzymogels and free enzyme was 5.0 and 4.4, respectively, while both free β-glucosidase and enzymogels had an optimal pH near 4.4. Highest hydrolysis sugar concentrations with cellulase and β-glucosidase enzymogels were 69 and 53 % of those with free enzymes, respectively. Enzyme recovery using enzymogels decreased with increasing pH, but cellulase recovery remained greater than 88 % throughout the operating range of pH values less than 5.0 and was greater than 95 % at pH values below 4.3. Recovery of β-glucosidase enzymogels was not affected by temperature and had little impact on cellulase recovery.

Modification of enzymes by use of high-pressure homogenization.

PubMed

Dos Santos Aguilar, Jessika Gonçalves; Cristianini, Marcelo; Sato, Helia Harumi

2018-07-01

High-pressure is an emerging and relatively new technology that can modify various molecules. High-pressure homogenization (HPH) has been used in several studies on protein modification, especially in enzymes used or found in food, from animal, plant or microbial resources. According to the literature, the enzymatic activity can be modulated under pressure causing inactivation, stabilization or activation of the enzymes, which, depending on the point of view could be very useful. Homogenization can generate changes in the structure of the enzyme modifying various chemical bonds (mainly weak bonds) causing different denaturation levels and, consequently, affecting the catalytic activity. This review aims to describe the various alterations due to HPH treatment in enzymes, to show the influence of high-pressure on proteins and to report the HPH effects on the enzymatic activity of different enzymes employed in the food industry and research. Copyright © 2018 Elsevier Ltd. All rights reserved.

The relationship between 25-hydroxyvitamin D concentration and liver enzymes in overweight or obese adults: Cross-sectional and interventional outcomes.

PubMed

Naderpoor, Negar; Mousa, Aya; de Courten, Maximilian; Scragg, Robert; de Courten, Barbora

2018-03-01

Vitamin D deficiency is prevalent in individuals with non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis. However, there is limited and inconsistent data on the effect of vitamin D supplementation on liver function. Hepatic enzymes have been used as surrogate markers for NAFLD and have been associated with metabolic syndrome. We examined the relationships between 25-hydroxyvitamin D (25(OH)D) and γ-glutamyl transferase (GGT), alanine aminotransferase (ALT), alkaline phosphatase (ALP) in 120 drug-naïve individuals with no history of liver disease. In addition, the effect of vitamin D supplementation (100,000 loading dose of cholecalciferol followed by 4000IU daily for 16 weeks) on hepatic enzymes was investigated in a subgroup of 54 vitamin D-deficient overweight or obese individuals (28 randomised to cholecalciferol and 26 to placebo). Hepatic enzymes, anthropometric parameters, lipid profile, insulin sensitivity (hyperinsulinaemic-euglycaemic clamp, M value) and high sensitivity C-reactive protein (hs-CRP) were measured before and after the intervention. In the cross-sectional study, levels of GGT and ALT were higher in men compared to women (both p=0.001). There were no significant differences in GGT, ALT and ALP between vitamin D categories (25(OH)D<25nmol/L, 25-50nmol/L, and >50nmol/L) and no relationships were found between the three enzymes and 25(OH)D before and after adjustment for age, sex, BMI, WHR, and insulin sensitivity (all p>0.5). In the randomised trial, 25(OH)D concentrations increased in the vitamin D group (mean change 57.0±21.3nmol/L) compared to the placebo group (mean change 1.9±15.1nmol/L). Mean changes in GGT, ALT and ALP were not significantly different between vitamin D and placebo groups (all p>0.2). Change in 25(OH)D concentration was not correlated with changes in GGT, ALT and ALP before and after adjustments for age and sex (all p>0.1). In summary, 25(OH)D concentrations were not related to hepatic enzymes in drug-naive adults with no history of liver disease, and vitamin D supplementation had no effect on the serum levels of hepatic enzymes in vitamin D-deficient and overweight or obese, otherwise healthy individuals. Hence, vitamin D supplementation is unlikely to prevent incident NAFLD. Copyright © 2017 Elsevier Ltd. All rights reserved.

Cinnamon extract inhibits α-glucosidase activity and dampens postprandial glucose excursion in diabetic rats

PubMed Central

2011-01-01

Background α-glucosidase inhibitors regulate postprandial hyperglycemia (PPHG) by impeding the rate of carbohydrate digestion in the small intestine and thereby hampering the diet associated acute glucose excursion. PPHG is a major risk factor for diabetic vascular complications leading to disabilities and mortality in diabetics. Cinnamomum zeylanicum, a spice, has been used in traditional medicine for treating diabetes. In this study we have evaluated the α-glucosidase inhibitory potential of cinnamon extract to control postprandial blood glucose level in maltose, sucrose loaded STZ induced diabetic rats. Methods The methanol extract of cinnamon bark was prepared by Soxhlet extraction. Phytochemical analysis was performed to find the major class of compounds present in the extract. The inhibitory effect of cinnamon extract on yeast α-glucosidase and rat-intestinal α-glucosidase was determined in vitro and the kinetics of enzyme inhibition was studied. Dialysis experiment was performed to find the nature of the inhibition. Normal male Albino wistar rats and STZ induced diabetic rats were treated with cinnamon extract to find the effect of cinnamon on postprandial hyperglycemia after carbohydrate loading. Results Phytochemical analysis of the methanol extract displayed the presence of tannins, flavonoids, glycosides, terpenoids, coumarins and anthraquinones. In vitro studies had indicated dose-dependent inhibitory activity of cinnamon extract against yeast α-glucosidase with the IC 50 value of 5.83 μg/ml and mammalian α-glucosidase with IC 50 value of 670 μg/ml. Enzyme kinetics data fit to LB plot pointed out competitive mode of inhibition and the membrane dialysis experiment revealed reversible nature of inhibition. In vivo animal experiments are indicative of ameliorated postprandial hyperglycemia as the oral intake of the cinnamon extract (300 mg/kg body wt.) significantly dampened the postprandial hyperglycemia by 78.2% and 52.0% in maltose and sucrose loaded STZ induced diabetic rats respectively, compared to the control. On the other hand, in rats that received glucose and cinnamon extract, postprandial hyperglycemia was not effectively suppressed, which indicates that the observed postprandial glycemic amelioration is majorly due to α-glucosidase inhibition. Conclusions The current study demonstrates one of the mechanisms in which cinnamon bark extract effectively inhibits α-glucosidase leading to suppression of postprandial hyperglycemia in STZ induced diabetic rats loaded with maltose, sucrose. This bark extract shows competitive, reversible inhibition on α-glucosidase enzyme. Cinnamon extract could be used as a potential nutraceutical agent for treating postprandial hyperglycemia. In future, specific inhibitor has to be isolated from the crude extract, characterized and therapeutically exploited. PMID:21711570

Perceptual Load Affects Eyewitness Accuracy and Susceptibility to Leading Questions.

PubMed

Murphy, Gillian; Greene, Ciara M

2016-01-01

Load Theory (Lavie, 1995, 2005) states that the level of perceptual load in a task (i.e., the amount of information involved in processing task-relevant stimuli) determines the efficiency of selective attention. There is evidence that perceptual load affects distractor processing, with increased inattentional blindness under high load. Given that high load can result in individuals failing to report seeing obvious objects, it is conceivable that load may also impair memory for the scene. The current study is the first to assess the effect of perceptual load on eyewitness memory. Across three experiments (two video-based and one in a driving simulator), the effect of perceptual load on eyewitness memory was assessed. The results showed that eyewitnesses were less accurate under high load, in particular for peripheral details. For example, memory for the central character in the video was not affected by load but memory for a witness who passed by the window at the edge of the scene was significantly worse under high load. High load memories were also more open to suggestion, showing increased susceptibility to leading questions. High visual perceptual load also affected recall for auditory information, illustrating a possible cross-modal perceptual load effect on memory accuracy. These results have implications for eyewitness memory researchers and forensic professionals.

Perceptual Load Affects Eyewitness Accuracy and Susceptibility to Leading Questions

PubMed Central

Murphy, Gillian; Greene, Ciara M.

2016-01-01

Load Theory (Lavie, 1995, 2005) states that the level of perceptual load in a task (i.e., the amount of information involved in processing task-relevant stimuli) determines the efficiency of selective attention. There is evidence that perceptual load affects distractor processing, with increased inattentional blindness under high load. Given that high load can result in individuals failing to report seeing obvious objects, it is conceivable that load may also impair memory for the scene. The current study is the first to assess the effect of perceptual load on eyewitness memory. Across three experiments (two video-based and one in a driving simulator), the effect of perceptual load on eyewitness memory was assessed. The results showed that eyewitnesses were less accurate under high load, in particular for peripheral details. For example, memory for the central character in the video was not affected by load but memory for a witness who passed by the window at the edge of the scene was significantly worse under high load. High load memories were also more open to suggestion, showing increased susceptibility to leading questions. High visual perceptual load also affected recall for auditory information, illustrating a possible cross-modal perceptual load effect on memory accuracy. These results have implications for eyewitness memory researchers and forensic professionals. PMID:27625628

Biocatalytic material comprising multilayer enzyme coated fiber

DOEpatents

Kim, Jungbae [Richland, WA; Kwak, Ja Hun [Richland, WA; Grate, Jay W [West Richland, WA

2009-11-03

The present invention relates generally to high stability, high activity biocatalytic materials and processes for using the same. The materials comprise enzyme aggregate coatings having high biocatalytic activity and stability useful in heterogeneous environment. These new materials provide a new biocatalytic immobilized enzyme system with applications in bioconversion, bioremediation, biosensors, and biofuel cells.

An easily regenerable enzyme reactor prepared from polymerized high internal phase emulsions.

PubMed

Ruan, Guihua; Wu, Zhenwei; Huang, Yipeng; Wei, Meiping; Su, Rihui; Du, Fuyou

2016-04-22

A large-scale high-efficient enzyme reactor based on polymerized high internal phase emulsion monolith (polyHIPE) was prepared. First, a porous cross-linked polyHIPE monolith was prepared by in-situ thermal polymerization of a high internal phase emulsion containing styrene, divinylbenzene and polyglutaraldehyde. The enzyme of TPCK-Trypsin was then immobilized on the monolithic polyHIPE. The performance of the resultant enzyme reactor was assessed according to the conversion ability of Nα-benzoyl-l-arginine ethyl ester to Nα-benzoyl-l-arginine, and the protein digestibility of bovine serum albumin (BSA) and cytochrome (Cyt-C). The results showed that the prepared enzyme reactor exhibited high enzyme immobilization efficiency and fast and easy-control protein digestibility. BSA and Cyt-C could be digested in 10 min with sequence coverage of 59% and 78%, respectively. The peptides and residual protein could be easily rinsed out from reactor and the reactor could be regenerated easily with 4 M HCl without any structure destruction. Properties of multiple interconnected chambers with good permeability, fast digestion facility and easily reproducibility indicated that the polyHIPE enzyme reactor was a good selector potentially applied in proteomics and catalysis areas. Copyright © 2016 Elsevier Inc. All rights reserved.

Vaccination of rhesus macaques with a vif-deleted simian immunodeficiency virus proviral DNA vaccine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sparger, Ellen E.; Dubie, Robert A.; Shacklett, Barbara L.

2008-05-10

Studies in non-human primates, with simian immunodeficiency virus (SIV) and simian/human immunodeficiency virus (SHIV) have demonstrated that live-attenuated viral vaccines are highly effective; however these vaccine viruses maintain a low level of pathogenicity. Lentivirus attenuation associated with deletion of the viral vif gene carries a significantly reduced risk for pathogenicity, while retaining the potential for virus replication of low magnitude in the host. This report describes a vif-deleted simian immunodeficiency virus (SIV)mac239 provirus that was tested as an attenuated proviral DNA vaccine by inoculation of female rhesus macaques. SIV-specific interferon-{gamma} enzyme-linked immunospot responses of low magnitude were observed after immunizationmore » with plasmid containing the vif-deleted SIV provirus. However, vaccinated animals displayed strong sustained virus-specific T cell proliferative responses and increasing antiviral antibody titers. These immune responses suggested either persistent vaccine plasmid expression or low level replication of vif-deleted SIV in the host. Immunized and unvaccinated macaques received a single high dose vaginal challenge with pathogenic SIVmac251. A transient suppression of challenge virus load and a greater median survival time was observed for vaccinated animals. However, virus loads for vaccinated and unvaccinated macaques were comparable by twenty weeks after challenge and overall survival curves for the two groups were not significantly different. Thus, a vif-deleted SIVmac239 proviral DNA vaccine is immunogenic and capable of inducing a transient suppression of pathogenic challenge virus, despite severe attenuation of the vaccine virus.« less

Physical Weight Loading Induces Expression of Tryptophan Hydroxylase 2 in the Brain Stem

PubMed Central

Shim, Joon W.; Dodge, Todd R.; Hammond, Max A.; Wallace, Joseph M.; Zhou, Feng C.; Yokota, Hiroki

2014-01-01

Sustaining brain serotonin is essential in mental health. Physical activities can attenuate mental problems by enhancing serotonin signaling. However, such activity is not always possible in disabled individuals or patients with dementia. Knee loading, a form of physical activity, has been found to mimic effects of voluntary exercise. Focusing on serotonergic signaling, we addressed a question: Does local mechanical loading to the skeleton elevate expression of tryptophan hydroxylase 2 (tph2) that is a rate-limiting enzyme for brain serotonin? A 5 min knee loading was applied to mice using 1 N force at 5 Hz for 1,500 cycles. A 5-min treadmill running was used as an exercise (positive) control, and a 90-min tail suspension was used as a stress (negative) control. Expression of tph2 was determined 30 min – 2 h in three brain regions ––frontal cortex (FC), ventromedial hypothalamus (VMH), and brain stem (BS). We demonstrated for the first time that knee loading and treadmill exercise upregulated the mRNA level of tph2 in the BS, while tail suspension downregulated it. The protein level of tph2 in the BS was also upregulated by knee loading and downregulated by tail suspension. Furthermore, the downregulation of tph2 mRNA by tail suspension can be partially suppressed by pre-application of knee loading. The expression of tph2 in the FC and VMH was not significantly altered with knee loading. In this study we provided evidence that peripheral mechanical loading can activate central tph2 expression, suggesting that physical cues may mediate tph2-cathalyzed serotonergic signaling in the brain. PMID:24416346

High activity CAZyme cassette for improving biomass degradation in thermophiles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brunecky, Roman; Chung, Daehwan; Sarai, Nicholas S.

Currently, Thermophilic microorganisms and their enzymes offer several advantages for industrial application over their mesophilic counterparts. For example, a hyperthermophilic anaerobe, Caldicellulosiruptor bescii, was recently isolated from hot springs in Kamchatka, Siberia, and shown to have very high cellulolytic activity. Additionally, it is one of a few microorganisms being considered as viable candidates for consolidated bioprocessing applications. Moreover, C. bescii is capable of deconstructing plant biomass without enzymatic or chemical pretreatment. This ability is accomplished by the production and secretion of free, multi-modular and multi-functional enzymes, one of which, CbCel9A/Cel48A also secretion of free, multi-modular and multi-functional enzymes, one ofmore » which, CbCel9A/Cel48A also known as CelA, is able to outperform enzymes found in commercial enzyme preparations. Furthermore, the complete C. bescii exoproteome is extremely thermostable and highly active at elevated temperatures, unlike commercial fungal cellulases. Understanding the functional diversity of enzymes in the C. bescii exoproteome and how inter-molecular synergy between them confers C. bescii with its high cellulolytic activity is an important endeavor to enable the production more efficient biomass degrading enzyme formulations and in turn, better cellulolytic industrial microorganisms. We found that the combination of three or four of the most highly expressed enzymes in the C. bescii exoproteome exhibits such synergistic activity. For example, some discrete combinations of these enzymes mimic and even improve upon the activity of the exoproteome, even though some of the enzymes lack significant activity on their own. We have demonstrated that it is possible to replicate the cellulolytic activity of the native C. bescii exoproteome utilizing a minimal gene set, and that these minimal gene sets are more active than the whole exoproteome. In the future, this may lead to more simplified and efficient cellulolytic enzyme preparations or yield improvements when these enzymes are expressed in microorganisms engineered for consolidated bioprocessing.« less

High activity CAZyme cassette for improving biomass degradation in thermophiles

DOE PAGES

Brunecky, Roman; Chung, Daehwan; Sarai, Nicholas S.; ...

2018-02-01

Currently, Thermophilic microorganisms and their enzymes offer several advantages for industrial application over their mesophilic counterparts. For example, a hyperthermophilic anaerobe, Caldicellulosiruptor bescii, was recently isolated from hot springs in Kamchatka, Siberia, and shown to have very high cellulolytic activity. Additionally, it is one of a few microorganisms being considered as viable candidates for consolidated bioprocessing applications. Moreover, C. bescii is capable of deconstructing plant biomass without enzymatic or chemical pretreatment. This ability is accomplished by the production and secretion of free, multi-modular and multi-functional enzymes, one of which, CbCel9A/Cel48A also secretion of free, multi-modular and multi-functional enzymes, one ofmore » which, CbCel9A/Cel48A also known as CelA, is able to outperform enzymes found in commercial enzyme preparations. Furthermore, the complete C. bescii exoproteome is extremely thermostable and highly active at elevated temperatures, unlike commercial fungal cellulases. Understanding the functional diversity of enzymes in the C. bescii exoproteome and how inter-molecular synergy between them confers C. bescii with its high cellulolytic activity is an important endeavor to enable the production more efficient biomass degrading enzyme formulations and in turn, better cellulolytic industrial microorganisms. We found that the combination of three or four of the most highly expressed enzymes in the C. bescii exoproteome exhibits such synergistic activity. For example, some discrete combinations of these enzymes mimic and even improve upon the activity of the exoproteome, even though some of the enzymes lack significant activity on their own. We have demonstrated that it is possible to replicate the cellulolytic activity of the native C. bescii exoproteome utilizing a minimal gene set, and that these minimal gene sets are more active than the whole exoproteome. In the future, this may lead to more simplified and efficient cellulolytic enzyme preparations or yield improvements when these enzymes are expressed in microorganisms engineered for consolidated bioprocessing.« less