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.

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

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

Nutrients removal and substrate enzyme activities in vertical subsurface flow constructed wetlands for mariculture wastewater treatment: Effects of ammonia nitrogen loading rates and salinity levels.

PubMed

Li, Meng; Liang, Zhenlin; Callier, Myriam D; Roque d'orbcastel, Emmanuelle; Sun, Guoxiang; Ma, Xiaona; Li, Xian; Wang, Shunkui; Liu, Ying; Song, Xiefa

2018-06-01

This study aims to investigate the effects of ammonia nitrogen loading rates and salinity levels on nutrients removal rates and substrate enzyme activities of constructed wetland (CW) microcosms planted with Salicornia bigelovii treating mariculture wastewater. Activities of urease (UA), dehydrogenase (DA), protease (PrA) and phosphatase (PA) were considered. Using principal component analysis (PCA), nutrient removal index (NRI) and enzyme activity index (EAI) were developed to evaluate the effects. The results revealed that increasing ammonia nitrogen loading rates had positive effects on nitrogen removal rates (i.e. NH 4 -N and DIN) and enhanced substrate enzyme activities. Compared with low salinity (i.e. 15 and 22), high salinity levels (i.e. 29 and 36) enhanced nutrients removal rates, DA and UA, but weaken PA and PrA. In conclusion, CW microcosms with Salicornia bigelovii can be used for the removal of nutrients under a range of ammonia nitrogen loadings and high salinity levels. Copyright © 2018 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

Process for preparing multilayer enzyme coating on a fiber

DOEpatents

Kim, Jungbae [Richland, WA; Kwak, Ja Hun [Richland, WA; Grate, Jay W [West Richland, WA

2009-11-03

A process for preparing high stability, high activity biocatalytic materials is disclosed and processes for using the same. The process involves coating of a material or fiber with enzymes and enzyme aggregate providing a material or fiber with high biocatalytic activity and stability useful in heterogeneous environments. In one illustrative approach, enzyme "seeds" are covalently attached to polymer nanofibers followed by treatment with a reagent that crosslinks additional enzyme molecules to the seed enzymes forming enzyme aggregates thereby improving biocatalytic activity due to increased enzyme loading and enzyme stability. This approach creates a useful new biocatalytic immobilized enzyme system with potential applications in bioconversion, bioremediation, biosensors, and biofuel cells.

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

Non-ionic Surfactants and Non-Catalytic Protein Treatment on Enzymatic Hydrolysis of Pretreated Creeping Wild Ryegrass

NASA Astrophysics Data System (ADS)

Zheng, Yi; Pan, Zhongli; Zhang, Ruihong; Wang, Donghai; Jenkins, Bryan

Our previous research has shown that saline Creeping Wild Ryegrass (CWR), Leymus triticoides, has a great potential to be used for bioethanol production because of its high fermentable sugar yield, up to 85% cellulose conversion of pretreated CWR. However, the high cost of enzyme is still one of the obstacles making large-scale lignocellulosic bioethanol production economically difficult. It is desirable to use reduced enzyme loading to produce fermentable sugars with high yield and low cost. To reduce the enzyme loading, the effect of addition of non-ionic surfactants and non-catalytic protein on the enzymatic hydrolysis of pretreated CWR was investigated in this study. Tween 20, Tween 80, and bovine serum albumin (BSA) were used as additives to improve the enzymatic hydrolysis of dilute sulfuric-acid-pretreated CWR. Under the loading of 0.1 g additives/g dry solid, Tween 20 was the most effective additive, followed by Tween 80 and BSA. With the addition of Tween 20 mixed with cellulase loading of 15 FPU/g cellulose, the cellulose conversion increased 14% (from 75 to 89%), which was similar to that with cellulase loading of 30 FPU/g cellulose and without additive addition. The results of cellulase and BSA adsorption on the Avicel PH101, pretreated CWR, and lignaceous residue of pretreated CWR support the theory that the primary mechanism behind the additives is prevention of non-productive adsorption of enzymes on lignaceous material of pretreated CWR. The addition of additives could be a promising technology to improve the enzymatic hydrolysis by reducing the enzyme activity loss caused by non-productive adsorption.

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.

Crosslinked Enzyme Aggregates in Hierarchically-Ordered Mesoporous Silica: A Simple and Effective Method for Enzyme Stabilization

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

Kim, Moon Il; Kim, Jungbae; Lee, Jinwoo

2007-02-01

alpha-chymotrypsin (CT) and lipase (LP) were immobilized in hierarchically-ordered mesocellular mesoporous silica (HMMS) in a simple but effective way for the enzyme stabilization, which was achieved by the enzyme adsorption followed by glutaraldehyde (GA) crosslinking. This resulted in the formation of nanometer scale crosslinked enzyme aggregates (CLEAs) entrapped in the mesocellular pores of HMMS (37 nm), which did not leach out of HMMS through narrow mesoporous channels (13 nm). CLEA of alpha-chymotrypsin (CLEA-CT) in HMMS showed a high enzyme loading capacity and significantly increased enzyme stability. No activity decrease of CLEA-CT was observed for two weeks under even rigorously shakingmore » condition, while adsorbed CT in HMMS and free CT showed a rapid inactivation due to the enzyme leaching and presumably autolysis, respectively. With the CLEA-CT in HMMS, however, there was no tryptic digestion observed suggesting that the CLEA-CT is not susceptible to autolysis. Moreover, CLEA of lipase (CLEA-LP) in HMMS retained 30% specific activity of free lipase with greatly enhanced stability. This work demonstrates that HMMS can be efficiently employed as host materials for enzyme immobilization leading to highly enhanced stability of the immobilized enzymes with high enzyme loading and activity.« less

Combined of ultrasound irradiation with high hydrostatic pressure (US/HHP) as a new method to improve immobilization of dextranase onto alginate gel.

PubMed

Bashari, Mohanad; Abbas, Shabbar; Xu, Xueming; Jin, Zhengyu

2014-07-01

In this research work, dextranase was immobilized onto calcium alginate beads by the combination of ultrasonic irradiation and high hydrostatic pressure (US/HHP) treatments. Effects of US/HHP treatments on loading efficiency and immobilization yield of dextranase enzyme onto calcium alginate beads were investigated. Furthermore, the activities of immobilized enzymes prepared with and without US/HHP treatments and that prepared with ultrasonic irradiation (US) and high hydrostatic pressure (HHP), as a function of pH, temperature, recyclability and enzyme kinetic parameters, were compared with that for free enzyme. The maximum loading efficiency and the immobilization yield were observed when the immobilized dextranase was prepared with US (40 W at 25 kHz for 15 min) combined with HHP (400 MPa for 15 min), under which the loading efficiency and the immobilization yield increased by 88.92% and 80.86%, respectively, compared to immobilized enzymes prepared without US/HHP treatment. On the other hand, immobilized enzyme prepared with US/HHP treatment showed Vmax, KM, catalytic and specificity constants values higher than that for the immobilized enzyme prepared with HHP treatment, indicated that, this new US/HHP method improved the catalytic kinetics activity of immobilized dextranase at all the reaction conditions studied. Compared to immobilized enzyme prepared either with US or HHP, the immobilized enzymes prepared with US/HHP method exhibited a higher: pH optimum, optimal reaction temperature, thermal stability and recyclability, and lower activation energy, which, illustrating the effectiveness of the US/HHP method. These results indicated that, the combination of US and HHP treatments could be an effective method for improving the immobilization of enzymes in polymers. Copyright © 2014 Elsevier B.V. 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.

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.

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.

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.

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.

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.

Open-mouthed hybrid microcapsules with elevated enzyme loading and enhanced catalytic activity.

PubMed

Shi, Jiafu; Zhang, Shaohua; Wang, Xiaoli; Jiang, Zhongyi

2014-10-25

Open-mouthed hybrid microcapsules (HMCs) are synthesized through a hard-templating method. When utilized for enzyme immobilization and enzymatic catalysis, the open-mouthed HMCs show high enzyme loading capability, enhanced catalytic activity and desirable recycling stability, due to their fully exposed outer and inner surfaces.

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.

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.

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.

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.

Development of rapid bioconversion with integrated recycle technology for ethanol production from extractive ammonia pretreated corn stover.

PubMed

Jin, Mingjie; Liu, Yanping; da Costa Sousa, Leonardo; Dale, Bruce E; Balan, Venkatesh

2017-08-01

High enzyme loading and low productivity are two major issues impeding low cost ethanol production from lignocellulosic biomass. This work applied rapid bioconversion with integrated recycle technology (RaBIT) and extractive ammonia (EA) pretreatment for conversion of corn stover (CS) to ethanol at high solids loading. Enzymes were recycled via recycling unhydrolyzed solids. Enzymatic hydrolysis with recycled enzymes and fermentation with recycled yeast cells were studied. Both enzymatic hydrolysis time and fermentation time were shortened to 24 h. Ethanol productivity was enhanced by two times and enzyme loading was reduced by 30%. Glucan and xylan conversions reached as high as 98% with an enzyme loading of as low as 8.4 mg protein per g glucan. The overall ethanol yield was 227 g ethanol/kg EA-CS (191 g ethanol/kg untreated CS). Biotechnol. Bioeng. 2017;114: 1713-1720. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Functionalization of a membrane sublayer using reverse filtration of enzymes and dopamine coating.

PubMed

Luo, Jianquan; Meyer, Anne S; Mateiu, R V; Kalyani, Dayanand; Pinelo, Manuel

2014-12-24

High permeability, high enzyme loading, and strong antifouling ability are the desired features for a biocatalytic membrane to be used in an enzymatic membrane reactor (EMR). To achieve these goals, the membrane sublayer was enriched with laccase by reverse filtration in this case, and the resulting enzyme-loaded sublayer was covered with a dopamine coating. After membrane reversal, the virgin membrane skin layer was facing the feed and the enzymes were entrapped by a polydopamine network in the membrane sublayer. Thus, the membrane sublayer was functionalized as a catalytically active layer. The effects of the original membrane properties (i.e., materials, pore size, and structure), enzyme type (i.e., laccase and alcohol dehydrogenase), and coating conditions (i.e., time and pH) on the resulting biocatalytic membrane permeability, enzyme loading, and activity were investigated. Using a RC10 kDa membrane with sponge-like sublayer to immobilize laccase with dopamine coating, the trade-off between permeability and enzyme loading was broken, and enzyme loading reached 44.5% without any permeability loss. After 85 days of storage and reuse 14 times, more than 80% of the immobilized laccase activity was retained for the membrane with a dopamine coating, while the relative activity was less than 40% without the coating. The resistance to high temperature and acidic/alkaline pH was also improved by the dopamine coating for the immobilized laccase. Moreover, this biocatalytic membrane could resist mild hydrodynamic cleaning (e.g., back-flushing), but the catalytic ability was reduced by chemical cleaning at extreme pH (e.g., 1.5 and 11.5). Since the immobilized enzyme is not directly facing the bulk of EMRs and the substrate can be specifically selected by the separation skin layer, this biocatalytic membrane is promising for cascade catalytic reactions.

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

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.

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.

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.

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

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.

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.

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.

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

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.

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.

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.

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.

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.

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.

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.

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.

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...

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.

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.

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

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.

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.

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.

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.

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).

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.

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

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.

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.

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.

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.

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.

Encapsulation of enzyme via one-step template-free formation of stable organic-inorganic capsules: A simple and efficient method for immobilizing enzyme with high activity and recyclability.

PubMed

Huang, Renliang; Wu, Mengyun; Goldman, Mark J; Li, Zhi

2015-06-01

Enzyme encapsulation is a simple, gentle, and general method for immobilizing enzyme, but it often suffers from one or more problems regarding enzyme loading efficiency, enzyme leakage, mechanical stability, and recyclability. Here we report a novel, simple, and efficient method for enzyme encapsulation to overcome these problems by forming stable organic-inorganic hybrid capsules. A new, facile, one-step, and template-free synthesis of organic-inorganic capsules in aqueous phase were developed based on PEI-induced simultaneous interfacial self-assembly of Fmoc-FF and polycondensation of silicate. Addition of an aqueous solution of Fmoc-FF and sodium silicate into an aqueous solution of PEI gave a new class of organic-inorganic hybrid capsules (FPSi) with multi-layered structure in high yield. The capsules are mechanically stable due to the incorporation of inorganic silica. Direct encapsulation of enzyme such as epoxide hydrolase SpEH and BSA along with the formation of the organic-inorganic capsules gave high yield of enzyme-containing capsules (∼1.2 mm in diameter), >90% enzyme loading efficiency, high specific enzyme loading (158 mg protein g(-1) carrier), and low enzyme leakage (<3% after 48 h incubation). FPSi-SpEH capsules catalyzed the hydrolysis of cyclohexene oxide to give (1R, 2R)-cyclohexane-1,2-diol in high yield and concentration, with high specific activity (6.94 U mg(-1) protein) and the same high enantioselectivity as the free enzyme. The immobilized SpEH demonstrated also excellent operational stability and recyclability: retaining 87% productivity after 20 cycles with a total reaction time of 80 h. The new enzyme encapsulation method is efficient, practical, and also better than other reported encapsulation methods. © 2015 Wiley Periodicals, Inc.

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 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.

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.

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.

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.

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.

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.

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

Capability of parasulfonato calix[6]arene, as an anion dopant, and organic solvents in enhancing the sensitivity and loading of glucose oxidase (GOx) on polypyrrole film in a biosensor: a comparative study.

PubMed

Safarnavadeh, Vahideh; Zare, Karim; Fakhari, Ali Reza

2013-11-15

In this study, the effects of two solvents (acetonitrile and water) and an anion dopant (para sulfonato calix[6]arene ((C[6]S)(-6))), on the manufacturing and properties of a polypyrrole (Ppy)-based, glucose oxidase amperometric biosensor were studied. Pyrrole was polymerized using galvanostatic mode in two different solvents, and the effect of (C[6]S)(-6) was studied in aqueous solution. The morphology of the obtained polypyrrole films was studied by scanning electron microscopy (SEM). Glucose oxidase (GOx) was adsorbed on the Ppy films via cross-linking method. Then the amperometric responses of the Pt/Ppy/GOx electrodes were measured using the amperometric method at the potential of 0.7 V in steps of adding a glucose solution to a potassium phosphate buffer. We found that acetonitrile and (C[6]S)(-6) increase the sensitivity of the enzyme electrode up to 79.30 µA M(-1)cm(-2) in comparison with 31.60 μA M(-1)cm(-2) for the electrode synthesized in calixarene free aqueous solvent. Also (C[6]S)(-6) has the main role in preventing leaching the enzyme from the electrode. This fact increases loading of the enzyme and stability of the biosensor. So that the steady state current density of the aforementioned electrode increases linearly with increasing glucose concentration up to 190 mM. Whereas the linearity was observed up to 61 mM and 80 mM for the electrodes made using calixarene free acetonitrile and aqueous solutions, respectively. Copyright © 2013 Elsevier B.V. All rights reserved.

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

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

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.

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.

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.

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.

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

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.

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.

Thermal assisted alkaline pretreatment of rice husk for enhanced biomass deconstruction and enzymatic saccharification: Physico-chemical and structural characterization.

PubMed

Shahabazuddin, Md; Sarat Chandra, T; Meena, S; Sukumaran, R K; Shetty, N P; Mudliar, S N

2018-04-21

Thermal assisted alkaline pretreatment (TAAP) of rice husk (RH) was investigated to facilitate enzymatic saccharification by enhancing the enzyme accessibility to cellulosic components. Statistically guided experiments based on the Box-Behnken design involving four factors viz. biomass loading, particle size, NaOH loading and reaction time was considered for optimization. The maximum sugar yield of 371 mg g -1 biomass was obtained at optimized pretreatment condition [biomass loading (10% w/w), particle size (0.25-0.625 mm), NaOH loading (2% w/w), and reaction time (40 min)]. The TAAP of RH resulted in the efficient removal of lignin (14.9-54% (w/w)) with low hemicellulose solubilization [10.7-33.1% (w/w)] and with a simultaneous increase in cellulose concentration [32.65-51.65% (w/w)]. The SEM analysis indicated increased porosity and biomass disruption during TAAP. The FTIR analysis showed progressive removal of noncellulosic constituents, and XRD analysis revealed an increase in cellulose crystallinity post-TAAP indicating the effectiveness of pretreatment. Copyright © 2018 Elsevier Ltd. All rights reserved.

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.

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.

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

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

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

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.

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.

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.

Paper sludge (PS) to bioethanol: Evaluation of virgin and recycle mill sludge for low enzyme, high-solids fermentation.

PubMed

Boshoff, Sonja; Gottumukkala, Lalitha Devi; van Rensburg, Eugéne; Görgens, Johann

2016-03-01

Paper sludge (PS) from the paper and pulp industry consists primarily of cellulose and ash and has significant potential for ethanol production. Thirty-seven PS samples from 11 South African paper and pulp mills exhibited large variation in chemical composition and resulting ethanol production. Simultaneous saccharification and fermentation (SSF) of PS in fed-batch culture was investigated at high solid loadings and low enzyme dosages. Water holding capacity and viscosity of the PS influenced ethanol production at elevated solid loadings of PS. High viscosity of PS from virgin pulp mills restricted the solid loading to 18% (w/w) at an enzyme dosage of 20 FPU/gram dry PS (gdPS), whereas an optimal solid loading of 27% (w/w) was achieved with corrugated recycle mill PS at 11 FPU/gdPS. Ethanol concentration and yield of virgin pulp and corrugated recycle PS were 34.2g/L at 66.9% and 45.5 g/L at 78.2%, respectively. Copyright © 2015 Elsevier Ltd. All rights reserved.

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.

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

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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

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.

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

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.

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.

Effect of sodium tripolyphosphate concentration and simulated gastrointestinal fluids on release profile of paracetamol from chitosan microsphere

NASA Astrophysics Data System (ADS)

Mulia, Kamarza; Andrie; Krisanti, Elsa A.

2018-03-01

The problem to overcome in oral drug administration is the significant pH changes present in the human digestive system. In this study, ionotropic gelation method employing 2-8% (w/v) tripolyphosphate solutions were used to crosslink chitosan microspheres for a controlled release of paracetamol as a model drug. The release profiles of paracetamol from chitosan microspheres were determined using simulated gastrointestinal fluids having pH values of 1.2, 6.8, and 7.4. The results showed that the paracetamol loading and the encapsulation efficiency values increased with increasing concentration of tripolyphosphate solutions used in the preparation step. Paracetamol released at pH 1.2 and 6.8 buffer solutions was significantly higher than that at pH 7.4; also, more paracetamol was released in the presence of α-amylase and β-glucosidase enzymes. The release profiles showed zero-order release behaviour up to 8 hours where the highest drug release was 39% of the paracetamol loaded in the chitosan microspheres, indicating a strong crosslinking between chitosan and TPP anions. The relatively low accumulated drug release could be compensated by employing suitable enzymes, lower TPP solution concentration, and addition of other biodegradable polymer to reduce the TPP crosslink.

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.

Fabrication of glycopolymer/MWCNTs composite nanofibers and its enzyme immobilization applications.

PubMed

Quan, Jing; Liu, Zhongqing; Branford-White, Christopher; Nie, Huali; Zhu, Limin

2014-09-01

Glycopolymer (poly(AN-co-OVSEG))/MWCNTs (multiwalled carbon nanotubes) composite nanofibers are fabricated using a facile approach combining enzymatic synthesis, radical polymerization and electrospinning. The structure of the glycopolymer was confirmed by FT-IR and (1)H NMR. Poly(AN-co-OVSEG)/MWCNTs composite nanofibers were prepared using electrospinning and characterized by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The hydrophilic properties of the composite nanofibers surfaces were increased since the contact angle of poly(AN-co-OVSEG)/MWCNTs composite was reduced from 65.5° to 37° compared to (PAN). As an enzymatic model catalase (CAT) was loaded (ca. 55.0mg/g) to the poly(AN-co-OVSEG)/MWCNTs nanofibers. The optimum temperature for poly(AN-co-OVSEG)/MWCNTs nanofibers increased from 25°C to 45°C compared to free CAT. The covalently immobilized enzymes conjugate exhibited 60% activity at 60°C, while the free enzyme was entirely inactivity after 5min heat treatment. The immobilized CAT retained 70% of its initial activity after 5 cycles of decomposition of hydrogen peroxide. Copyright © 2014 Elsevier B.V. All rights reserved.

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.

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.

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.

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.

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.

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.

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.

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.

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

A SENSITIVE FLUORESCENCE-BASED ASSAY FOR MONITORING GM2 GANGLIOSIDE HYDROLYSIS IN LIVE PATIENT CELLS AND THEIR LYSATES

PubMed Central

Tropak, Michael B.; Bukovac, Scott W.; Rigat, Brigitte A.; Yonekawa, Sayuri; Wakarchuk, Warren; Mahuran, Don J.

2010-01-01

Enzyme enhancement therapy, utilizing small molecules as pharmacological chaperones, is anattractive approach for the treatment of lysosomal storage diseases that are associated with protein misfolding. However, pharmacological chaperones are alsoinhibitors of their target enzyme. Thus, a major concern with this approach is that, despite enhancing protein folding within, and intracellular transport of the functional mutant enzyme out of the endoplasmic reticulum, the chaperone will continue to inhibit the enzyme in the lysosome, preventing substrate clearance. Herewe demonstrate that the in vitro hydrolysis of a fluorescent derivative of lyso-GM2 ganglioside, like natural GM2 ganglioside, is specifically carried out by the β-hexosaminidase A isozyme, requires the GM2 activator protein as a co-factor, increases when the derivative is incorporated into anionic liposomes and follows similar Michaelis-Menten kinetics. This substrate can also be used to differentiate between lysates from normal and GM2 activator-deficient cells. When added to the growth medium of cells, the substrate is internalized and primarily incorporated into lysosomes. Utilizing adult Tay-Sachs fibroblasts that have been pre-treated with the pharmacological chaperone Pyrimethamine and subsequently loaded with this substrate, we demonstrate an increase in both the levels of mutant β-hexosaminidase A and substrate-hydrolysis as compared to mock treated cells. PMID:19917668

A sensitive fluorescence-based assay for monitoring GM2 ganglioside hydrolysis in live patient cells and their lysates.

PubMed

Tropak, Michael B; Bukovac, Scott W; Rigat, Brigitte A; Yonekawa, Sayuri; Wakarchuk, Warren; Mahuran, Don J

2010-03-01

Enzyme enhancement therapy, utilizing small molecules as pharmacological chaperones, is an attractive approach for the treatment of lysosomal storage diseases that are associated with protein misfolding. However, pharmacological chaperones are also inhibitors of their target enzyme. Thus, a major concern with this approach is that, despite enhancing protein folding within, and intracellular transport of the functional mutant enzyme out of the endoplasmic reticulum, the chaperone will continue to inhibit the enzyme in the lysosome, preventing substrate clearance. Here we demonstrate that the in vitro hydrolysis of a fluorescent derivative of lyso-GM2 ganglioside, like natural GM2 ganglioside, is specifically carried out by the beta-hexosaminidase A isozyme, requires the GM2 activator protein as a co-factor, increases when the derivative is incorporated into anionic liposomes and follows similar Michaelis-Menten kinetics. This substrate can also be used to differentiate between lysates from normal and GM2 activator-deficient cells. When added to the growth medium of cells, the substrate is internalized and primarily incorporated into lysosomes. Utilizing adult Tay-Sachs fibroblasts that have been pre-treated with the pharmacological chaperone Pyrimethamine and subsequently loaded with this substrate, we demonstrate an increase in both the levels of mutant beta-hexosaminidase A and substrate-hydrolysis as compared to mock-treated cells.

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.

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

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.

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.

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.

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.

Three Dimensional Immobilization of Beta-Galactosidase on a Silicon Surface (Preprint)

DTIC Science & Technology

2006-12-01

initial activity after 10 days at 24°C. The ability to generate three- dimensional structures with enhanced loading capacity for biosensing molecules...dimensional structures for biosensors (Charles et al. 2004). Silicon samples that had been washed but not activated with APTS did not retain any enzyme...preparation. The use of silica particles to build a 3-dimensional structure not only provides an increased capacity for the immobilization of β

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

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

Dissecting the effect of chemical additives on the enzymatic hydrolysis of pretreated wheat straw.

PubMed

Monschein, Mareike; Reisinger, Christoph; Nidetzky, Bernd

2014-10-01

Chemical additives were examined for ability to increase the enzymatic hydrolysis of thermo-acidically pretreated wheat straw by Trichoderma reesei cellulase at 50 °C. Semi-empirical descriptors derived from the hydrolysis time courses were applied to compare influence of these additives on lignocellulose bioconversion on a kinetic level, presenting a novel view on their mechanism of action. Focus was on rate retardation during hydrolysis, substrate conversion and enzyme adsorption. PEG 8000 enabled a reduction of enzyme loading by 50% while retaining the same conversion of 67% after 24h. For the first time, a beneficial effect of urea is reported, increasing the final substrate conversion after 48 h by 16%. The cationic surfactant cetyl-trimethylammonium bromide (CTAB) enhanced the hydrolysis rate at extended reaction time (rlim) by 34% and reduced reaction time by 28%. A combination of PEG 8000 and urea increased sugar release more than additives used individually. Copyright © 2014 Elsevier Ltd. All rights reserved.

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.

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.

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

Differential release of manure-borne bioactive phosphorus forms to runoff and leachate under simulated rain.

PubMed

Blaustein, R A; Dao, Thanh H; Pachepsky, Y A; Shelton, D R

2017-05-01

Limited information exists on the unhindered release of bioactive phosphorus (P) from a manure layer to model the partitioning and transport of component P forms before they reach an underlying soil. Rain simulations were conducted to quantify effects of intensity (30, 60, and 90 mm h -1 ) on P release from an application of 60 Mg ha -1 of dairy manure. Runoff contained water-extractable- (WEP), exchangeable and enzyme-labile bioactive P (TBIOP), in contrast to the operationally defined "dissolved-reactive P" form. The released P concentrations and flow-weighed mass loads were described by the log-normal probability density function. At a reference condition of 30 mm h -1 and maintaining the surface at a 5% incline, runoff was minimal, and WEP accounted for 20.9% of leached total P (TP) concentrations, with an additional 25-30% as exchangeable and enzyme-labile bioactive P over the 1-h simulation. On a 20% incline, increased intensity accelerated occurrence of concentration max and shifted the skewed P concentration distribution more to the left. Differences in trends of WEP, TBIOP, or net enzyme-labile P (PHP o ) cumulative mass released per unit mass of manure between intensities were attributable to the higher frequency of raindrops striking the manure layer, thus increasing detachment and load of colloidal PHP o of the water phases. Thus, detailed knowledge of manure physical characteristics, bioactive P distribution in relation to rain intensity, and attainment of steady-state of water fluxes were critical factors in improved prediction of partitioning and movement of manure-borne P under rainfall. Published by Elsevier Ltd.

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.

Thiol peroxidase deficiency leads to increased mutational load and decreased fitness in Saccharomyces cerevisiae.

PubMed

Kaya, Alaattin; Lobanov, Alexei V; Gerashchenko, Maxim V; Koren, Amnon; Fomenko, Dmitri E; Koc, Ahmet; Gladyshev, Vadim N

2014-11-01

Thiol peroxidases are critical enzymes in the redox control of cellular processes that function by reducing low levels of hydroperoxides and regulating redox signaling. These proteins were also shown to regulate genome stability, but how their dysfunction affects the actual mutations in the genome is not known. Saccharomyces cerevisiae has eight thiol peroxidases of glutathione peroxidase and peroxiredoxin families, and the mutant lacking all these genes (∆8) is viable. In this study, we employed two independent ∆8 isolates to analyze the genome-wide mutation spectrum that results from deficiency in these enzymes. Deletion of these genes was accompanied by a dramatic increase in point mutations, many of which clustered in close proximity and scattered throughout the genome, suggesting strong mutational bias. We further subjected multiple lines of wild-type and ∆8 cells to long-term mutation accumulation, followed by genome sequencing and phenotypic characterization. ∆8 lines showed a significant increase in nonrecurrent point mutations and indels. The original ∆8 cells exhibited reduced growth rate and decreased life span, which were further reduced in all ∆8 mutation accumulation lines. Although the mutation spectrum of the two independent isolates was different, similar patterns of gene expression were observed, suggesting the direct contribution of thiol peroxidases to the observed phenotypes. Expression of a single thiol peroxidase could partially restore the growth phenotype of ∆8 cells. This study shows how deficiency in nonessential, yet critical and conserved oxidoreductase function, leads to increased mutational load and decreased fitness. Copyright © 2014 by the Genetics Society of America.

Dynamic and Static Exercises Differentially Affect Plasma Cytokine Content in Elite Endurance- and Strength-Trained Athletes and Untrained Volunteers

PubMed Central

Kapilevich, Leonid V.; Zakharova, Anna N.; Kabachkova, Anastasia V.; Kironenko, Tatyana A.; Orlov, Sergei N.

2017-01-01

Extensive exercise increases the plasma content of IL-6, IL-8, IL-15, leukemia inhibitory factor (LIF), and several other cytokines via their augmented transcription in skeletal muscle cells. However, the relative impact of aerobic and resistant training interventions on cytokine production remains poorly defined. In this study, we compared effects of dynamic and static load on cytokine plasma content in elite strength- and endurance-trained athletes vs. healthy untrained volunteers. The plasma cytokine content was measured before, immediately after, and 30 min post-exercise using enzyme-linked immunosorbent assay. Pedaling on a bicycle ergometer increased IL-6 and IL-8 content in the plasma of trained athletes by about 4- and 2-fold, respectively. In contrast to dynamic load, weightlifting had negligible impact on these parameters in strength exercise-trained athletes. Unlike IL-6 and IL-8, dynamic exercise had no impact on IL-15 and LIF, whereas static load increases the content of these cytokines by ~50%. Two-fold increment of IL-8 content seen in athletes subjected to dynamic exercise was absent in untrained individuals, whereas the ~50% increase in IL-15 triggered by static load in the plasma of weightlifting athletes was not registered in the control group. Thus, our results show the distinct impact of static and dynamic exercises on cytokine content in the plasma of trained athletes. They also demonstrate that both types of exercises differentially affect cytokine content in plasma of athletes and untrained persons. PMID:28194116

Rivastigmine-loaded in situ gelling nanostructured lipid carriers for nose to brain delivery.

PubMed

Wavikar, Preeti R; Vavia, Pradeep R

2015-01-01

In the current research work, rivastigmine (RV)-loaded in situ gelling nanostructured lipid carriers (NLCs) were developed for nose to brain delivery. NLCs were fabricated by ethanol injection method using glyceryl monosterate, Capmul MCM C8, Lecithin and Tween 80. NLCs showed average particle size of 123.2 ± 2.3 nm with entrapment efficiency of 68.34 ± 3.4%. DSC, XRD and IR studies showed complete amorphization and incorporation of the drug into nanoparticles. NLCs were incorporated into an in situ gelling system using 0.8% gellan gum and 15% Lutrol F 127. RV in situ gel showed excellent elasticity, rheology, mucoadhesion and adhesiveness to facilitate its adhesion to the upper nasal mucosa. NLC-based in situ gel showed a 2-fold increase in nasal permeation of the drug over plain RV solution. In situ gelling NLCs showed a 3-fold increase in enzyme inhibition efficacy.

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.

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.

Thermomyces lanuginosus lipase-catalyzed synthesis of natural flavor esters in a continuous flow microreactor.

PubMed

Gumel, Ahmad Mohammed; Annuar, M S M

2016-06-01

Enzymatic catalysis is considered to be among the most environmental friendly processes for the synthesis of fine chemicals. In this study, lipase from Thermomyces lanuginosus (Lecitase Ultra™) was used to catalyze the synthesis of flavor esters, i.e., methyl butanoate and methyl benzoate by esterification of the acids with methanol in a microfluidic system. Maximum reaction rates of 195 and 115 mM min -1 corresponding to catalytic efficiencies (k cat /K M ) of 0.30 and 0.24 min -1  mM -1 as well as yield conversion of 54 and 41 % were observed in methyl butanoate and methyl benzoate synthesis, respectively. Catalytic turnover (k cat ) was higher for methyl butanoate synthesis. Rate of synthesis and yield decreased with increasing flow rates. For both esters, increase in microfluidic flow rate resulted in increased advective transport over molecular diffusion and reaction rate, thus lower conversion. In microfluidic synthesis using T. lanuginosus lipase, the following reaction conditions were 40 °C, flow rate 0.1 mL min -1 , and 123 U g -1 enzyme loading found to be the optimum operating limits. The work demonstrated the application of enzyme(s) in a microreactor system for the synthesis of industrially important esters.

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

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.

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.

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.

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

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...

Phosphate binding reduces aortic angiotensin-converting enzyme and enhances nitric oxide bioactivity in experimental renal insufficiency.

PubMed

Eräranta, Arttu; Törmänen, Suvi; Kööbi, Peeter; Vehmas, Tuija I; Lakkisto, Päivi; Tikkanen, Ilkka; Moilanen, Eeva; Niemelä, Onni; Mustonen, Jukka; Pörsti, Ilkka

2014-01-01

Disturbed calcium-phosphorus metabolism is associated with increased kidney angiotensin-converting enzyme (ACE) in experimental chronic renal insufficiency (CRI). However, information about the effects of phosphate binding and loading on vascular ACE is lacking. Fifteen weeks after 5/6 nephrectomy (NX), rats were placed on a phosphate-binding (NX+Ca, 3.0% Ca), phosphate-loading (NX+Pi, 1.5% Pi), or control diet for 12 weeks (NX and sham). Aortic ACE, blood pressure, plasma phosphate, and parathyroid hormone were increased in the NX and NX+Pi groups, but were reduced with phosphate binding. Endothelium-mediated relaxations of isolated mesenteric conduit artery rings to acetylcholine were impaired in the NX and NX+Pi groups, but did not differ from sham in NX+Ca rats. Experiments with nitric oxide (NO) synthase inhibition in vitro suggested that the NO-mediated component of acetylcholine response was lower in the NX and NX+Pi groups, but did not differ from sham in NX+Ca rats. In all NX groups, aortic endothelial NO synthase (eNOS) was reduced, while plasma and urine concentrations of NO metabolites were increased. Aortic nitrated proteins and calcification were increased in the NX and NX+Pi groups when compared with the NX+Ca and sham groups. Hypertension in the NX model of CRI was associated with reduced vasorelaxation, decreased eNOS, and increased ACE and nitrated proteins in the aorta. Phosphate binding with calcium carbonate enhanced vasorelaxation via endogenous NO and suppressed elevation of ACE and nitrated proteins, suggesting reduced vascular oxidative stress. Our findings support the view that correction of the calcium-phosphorus balance prevents CRI-induced vascular pathophysiology.

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.

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

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.

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

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.

Digestive enzyme expression and epithelial structure of small intestine in neonatal rats after 16 days spaceflight

NASA Astrophysics Data System (ADS)

Miyake, M.; Yamasaki, M.; Hazama, A.; Ijiri, K.; Shimizu, T.

It is important to assure whether digestive system can develop normally in neonates during spaceflight. Because the small intestine changes its function and structure drastically around weaning known as redifferentiation. Lactase expression declines and sucrase increases in small intestine for digestion of solid food before weaning. In this paper, we compared this enzyme transition and structural development of small intestine in neonatal rats after spaceflight. To find digestive genes differentially expressed in fight rats, DNA membrane macroarray was also used. Eight-day old rats were loaded to Space Shuttle Columbia, and housed in the animal facility for 16 days in space (STS-90, Neurolab mission). Two control groups (AGC; asynchronous ground control and VIV; vivarium) against flight group (FLT) were prepared. There was no difference in structure (crypt depth) and cell differentiation of epithelium between FLT and AGC by immunohistochemical analysis. We found that the amount of sucrase mRNA compared to lactase was decreased in FLT by RT-PCR. It reflected the enzyme transition was inhibited. Increase of 5 genes (APO A-I, APO A-IV, ACE, aFABP and aminopeptidase M) and decrease of carboxypeptidase-D were detected in FLT using macroarray. We think nutrition differences (less nourishment and late weaning) during spaceflight may cause inhibition of enzyme transition at least partly. The weightlessness might contribute to the inhibition through behavioral change.

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.

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.

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

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-...

Placental sulfatase deficiency: maternal and fetal expression of steroid sulfatase deficiency and X-linked ichthyosis.

PubMed

Bradshaw, K D; Carr, B R

1986-07-01

PSD-X-linked ichthyosis are manifestations of a similar disorder of an inborn error of metabolism characterized by a deficiency of steroid sulfatase. The decreased enzyme activity is due to the absence of the expression of enzyme (steroid sulfatase) protein. Affected individuals with this disorder are males (X-linked inheritance) with a frequency of 1/2000 to 1/6000 births. Homozygous females from cosanguineous marriages have been reported with this disorder. The diagnosis is suspected and confirmed by: Low estriol excretion; Negative DHEAS loading test Increased DHEAS in amnionic fluid; Normal DHEAS in cord plasma; Possible delayed or abnormal labor patterns; Decreased sulfatase activity in the placenta, fibroblast, erythrocytes, lymphocytes or leukocytes of affected individuals; Development of ichthyosis in male infants at 2 to 3 months of age.

Effects of laccase on lignin depolymerization and enzymatic hydrolysis of ensiled corn stover.

PubMed

Chen, Qin; Marshall, Megan N; Geib, Scott M; Tien, Ming; Richard, Tom L

2012-08-01

The aim of this study was to explore the synergies of laccase, a ligninolytic enzyme, with cellulose and hemicellulase amendments on ensiled corn stover. Molecular signals of lignin decomposition were observed by tetramethylammonium hydroxide thermochemolysis and gas chromatography-mass spectroscopy (TMAH-GC-MS) analysis. The significant findings suggest that ensilage might provide a platform for biological pretreatment. By partially hydrolyzing cellulose and hemicellulose into soluble sugars, ensilage facilitates laccase penetration into the lignocellulose complex to enhance lignin degradation. Downstream cellulose hydrolysis was improved 7% with increasing laccase loading rate. These results demonstrate the potential of enzymes, either directly amended or expressed by microbes during ensilage, to maximize utilization of corn stover for cellulosic biofuels and other downstream fermentations. Copyright © 2012. Published by Elsevier Ltd.

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.

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.

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.

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.

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

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.

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 ...

Effect of bovine serum albumin (BSA) on enzymatic cellulose hydrolysis.

PubMed

Wang, Hui; Mochidzuki, Kazuhiro; Kobayashi, Shinichi; Hiraide, Hatsue; Wang, Xiaofen; Cui, Zongjun

2013-06-01

Bovine serum albumin (BSA) was added to filter paper during the hydrolysis of cellulase. Adding BSA before the addition of the cellulase enhances enzyme activity in the solution, thereby increasing the conversion rate of cellulose. After 48 h of BSA treatment, the BSA adsorption quantities are 3.3, 4.6, 7.8, 17.2, and 28.3 mg/g substrate, each with different initial BSA concentration treatments at 50 °C; in addition, more cellulase was adsorbed onto the filter paper at 50 °C compared with 35 °C. After 48 h of hydrolysis, the free-enzyme activity could not be measured without the BSA treatment, whereas the remaining activity of the filter paper activity was approximately 41 % when treated with 1.0 mg/mL BSA. Even after 96 h of hydrolysis, 25 % still remained. Meanwhile, after 48 h of incubation without substrate, the remaining enzyme activities were increased 20.7 % (from 43.7 to 52.7 %) and 94.8 % (from 23.3 to 45.5 %) at 35 and 50 °C, respectively. Moreover, the effect of the BSA was more obvious at 35 °C compared with 50 °C. When using 15 filter paper cellulase units per gram substrate cellulase loading at 50 °C, the cellulose conversion was increased from 75 % (without BSA treatment) to ≥90 % when using BSA dosages between 0.1 and 1.5 mg/mL. Overall, these results suggest that there are promising strategies for BSA treatment in the reduction of enzyme requirements during the hydrolysis of cellulose.

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.

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-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.

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.

Structural changes of corn stover lignin during acid pretreatment.

PubMed

Moxley, Geoffrey; Gaspar, Armindo Ribeiro; Higgins, Don; Xu, Hui

2012-09-01

In this study, raw corn stover was subjected to dilute acid pretreatments over a range of severities under conditions similar to those identified by the National Renewable Energy Laboratory (NREL) in their techno-economic analysis of biochemical conversion of corn stover to ethanol. The pretreated corn stover then underwent enzymatic hydrolysis with yields above 70 % at moderate enzyme loading conditions. The enzyme exhausted lignin residues were characterized by ³¹P NMR spectroscopy and functional moieties quantified and correlated to enzymatic hydrolysis yields. Results from this study indicated that both xylan solubilization and lignin degradation are important for improving the enzyme accessibility and digestibility of dilute acid pretreated corn stover. At lower pretreatment temperatures, there is a good correlation between xylan solubilization and cellulose accessibility. At higher pretreatment temperatures, lignin degradation correlated better with cellulose accessibility, represented by the increase in phenolic groups. During acid pretreatment, the ratio of syringyl/guaiacyl functional groups also gradually changed from less than 1 to greater than 1 with the increase in pretreatment temperature. This implies that more syringyl units are released from lignin depolymerization of aryl ether linkages than guaiacyl units. The condensed phenolic units are also correlated with the increase in pretreatment temperature up to 180 °C, beyond which point condensation reactions may overtake the hydrolysis of aryl ether linkages as the dominant reactions of lignin, thus leading to decreased cellulose accessibility.

Protein immobilization on epoxy-activated thin polymer films: effect of surface wettability and enzyme loading.

PubMed

Chen, Bo; Pernodet, Nadine; Rafailovich, Miriam H; Bakhtina, Asya; Gross, Richard A

2008-12-02

A series of epoxy-activated polymer films composed of poly(glycidyl methacrylate/butyl methacrylate/hydroxyethyl methacrylate) were prepared. Variation in comonomer composition allowed exploration of relationships between surface wettability and Candida antartica lipase B (CALB) binding to surfaces. By changing solvents and polymer concentrations, suitable conditions were developed for preparation by spin-coating of uniform thin films. Film roughness determined by AFM after incubation in PBS buffer for 2 days was less than 1 nm. The occurrence of single CALB molecules and CALB aggregates at surfaces was determined by AFM imaging and measurements of volume. Absolute numbers of protein monomers and multimers at surfaces were used to determine values of CALB specific activity. Increased film wettability, as the water contact angle of films increased from 420 to 550, resulted in a decreased total number of immobilized CALB molecules. With further increases in the water contact angle of films from 55 degrees to 63 degrees, there was an increased tendency of CALB molecules to form aggregates on surfaces. On all flat surfaces, two height populations, differing by more than 30%, were observed from height distribution curves. They are attributed to changes in protein conformation and/or orientation caused by protein-surface and protein-protein interactions. The fraction of molecules in these populations changed as a function of film water contact angle. The enzyme activity of immobilized films was determined by measuring CALB-catalyzed hydrolysis of p-nitrophenyl butyrate. Total enzyme specific activity decreased by decreasing film hydrophobicity.

[Molecular mechanisms of skeletal muscle hypertrophy].

PubMed

Astratenkova, I V; Rogozkin, V A

2014-06-01

Enzymes Akt, AMPK, mTOR, S6K and PGC-1a coactivator take part in skeletal muscles in the regulation of synthesis of proteins. The expression of these proteins is regulated by growth factors, hormones, nutrients, mechanical loading and leads to an increase in muscle mass and skeletal muscle hypertrophy. The review presents the results of studies published in the past four years, which expand knowledge on the effects of various factors on protein synthesis in skeletal muscle. The attention is focused on the achievements that reveal and clarify the signaling pathways involved in the regulation of protein synthesis in skeletal muscle. The central place is taken by mTOR enzyme which controls and regulates the main stages of the cascade of reactions of muscle proteins providing synthesis in the conditions of human life. coactivator PGC-1a.

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.

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.

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.

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.

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.

Responsive polymer-based colloids for drug delivery and bioconversion

NASA Astrophysics Data System (ADS)

Kudina, Olena

Responsive polymer-based colloids (RPBC) are the colloidal structures containing responsive polymeric component which is able to adapt its physico-chemical properties to the environment by undergoing chemical and/or conformational changes. The goal of the dissertation is to develop and characterize several groups of RPBC with different morphological complexity and explore their potential in drug delivery and bioconversion. The role of RPBC morphology for these specific applications is discussed in details. Three groups of RPBC were fabricated: i. polymeric micelles; ii. mixed polymeric micelles; iii. hybrid polymer-inorganic particles. All fabricated RPBCs contain polymeric component in their structure. The dissertation investigates how the changes of the responsive polymeric component properties are reflected in morphologies of RPBC. The first group of RPBC, polymeric micelles, was formed by the self-assembly of amphiphilic invertible polymers (AIPs) synthesized in our group. AIPs self-assemble into invertible micellar assemblies (IMAs) in solvents of different polarity. In this work, IMAs ability to invert the structure as a response to the change in solvent polarity was demonstrated using 1H NMR spectroscopy and SANS. It was shown that the IMAs incorporate hydrophobic cargo either in the core or in the shell, depending on the chemical structure of cargo molecules. Following in vitro study demonstrates that loaded with drug (curcumin) IMAs are cytotoxic to osteosarcoma cells. Mixed polymeric micelles represent another, more complex, RPBC morphologies studied in the dissertation. Mixed micelles were fabricated from AIPs and amphiphilic oligomers synthesized from pyromellitic dianhydride, polyethylene glycol methyl ethers, and alkanols/cholesterol. The combination of selected AIP and oligomers based on cholesterol results in mixed micelles with an increased drug-loading capacity (from 10% w/w loaded curcumin in single component IMAs to 26%w/w in mixed micelles). Even more complex colloids are hybrid polymer-inorganic particles, the third RPBC group studied in dissertation. Material was designed as core--shell particles with superparamagnetic core engulfed by grafted polymer brushes. These particles were loaded with enzymes (cellulases), thus, are turned into enzymogels for cellulose bioconversion. The study demonstrates that such RPBCs can be used multiple times during hydrolysis and provide an about four-fold increase in glucose production in comparison to free enzymes.

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.

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.

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.

Nitrogen-Doped Carbon Nanotubes Supported by Macroporous Carbon as an Efficient Enzymatic Biosensing Platform for Glucose.

PubMed

Song, Yonghai; Lu, Xingping; Li, Yi; Guo, Qiaohui; Chen, Shuiliang; Mao, Lanqun; Hou, Haoqing; Wang, Li

2016-01-19

Effective immobilization of enzymes/proteins on an electrode surface is very essential for biosensor development, but it still remains challenging because enzymes/proteins tend to form close-packed structures on the electrode surface. In this work, nitrogen-doped carbon nanotubes (NCNTs) supported by three-dimensional Kenaf Stem-derived porous carbon (3D-KSC) (denoted as 3D-KSC/NCNTs) nanocomposites were constructed as the supporting matrix to load glucose oxidase (GOD) for preparing integrated glucose biosensors. These NCNTs are vertically arrayed on the channel walls of the 3D-KSC via the chemical vapor deposition method, which could noticeably increase the effective surface area, mechanical stability, and active sites (originating from the doped nitrogen) of the nanocomposites. The integrated glucose biosensor exhibits some advantages over the traditional GOD electrodes in terms of the capability to promote the direct electron transfer of GOD, enhance the mechanical stability of the biosensor attributed to the strong interaction between NCNTs and GOD, and enlarge the specific surface area to efficiently load a large number of GODs. The as-prepared biosensor shows a good performance toward both oxygen reduction and glucose biosensing. This study essentially offers a novel approach for the development of biosensors with excellent analytical properties.

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.

Systems Analysis of the Response of Photosynthesis, Metabolism, and Growth to an Increase in Irradiance in the Photosynthetic Model Organism Chlamydomonas reinhardtii[C][W][OPEN

PubMed Central

Mettler, Tabea; Mühlhaus, Timo; Hemme, Dorothea; Schöttler, Mark-Aurel; Rupprecht, Jens; Idoine, Adam; Veyel, Daniel; Pal, Sunil Kumar; Yaneva-Roder, Liliya; Winck, Flavia Vischi; Sommer, Frederik; Vosloh, Daniel; Seiwert, Bettina; Erban, Alexander; Burgos, Asdrubal; Arvidsson, Samuel; Schönfelder, Stephanie; Arnold, Anne; Günther, Manuela; Krause, Ursula; Lohse, Marc; Kopka, Joachim; Nikoloski, Zoran; Mueller-Roeber, Bernd; Willmitzer, Lothar; Bock, Ralph; Schroda, Michael; Stitt, Mark

2014-01-01

We investigated the systems response of metabolism and growth after an increase in irradiance in the nonsaturating range in the algal model Chlamydomonas reinhardtii. In a three-step process, photosynthesis and the levels of metabolites increased immediately, growth increased after 10 to 15 min, and transcript and protein abundance responded by 40 and 120 to 240 min, respectively. In the first phase, starch and metabolites provided a transient buffer for carbon until growth increased. This uncouples photosynthesis from growth in a fluctuating light environment. In the first and second phases, rising metabolite levels and increased polysome loading drove an increase in fluxes. Most Calvin-Benson cycle (CBC) enzymes were substrate-limited in vivo, and strikingly, many were present at higher concentrations than their substrates, explaining how rising metabolite levels stimulate CBC flux. Rubisco, fructose-1,6-biosphosphatase, and seduheptulose-1,7-bisphosphatase were close to substrate saturation in vivo, and flux was increased by posttranslational activation. In the third phase, changes in abundance of particular proteins, including increases in plastidial ATP synthase and some CBC enzymes, relieved potential bottlenecks and readjusted protein allocation between different processes. Despite reasonable overall agreement between changes in transcript and protein abundance (R2 = 0.24), many proteins, including those in photosynthesis, changed independently of transcript abundance. PMID:24894045

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.

Maternal loading with very low-density lipoproteins stimulates fetal surfactant synthesis.

PubMed

Ryan, Alan J; Medh, Jheem D; McCoy, Diann M; Salome, Ronald G; Mallampalli, Rama K

2002-08-01

We examined whether administration of very low-density lipoproteins (VLDL) to pregnant rats increases surfactant phosphatidylcholine (PtdCho) content in fetal pre-type II alveolar epithelial cells. VLDL-triglycerides are hydrolyzed to fatty acids by lipoprotein lipase (LPL), an enzyme activated by heparin. Fatty acids released by LPL can incorporate into the PtdCho molecule or activate the key biosynthetic enzyme cytidylyltransferase (CCT). Dams were given BSA, heparin, VLDL, or VLDL with heparin intravenously. Radiolabeled VLDL given to the pregnant rat crossed the placenta and was distributed systemically in the fetus and incorporated into disaturated PtdCho (DSPtdCho) in pre-type II cells. Maternal administration of VLDL with heparin increased DSPtdCho content in cells by 45% compared with control (P < 0.05). VLDL produced a dose-dependent, saturable, and selective increase in CCT activity. VLDL did not significantly alter immunoreactive CCT content but increased palmitic, stearic, and oleic acids in pre-type II cells. Furthermore, hypertriglyceridemic apolipoprotein E knockout mice contained significantly greater levels of DSPtdCho content in alveolar lavage and CCT activity compared with either LDL receptor knockout mice or wild-type controls that have normal serum triglycerides. Thus the nutritional or genetic modulation of serum VLDL-triglycerides provides specific fatty acids that stimulate PtdCho synthesis and CCT activity thereby increasing surfactant content.

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.

[The significance of free radicals and antioxidants due to the load induced by sport activity].

PubMed

Holecek, V; Liska, J; Racek, J; Rokyta, R

2004-01-01

Sport performance is followed by a high production of free radicals. The main reasons are reperfusion after the previous imbalance between the increased need of the organism and the ability of blood supply by oxygen, increased production of ATP, decomposition of the cells particularly white blood cells, oxidation of the purin basis from DNA, stress, output of epinephrine release of free iron, increased temperature in the muscle and its inflammation, and the reception of free radicals from external environment. Peroxidation of lipids, proteins, DNA and other compounds follows the previous biochemical steps. Antioxidants are consumed by free radicals, antioxidative enzymes are released into blood plasma, intracellular calcium is increased, the production of nitric oxide rises, the levels of hydrogen peroxide and hypochlorous acid increase. These penetrate through the membranes and oxidatively damage the tissues. Training improves the ability of the organism to balance the increased load of free radicals. The damage can be lowered by the application of a mixture of antioxidants, the most important are vitamin C, A, E, glutathione, selenium, carnosine, eventually bioflavonoids and ginkgo biloba. The lack of antioxidants can significantly diminish the sport performance and therefore the supplementation with antioxidants is for top sportsmen but also for aged people advisable.

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

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

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.

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.

Dual function of tributyrin emulsion: solubilization and enhancement of anticancer effect of celecoxib.

PubMed

Kang, Sung Nam; Hong, Soon-Seok; Lee, Mi-Kyung; Lim, Soo-Jeong

2012-05-30

Tributyrin, a triglyceride analogue of butyrate, can act as a prodrug of an anticancer agent butyrate after being cleaved by intracellular enzymes. We recently demonstrated that the emulsion containing tributyrin as an inner oil phase possesses a potent anticancer activity. Herein we sought to develop tributyrin emulsion as a carrier of celecoxib, a poorly-water soluble drug with anticancer activity. Combined treatment of human HCT116 colon cancer cells with free celecoxib plus tributyrin emulsion inhibited the cellular proliferation more effectively than that of each drug alone, suggesting the possibility of tributyrin emulsion as a potential celecoxib carrier. The mean droplet size of emulsions tended to increase as the tributyrin content in emulsion increases and the concentration of celecoxib loaded in emulsions was affected by tributyrin content and the initial amount of celecoxib, but not by the total amount of surfactant mixture. The concentration of celecoxib required to inhibit the growth of HCT116 and B16-F10 cancer cells by 50% was 2.6- and 3.1-fold lowered by loading celecoxib in tributyrin emulsions, compared with free celecoxib. These data suggest that the anticancer activity of celecoxib was enhanced by loading in tributyrin emulsions, probably due to the solubilization capacity and anticancer activity of tributyrin emulsion. Copyright © 2012 Elsevier B.V. All rights reserved.

Cardiomyocyte exosomes regulate glycolytic flux in endothelium by direct transfer of GLUT transporters and glycolytic enzymes.

PubMed

Garcia, Nahuel A; Moncayo-Arlandi, Javier; Sepulveda, Pilar; Diez-Juan, Antonio

2016-03-01

Cardiomyocytes (CMs) and endothelial cells (ECs) have an intimate anatomical relationship, which is essential for maintaining the metabolic requirements of the heart. Little is known about the mechanisms that regulate nutrient flow from ECs to associated CMs, especially in situations of acute stress when local active processes are required to regulate endothelial transport. We examined whether CM-derived exosomes can modulate glucose transport and metabolism in ECs. In conditions of glucose deprivation, CMs increase the synthesis and secretion of exosomes. These exosomes are loaded with functional glucose transporters and glycolytic enzymes, which are internalized by ECs, leading to increased glucose uptake, glycolytic activity, and pyruvate production in recipient cells. These findings establish CM-derived exosomes as key components of the cardio-endothelial communication system which, through intercellular protein complementation, would allow a rapid response from ECs to increase glucose transport and a putative uptake of metabolic fuels from blood to CMs. This CM-EC protein complementation process might have implications for metabolic regulation in health and disease. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2015. For permissions please email: journals.permissions@oup.com.

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.

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

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.

Thapsigargin defines the roles of cellular calcium in secretagogue-stimulated enzyme secretion from pancreatic acini.

PubMed

Metz, D C; Patto, R J; Mrozinski, J E; Jensen, R T; Turner, R J; Gardner, J D

1992-10-15

In the present study we used thapsigargin (TG), an inhibitor of microsomal calcium ATPase, to evaluate the roles of free cytoplasmic calcium and intracellular stored calcium in secretagogue-stimulated enzyme secretion from rat pancreatic acini. Using microspectrofluorimetry of fura-2-loaded pancreatic acini, we found that TG caused a sustained increase in free cytoplasmic calcium by mobilizing calcium from inositol 1,4,5-trisphosphate-sensitive intracellular stores and by increasing influx of extracellular calcium. TG also caused a small increase in basal amylase secretion, inhibited the stimulation of amylase secretion caused by secretagogues that increase inositol 1,4,5-trisphosphate, and potentiated the stimulation of amylase secretion caused by 12-O-tetradecanoylphorbol-13-acetate or secretagogues that increase cyclic adenosine 3',5'-monophosphate. Bombesin, which like TG increased free cytoplasmic calcium, also potentiated the stimulation of amylase secretion caused by secretagogues that increase cyclic adenosine 3',5'-monophosphate, but did not inhibit the stimulation of amylase secretion caused by secretagogues that increase inositol 1,4,5-trisphosphate. Finally, TG inhibited the sustained phase of cholecystokinin-stimulated amylase secretion and potentiated the time course of vasoactive intestinal peptide-stimulated amylase secretion. The present findings indicate that stimulation of amylase secretion by secretagogues that increase inositol 1,4,5-trisphosphate does not depend on increased free cytoplasmic calcium per se. In contrast, TG-induced potentiation of the stimulation of secretagogues that increase cellular cyclic adenosine 3',5'-monophosphate appears to result from increased free cytoplasmic calcium per se.

Effect of Polymer Porosity on Aqueous Self-Healing Encapsulation of Proteins in PLGA Microspheres

PubMed Central

Reinhold, Samuel E.

2014-01-01

Self-healing (SH) poly(lactic-co-glycolic acid) (PLGA) microspheres are a unique class of functional biomaterials capable of microencapsulating process-sensitive proteins by simple mixing and heating the drug-free polymer in aqueous protein solution. Drug-free SH microspheres of PLGA 50/50 with percolating pore networks of varying porosity (ε = 0.49–73) encapsulate increasing lysozyme (~1–10% w/w) with increasing ε, with typically ~20–25% pores estimated assessible to entry by the enzyme from the external solution. Release kinetics of lysozyme under physiological conditions is continuous over > 2 weeks and most strongly influenced by ε and protein loading before reaching a lag phase until 28 days at the study completion. Recovered enzyme after release is typically predominantly monomeric and active. Formulations containing acid-neutralizing MgCO3 at >4.3% exhibit >97% monomeric and active protein after the release with full mass balance recovery. Hence, control of SH polymer ε is a key parameter to development of this new class of biomaterials. PMID:24285573

Macrophages offer a paradigm switch for CNS delivery of therapeutic proteins

PubMed Central

Klyachko, Natalia L; Haney, Matthew J; Zhao, Yuling; Manickam, Devika S; Mahajan, Vivek; Suresh, Poornima; Hingtgen, Shawn D; Mosley, R Lee; Gendelman, Howard E; Kabanov, Alexander V; Batrakova, Elena V

2013-01-01

Aims Active targeted transport of the nanoformulated redox enzyme, catalase, in macrophages attenuates oxidative stress and as such increases survival of dopaminergic neurons in animal models of Parkinson’s disease. Optimization of the drug formulation is crucial for the successful delivery in living cells. We demonstrated earlier that packaging of catalase into a polyion complex micelle (‘nanozyme’) with a synthetic polyelectrolyte block copolymer protected the enzyme against degradation in macrophages and improved therapeutic outcomes. We now report the manufacture of nanozymes with superior structure and therapeutic indices. Methods Synthesis, characterization and therapeutic efficacy of optimal cell-based nanoformulations are evaluated. Results A formulation design for drug carriers typically works to avoid entrapment in monocytes and macrophages focusing on small-sized nanoparticles with a polyethylene glycol corona (to provide a stealth effect). By contrast, the best nanozymes for delivery in macrophages reported in this study have a relatively large size (~200 nm), which resulted in improved loading capacity and release from macrophages. Furthermore, the cross-linking of nanozymes with the excess of a nonbiodegradable linker ensured their low cytotoxicity, and efficient catalase protection in cell carriers. Finally, the ‘alternatively activated’ macrophage phenotype (M2) utilized in these studies did not promote further inflammation in the brain, resulting in a subtle but statistically significant effect on neuronal regeneration and repair in vivo. Conclusion The optimized cross-linked nanozyme loaded into macrophages reduced neuroinflammatory responses and increased neuronal survival in mice. Importantly, the approach for nanoformulation design for cell-mediated delivery is different from the common requirements for injectable formulations. PMID:24237263

Macrophages offer a paradigm switch for CNS delivery of therapeutic proteins.

PubMed

Klyachko, Natalia L; Haney, Matthew J; Zhao, Yuling; Manickam, Devika S; Mahajan, Vivek; Suresh, Poornima; Hingtgen, Shawn D; Mosley, R Lee; Gendelman, Howard E; Kabanov, Alexander V; Batrakova, Elena V

2014-07-01

Active targeted transport of the nanoformulated redox enzyme, catalase, in macrophages attenuates oxidative stress and as such increases survival of dopaminergic neurons in animal models of Parkinson's disease. Optimization of the drug formulation is crucial for the successful delivery in living cells. We demonstrated earlier that packaging of catalase into a polyion complex micelle ('nanozyme') with a synthetic polyelectrolyte block copolymer protected the enzyme against degradation in macrophages and improved therapeutic outcomes. We now report the manufacture of nanozymes with superior structure and therapeutic indices. Synthesis, characterization and therapeutic efficacy of optimal cell-based nanoformulations are evaluated. A formulation design for drug carriers typically works to avoid entrapment in monocytes and macrophages focusing on small-sized nanoparticles with a polyethylene glycol corona (to provide a stealth effect). By contrast, the best nanozymes for delivery in macrophages reported in this study have a relatively large size (≈ 200 nm), which resulted in improved loading capacity and release from macrophages. Furthermore, the cross-linking of nanozymes with the excess of a nonbiodegradable linker ensured their low cytotoxicity, and efficient catalase protection in cell carriers. Finally, the 'alternatively activated' macrophage phenotype (M2) utilized in these studies did not promote further inflammation in the brain, resulting in a subtle but statistically significant effect on neuronal regeneration and repair in vivo. The optimized cross-linked nanozyme loaded into macrophages reduced neuroinflammatory responses and increased neuronal survival in mice. Importantly, the approach for nanoformulation design for cell-mediated delivery is different from the common requirements for injectable formulations.

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.

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.

Chromosome Duplication in Saccharomyces cerevisiae

PubMed Central

Bell, Stephen P.; Labib, Karim

2016-01-01

The accurate and complete replication of genomic DNA is essential for all life. In eukaryotic cells, the assembly of the multi-enzyme replisomes that perform replication is divided into stages that occur at distinct phases of the cell cycle. Replicative DNA helicases are loaded around origins of DNA replication exclusively during G1 phase. The loaded helicases are then activated during S phase and associate with the replicative DNA polymerases and other accessory proteins. The function of the resulting replisomes is monitored by checkpoint proteins that protect arrested replisomes and inhibit new initiation when replication is inhibited. The replisome also coordinates nucleosome disassembly, assembly, and the establishment of sister chromatid cohesion. Finally, when two replisomes converge they are disassembled. Studies in Saccharomyces cerevisiae have led the way in our understanding of these processes. Here, we review our increasingly molecular understanding of these events and their regulation. PMID:27384026

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

Dendritic cells induce specific cytotoxic T lymphocytes against prostate cancer TRAMP-C2 cells loaded with freeze- thaw antigen and PEP-3 peptide.

PubMed

Liu, Xiao-Qi; Jiang, Rong; Li, Si-Qi; Wang, Jing; Yi, Fa-Ping

2015-01-01

Prostate cancer is the most common cancer in men. In this study, we investigated immune responses of cytotoxic T lymphocytes (CTLs) against TRAMP-C2 prostate cancer cells after activation by dendritic cells (DCs) loaded with TRAMP-C2 freeze-thaw antigen and/or PEP-3 peptide in vitro. Bone marrow-derived DC from the bone marrow of the C57BL/6 were induced to mature by using the cytokine of rhGM-CSF and rhIL-4, and loaded with either the freeze-thaw antigen or PEP-3 peptide or both of them. Maturation of DCs was detected by flow cytometry. The killing efficiency of the CTLs on TRAMP-C2 cells were detected by flow cytometry, CCK8, colony formation, transwell migration, and wound-healing assay. The levels of the IFN-γ, TNF-β and IL-12 were measured by enzyme-linked immunosorbent assay (ELISA). Compared with the unloaded DCs, the loaded DCs had significantly increased expression of several phenotypes related to DC maturation. CTLs activated by DCs loaded with freeze-thaw antigen and PEP-3 peptide had more evident cytotoxicity against TRAMP-C2 cells in vitro. The secretion levels of IFN-γ, TNF-β and IL-12, secreted by DCs loaded with antigen and PEP-3 and interaction with T cells, were higher than in the other groups. Our results suggest that the CTLs activated by DCs loaded with TRAMP-C2 freeze-thaw antigen and PEP-3 peptide exert a remarkable killing efficiency against TRAMP-C2 cells in vitro.

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

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

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.

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.

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.

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.

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.

Aldehydic load and aldehyde dehydrogenase 2 profile during the progression of post-myocardial infarction cardiomyopathy: benefits of Alda-1

PubMed Central

Gomes, Katia M.S.; Bechara, Luiz R.G.; Lima, Vanessa M.; Ribeiro, Márcio A.C.; Campos, Juliane C.; Dourado, Paulo M.; Kowaltowski, Alicia J.; Mochly-Rosen, Daria; Ferreira, Julio C.B.

2015-01-01

Background/Objectives We previously demonstrated that reducing cardiac aldehydic load by aldehyde dehydrogenase 2 (ALDH2), a mitochondrial enzyme responsible for metabolizing the major lipid peroxidation product, protects against acute ischemia/reperfusion injury and chronic heart failure. However, time-dependent changes in ALDH2 profile, aldehydic load and mitochondrial bioenergetics during progression of post-myocardial infarction (post-MI) cardiomyopathy is unknown and should be established to determine the optimal time window for drug treatment. Methods Here we characterized cardiac ALDH2 activity and expression, lipid peroxidation, 4-hydroxy-2-nonenal (4-HNE) adduct formation, glutathione pool and mitochondrial energy metabolism and H2O2 release during the 4 weeks after permanent left anterior descending (LAD) coronary artery occlusion in rats. Results We observed a sustained disruption of cardiac mitochondrial function during the progression of post-MI cardiomyopathy, characterized by >50% reduced mitochondrial respiratory control ratios and up to 2 fold increase in H2O2 release. Mitochondrial dysfunction was accompanied by accumulation of cardiac and circulating lipid peroxides and 4-HNE protein adducts and down-regulation of electron transport chain complexes I and V. Moreover, increased aldehydic load was associated with a 90% reduction in cardiac ALDH2 activity and increased glutathione pool. Further supporting an ALDH2 mechanism, sustained Alda-1 treatment (starting 24hrs after permanent LAD occlusion surgery) prevented aldehydic overload, mitochondrial dysfunction and improved ventricular function in post-MI cardiomyopathy rats. Conclusion Taken together, our findings demonstrate a disrupted mitochondrial metabolism along with an insufficient cardiac ALDH2-mediated aldehyde clearance during the progression of ventricular dysfunction, suggesting a potential therapeutic value of ALDH2 activators during the progression of post-myocardial infarction cardiomyopathy. PMID:25464432

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.

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.

Development of immobilized cellulase through functionalized gold nano-particles for glucose production by continuous hydrolysis of waste bamboo chopsticks.

PubMed

Cheng, Cheanyeh; Chang, Kuo-Chung

2013-12-10

Cellulase immobilized on silica through the assistance of l-cysteine functionalized gold nano-particle was applied for the continuous hydrolysis of waste bamboo chopsticks powder to produce glucose. The optimal conditions for the continuous hydrolysis were pH 8.0, 50°C. A 4-day reaction with an initial 0.3 gL⁻¹ waste bamboo chopsticks powder, a feed containing 0.2 gL⁻¹ waste bamboo chopsticks powder at a continuous feed and draw rate of 0.5 mLmin⁻¹, and an enzyme loading of 40 mgcellulase(gsilica)⁻¹, has 72.0-76.6% conversion rates of repeated hydrolyses that correspond to a total production of 630.5-671.2mg glucose and are much better than batch hydrolyses. At higher enzyme loading (117 mgcellulase(gsilica)⁻¹), higher initial concentration (0.5 gL⁻¹), and higher feed concentration (0.42 gL⁻¹) the conversion rate increases to 82.9% and a total amount of 1418 mgglucose. The immobilized cellulase can be recovered easily by filtration and used repeatedly at least 6 times over a period more than 90 days with a recovered activity approximately the same as or better than previous reactions. Thus the process is promising for scaling up. Copyright © 2013 Elsevier Inc. All rights reserved.

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

Effect of low frequency, low amplitude magnetic fields on the permeability of cationic liposomes entrapping carbonic anhydrase: I. Evidence for charged lipid involvement.

PubMed

Ramundo-Orlando, A; Morbiducci, U; Mossa, G; D'Inzeo, G

2000-10-01

The influence of low frequency (4-16 Hz), low amplitude (25-75 mu T) magnetic fields on the diffusion processes in enzyme-loaded unilamellar liposomes as bioreactors was studied. Cationic liposomes containing dipalmitoylphosphatidylcholine, cholesterol, and charged lipid stearylamine (SA) at different molar ratios (6:3:1 or 5:3:2) were used. Previous kinetic experiments showed a very low self-diffusion rate of the substrate p-nitrophenyl acetate (p-NPA) across intact liposome bilayer. After 60 min of exposure to 7 Hz sinusoidal (50 mu T peak) and parallel static (50 mu T) magnetic fields the enzyme activity, as a function of increased diffusion rate of p-NPA, rose from 17 +/- 3% to 80 +/- 9% (P < .0005, n = 15) in the 5:3:2 liposomes. This effect was dependent on the SA concentration in the liposomes. Only the presence of combined sinusoidal (AC) and static (DC) magnetic fields affected the p-NPA diffusion rates. No enzyme leakage was observed. Such studies suggest a plausible link between the action of extremely low frequency magnetic field on charged lipids and a change of membrane permeability. Copyright 2000 Wiley-Liss, Inc.

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

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.

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.

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.

Conservation of body calcium by increased dietary intake of potassium: A potential measure to reduce the osteoporosis process during prolonged exposure to microgravity

NASA Technical Reports Server (NTRS)

Nechay, Bohdan R.

1989-01-01

During the 1988 NASA Summer Faculty Fellowship Program, it was proposed that the loss of skeletal calcium upon prolonged exposure to microgravity could be explained, in part, by a renal maladjustment characterized by an increased urinary excretion of calcium. It was theorized that because the conservation of body fluids and electrolytes depends upon the energy of adenosine triphosphate and enzymes that control the use of its energy for renal ion transport, an induction of renal sodium and potassium-dependent adenosine triphosphatase (Na + K ATPase) by oral loading with potassium would increase the reabsorption of sodium directly and that of calcium indirectly, leading to improved hydration and to reduced calcium loss. Preliminary studies showed the following. Rats drinking water containing 0.2 M potassium chloride for six to 13 days excreted in urine 22 muEq of calcium and 135 muEq of sodium per 100 grams of body weight per day. The corresponding values for control rats drinking tap water were 43 muEq and 269 muEq respectively. Renal Na + K ATPase activity in potassium loaded rats was higher than in controls. Thus, oral potassium loading resulted in increased Na + K ATPase activity and diminished urinary excretion of calcium and of sodium as predicted by the hypothesis. An extension of these studies to humans has the potential of resulting in development of harmless, non-invasive, drug-free, convenient measures to reduce bone loss and other electrolyte and fluid problems in space travelers exposed to prolonged periods of microgravity.

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

Developing procedures for the large-scale purification of human serum butyrylcholinesterase.

PubMed

Saxena, Ashima; Luo, Chunyuan; Doctor, Bhupendra P

2008-10-01

Human serum butyrylcholinesterase (Hu BChE) is the most viable candidate for the prophylactic treatment of organophosphate poisoning. A dose of 200 mg/70 kg is predicted to protect humans against 2x LD(50) of soman. Therefore, the aim of this study was to develop procedures for the purification of gram quantities of this enzyme from outdated human plasma or Cohn Fraction IV-4. The purification of Hu BChE was accomplished by batch adsorption on procainamide-Sepharose-CL-4B affinity gel followed by ion-exchange chromatography on a DEAE-Sepharose column. For the purification of enzyme from Cohn Fraction IV-4, it was resuspended in 25 mM sodium phosphate buffer, pH 8.0, and fat was removed by decantation, prior to batch adsorption on procainamide-Sepharose gel. In both cases, the procainamide gel was thoroughly washed with 25 mM sodium phosphate buffer, pH 8.0, containing 0.05 M NaCl, and the enzyme was eluted with the same buffer containing 0.1 M procainamide. The enzyme was dialyzed and the pH was adjusted to 4.0 before loading on the DEAE column equilibrated in sodium acetate buffer, pH 4.0. The column was thoroughly washed with 25 mM sodium phosphate buffer, pH 8.0 containing 0.05 M NaCl before elution with a gradient of 0.05-0.2M NaCl in the same buffer. The purity of the enzyme following these steps ranged from 20% to 40%. The purity of the enzyme increased to >90% by chromatography on an analytical procainamide affinity column. Results show that Cohn Fraction IV-4 is a much better source than plasma for the large-scale isolation of purified Hu BChE.

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.

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.

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.

A Profile of an Endosymbiont-enriched Fraction of the Coral Stylophora pistillata Reveals Proteins Relevant to Microbial-Host Interactions*

PubMed Central

Weston, Andrew J.; Dunlap, Walter C.; Shick, J. Malcolm; Klueter, Anke; Iglic, Katrina; Vukelic, Ana; Starcevic, Antonio; Ward, Malcolm; Wells, Mark L.; Trick, Charles G.; Long, Paul F.

2012-01-01

This study examines the response of Symbiodinium sp. endosymbionts from the coral Stylophora pistillata to moderate levels of thermal “bleaching” stress, with and without trace metal limitation. Using quantitative high throughput proteomics, we identified 8098 MS/MS events relating to individual peptides from the endosymbiont-enriched fraction, including 109 peptides meeting stringent criteria for quantification, of which only 26 showed significant change in our experimental treatments; 12 of 26 increased expression in response to thermal stress with little difference affected by iron limitation. Surprisingly, there were no significant increases in antioxidant or heat stress proteins; those induced to higher expression were generally involved in protein biosynthesis. An outstanding exception was a massive 114-fold increase of a viral replication protein indicating that thermal stress may substantially increase viral load and thereby contribute to the etiology of coral bleaching and disease. In the absence of a sequenced genome for Symbiodinium or other photosymbiotic dinoflagellate, this proteome reveals a plethora of proteins potentially involved in microbial-host interactions. This includes photosystem proteins, DNA repair enzymes, antioxidant enzymes, metabolic redox enzymes, heat shock proteins, globin hemoproteins, proteins of nitrogen metabolism, and a wide range of viral proteins associated with these endosymbiont-enriched samples. Also present were 21 unusual peptide/protein toxins thought to originate from either microbial consorts or from contamination by coral nematocysts. Of particular interest are the proteins of apoptosis, vesicular transport, and endo/exocytosis, which are discussed in context of the cellular processes of coral bleaching. Notably, the protein complement provides evidence that, rather than being expelled by the host, stressed endosymbionts may mediate their own departure. PMID:22351649

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.

State-of-the-art: Immunosuppression and biologic therapy.

PubMed

Sandborn, William J

2010-01-01

Azathioprine and 6-mercaptopurine are orally administered immunosuppressive drugs which are effective for the treatment of Crohn's disease and ulcerative colitis. Azathioprine is rapidly converted to 6-mercaptopurine after administration. 6-Mercaptopurine is then either converted to the putative active metabolites, the 6-thioguinine nucleotides, or inactivated by the enzyme xanthine oxidase to 6-thiouric acid or alternatively inactivated to 6-methylmercaptopurine by the enzyme thiopurine methyltransferase. Thiopurine methyltransferase activity is genetically determined, with one in 300 patients having low or absent enzyme activity, one in 10 patients having intermediate enzyme activity, and 9 in 10 patients having normal enzyme activity. Patients with intermediate or low thiopurine methyltransferase activity are at risk for early leukopenia. Higher erythrocyte 6-thioguinine nucleotide concentrations are associated with a greater likelihood of clinical response. Azathioprine is modestly effective for Crohn's disease and ulcerative colitis. Toxicity associated with azathioprine includes infection and lymphoma. Anti-TNF therapy with infliximab, adalimumab, and certolizumab pegol is effective for induction and maintenance treatment of Crohn's disease, and infliximab is effective for ulcerative colitis. Toxicity associated with anti-TNF therapy includes infection and lymphoma. Combination therapy with infliximab and azathioprine is more effective for inducing and maintaining steroid-free remission and mucosal healing then monotherapy with either drug alone. Strategies to reduce immunogenicity of anti-TNF agents include combination therapy with azathioprine and administration of a loading dose followed by systematic maintenance dosing. Higher serum trough concentrations of infliximab occur more frequently in patients receiving combination therapy with azathioprine and are associated with better clinical outcomes. Combination therapy is associated with an increased relative risk of opportunistic infection, but is not associated with an increased absolute risk of serious infection. Clinical practice should change such that combination therapy with an anti-TNF agent and azathioprine replace azathioprine in patients failing first line therapy with mesalamine and/or steroids. Copyright © 2010 S. Karger AG, Basel.

Expression of collagen and related growth factors in rat tendon and skeletal muscle in response to specific contraction types

PubMed Central

Heinemeier, K M; Olesen, J L; Haddad, F; Langberg, H; Kjaer, M; Baldwin, K M; Schjerling, P

2007-01-01

Acute exercise induces collagen synthesis in both tendon and muscle, indicating an adaptive response in the connective tissue of the muscle–tendon unit. However, the mechanisms of this adaptation, potentially involving collagen-inducing growth factors (such as transforming growth factor-β-1 (TGF-β-1)), as well as enzymes related to collagen processing, are not clear. Furthermore, possible differential effects of specific contraction types on collagen regulation have not been investigated. Female Sprague–Dawley rats were subjected to 4 days of concentric, eccentric or isometric training (n = 7–9 per group) of the medial gastrocnemius, by stimulation of the sciatic nerve. RNA was extracted from medial gastrocnemius and Achilles tendon tissue 24 h after the last training bout, and mRNA levels for collagens I and III, TGF-β-1, connective tissue growth factor (CTGF), lysyl oxidase (LOX), metalloproteinases (MMP-2 and -9) and their inhibitors (TIMP-1 and 2) were measured by Northern blotting and/or real-time PCR. In tendon, expression of TGF-β-1 and collagens I and III (but not CTGF) increased in response to all types of training. Similarly, enzymes/factors involved in collagen processing were induced in tendon, especially LOX (up to 37-fold), which could indicate a loading-induced increase in cross-linking of tendon collagen. In skeletal muscle, a similar regulation of gene expression was observed, but in contrast to the tendon response, the effect of eccentric training was significantly greater than the effect of concentric training on the expression of several transcripts. In conclusion, the study supports an involvement of TGF-β-1 in loading-induced collagen synthesis in the muscle–tendon unit and importantly, it indicates that muscle tissue is more sensitive than tendon to the specific mechanical stimulus. PMID:17540706

Expression of collagen and related growth factors in rat tendon and skeletal muscle in response to specific contraction types.

PubMed

Heinemeier, K M; Olesen, J L; Haddad, F; Langberg, H; Kjaer, M; Baldwin, K M; Schjerling, P

2007-08-01

Acute exercise induces collagen synthesis in both tendon and muscle, indicating an adaptive response in the connective tissue of the muscle-tendon unit. However, the mechanisms of this adaptation, potentially involving collagen-inducing growth factors (such as transforming growth factor-beta-1 (TGF-beta-1)), as well as enzymes related to collagen processing, are not clear. Furthermore, possible differential effects of specific contraction types on collagen regulation have not been investigated. Female Sprague-Dawley rats were subjected to 4 days of concentric, eccentric or isometric training (n = 7-9 per group) of the medial gastrocnemius, by stimulation of the sciatic nerve. RNA was extracted from medial gastrocnemius and Achilles tendon tissue 24 h after the last training bout, and mRNA levels for collagens I and III, TGF-beta-1, connective tissue growth factor (CTGF), lysyl oxidase (LOX), metalloproteinases (MMP-2 and -9) and their inhibitors (TIMP-1 and 2) were measured by Northern blotting and/or real-time PCR. In tendon, expression of TGF-beta-1 and collagens I and III (but not CTGF) increased in response to all types of training. Similarly, enzymes/factors involved in collagen processing were induced in tendon, especially LOX (up to 37-fold), which could indicate a loading-induced increase in cross-linking of tendon collagen. In skeletal muscle, a similar regulation of gene expression was observed, but in contrast to the tendon response, the effect of eccentric training was significantly greater than the effect of concentric training on the expression of several transcripts. In conclusion, the study supports an involvement of TGF-beta-1 in loading-induced collagen synthesis in the muscle-tendon unit and importantly, it indicates that muscle tissue is more sensitive than tendon to the specific mechanical stimulus.

Mitigating effect of chrysin loaded solid lipid nanoparticles against Amyloid β25-35 induced oxidative stress in rat hippocampal region: An efficient formulation approach for Alzheimer's disease.

PubMed

Vedagiri, Aishwarya; Thangarajan, Sumathi

2016-08-01

Alzheimer's disease (AD) is the most prevalent form of dementia. Amyloid-β25-35 (Aβ25-35), a well-established neurotoxicant, is reported to be involved in the etiology of AD. Chrysin (CN) with its wide range of biological activities in terms of reversing the neuronal damage once induced is limited due to its compromised bioavailability. Solid lipid nanoparticles (SLNs) on the other hand due to its improved protein stability, avoids proteolytic degradation, as well as sustained release of the incorporated molecules could be widely applied as a drug delivery vehicle. Hence, in the present investigation, we prepared CN loaded SLNs (CN-SLNs) and investigated its therapeutic role in alleviating Aβ25-35 administered neuronal damage. All the antioxidant enzymes and non-antioxidant enzyme in hippocampus were reduced significantly (P<0.01) in the Aβ25-35 injected group, whereas lipid peroxidation and acetylcholine esterase were increased significantly (P<0.01). These changes were restored significantly (P<0.01) by CN-SLNs (5mg/kg and 10mg/kg) and (P<0.05) by free CN (50mg/kg and 100mg/kg). Aβ25-35 also resulted in poor memory retention in behavioral tasks and histopathological sections of the hippocampal region showed the extent of neuronal loss which was thereby restored back on treatment with CN-SLNs and free CN. Our findings demonstrate that the therapeutic efficacy of CN could be attained at lower dose and also its oral bioavailability could be increased by encapsulating CN in SLNs. Thus the results suggest that CN-SLNs could be used as a potential therapeutic and a brain targeting strategy to combat the global burden of Alzheimer's disease. Copyright © 2016 Elsevier Ltd. All rights reserved.

Effect of Elastin Digestion on the Quasi-static Tensile Response of Medial Collateral Ligament

PubMed Central

Henninger, Heath B.; Underwood, Clayton J.; Romney, Steven J.; Davis, Grant L.; Weiss, Jeffrey A.

2014-01-01

Elastin is a structural protein that provides resilience to biological tissues. We examined the contributions of elastin to the quasi-static tensile response of porcine medial collateral ligament through targeted disruption of the elastin network with pancreatic elastase. Elastase concentration and treatment time were varied to determine a dose response. Whereas elastin content decreased with increasing elastase concentration and treatment time, the change in peak stress after cyclic loading reached a plateau above 1 U/ml elastase and 6 hr treatment. For specimens treated with 2 U/ml elastase for 6 hr, elastin content decreased approximately 35%. Mean peak tissue strain after cyclic loading (4.8%, p≥0.300), modulus (275 MPa, p≥0.114) and hysteresis (20%, p≥0.553) were unaffected by elastase digestion, but stress decreased significantly after treatment (up to 2 MPa, p≤0.049). Elastin degradation had no effect on failure properties, but tissue lengthened under the same pre-stress. Stiffness in the linear region was unaffected by elastase digestion, suggesting that enzyme treatment did not disrupt collagen. These results demonstrate that elastin primarily functions in the toe region of the stress-strain curve, yet contributes load support in the linear region. The increase in length after elastase digestion suggests that elastin may pre-stress and stabilize collagen crimp in ligaments. PMID:23553827

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.

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.

Acid-catalyzed autohydrolysis of wheat straw to improve sugar recovery.

PubMed

Ertas, Murat; Han, Qiang; Jameel, Hasan

2014-10-01

A comparison study of autohydrolysis and acid-catalyzed autohydrolysis of wheat straw was performed to understand the impact of acid addition on overall sugar recovery. Autohydrolysis combined with refining is capable of achieving sugar recoveries in the mid 70s. If the addition of a small amount of acid is capable of increasing the sugar recovery even higher it may be economically attractive. Acetic, sulfuric, hydrochloric and sulfurous acids were selected for acid-catalyzed autohydrolysis pretreatments. Autohydrolysis with no acid at 190 °C showed the highest total sugar in the prehydrolyzate. Enzymatic hydrolysis was performed for all the post-treated solids with and without refining at enzyme loadings of 4 and 10 FPU/g for 96 h. Acid-catalyzed autohydrolysis at 190 °C with sulfurous acid showed the highest total sugar recovery of 81.2% at 4 FPU/g enzyme charge compared with 64.3% at 190 °C autohydrolysis without acid. Copyright © 2014 Elsevier Ltd. All rights reserved.

Solid-phase extraction and on-disc derivatization of the major benzodiazepines in urine using enzyme hydrolysis and Toxi-Lab VC MP3 column.

PubMed

King, J W; King, L J

1996-01-01

Because of the increase in use of the newer benzodiazepines, we explored the opportunity to develop a gas chromatographic-mass spectrometric (GC-MS) method that encompasses most of the widely prescribed benzodiazepines in use today. The benzodiazepines included in our study are nordiazepam, oxazepam, temazepam, lorazepam, alpha-hydroxyalprazolam, alpha-hydroxytriazolam, desalkylflurazepam, and 2-hydroxyethylflurazepam. Using 1.0 mL of urine as the matrix, we added the enzyme Glusulase and incubated the specimens for 2 h to obtain the free drugs. The hydrolyzed samples were then loaded onto a Toxi-Lab Spec VC MP3 column containing a 15-mg disc. On-disc derivatization was accomplished by adding N-methyl-N-(t-butyldimethylsilyl) trifluroacetamide (MTBSTFA) with 1% TBDMSCI to the disc. The derivatives were then placed in a GC vial and analyzed by GC-MS in the selected ion monitoring mode. These results were then compared to confirmed positives by the traditional acid hydrolysis GC-MS method.

Improved performance of Yarrowia lipolytica lipase-catalyzed kinetic resolution of (R,S)-2-octanol by an integrated strategy of interfacial activation, bioimprinting and immobilization.

PubMed

Liu, Ying; Guo, Chen; Sun, Xi-Tong; Liu, Chun-Zhao

2013-08-01

Yarrowia lipolytica lipase (YLL) demonstrated an (R)-enantiopreference for efficient resolution of (R,S)-2-octanol. The activity, enantioselectivity, the ratio of substrate to enzyme, acetaldehyde tolerance, and operational stability of YLL were improved by an integrated strategy of interfacial activation, bioimprinting, and immobilization. In comparison with the control, both the enzymatic activity and enantioselectivity increased by a factor of 8.85 and 2.75 by the integrated strategy, respectively. Fifty-one percentage of conversion with 220 of enantioselectivity was obtained using the immobilized YLL prepared by the integrated strategy at a ratio of 104 of substrate to enzyme loaded. The immobilized YLL retained 97% of its initial activity without a decrease in enantioselectivity after 10 successive reuse cycles. Together these results will result in a promising strategy with the YYL for efficient resolution of (R,S)-2-octanol in practice. Copyright © 2013 Elsevier Ltd. All rights reserved.

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

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.

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.

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.

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.

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.

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).

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

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

Epichlorohydrin induced biochemical changes in the rose-ringed parakeet, Psittacula krameri Scopoli.

PubMed

Hans, B; Kaur, S; Sangha, G K

1999-08-01

Intraperitoneal administration of epichlorohydrin (ECH) at the dose level of 20 and 50 mg/kg body weight inhibited spermatogenesis in the testis of parakeet during breeding season. A total load of 60 mg/kg body weight of ECH given on 3 consecutive days proved to be lethal. Testicular proteins, nucleic acids (DNA and RNA), phospholipids and acid phosphatase activity were decreased, while the lipids, total cholesterol and alkaline phosphatase activity increased after ECH administration. The results suggest that the testicular atrophy caused by ECH was associated with an alteration in the activities of macromolecules and enzymes related to specific events of spermatogenesis.

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

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.

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.

Evaluation of bioavailability, efficacy, and safety profile of doxorubicin-loaded solid lipid nanoparticles

NASA Astrophysics Data System (ADS)

Patro, Nagaraju M.; Devi, Kshama; Pai, Roopa S.; Suresh, Sarasija

2013-12-01

We investigated the bioavailability, efficacy, and toxicity of doxorubicin-loaded solid lipid nanoparticles (DOX-SLNs) prepared by a simple modified double-emulsification method. A 3-factor, 3-level Box-Behnken statistical design was adopted in the optimization of DOX-SLN formulation considering dependent factors particle size and entrapment efficiency. Optimized SLN formulation composed of lipid (2 %) consisting of soya lecithin and Precirol ATO 5 (1:3) with Pluronic F68 (0.3 %) resulted in 217.36 ± 3.31 nm particle size and 59.45 ± 1.75 % entrapment efficiency. DOX-SLN exhibited significant enhancement ( p < 0.05) in bioavailability as compared with free DOX in Sprague-Dawley (SD) rats. DOX-SLN exhibited higher peak plasma concentration (6.761 ± 0.08 vs. 2.412 ± 0.04 μg/ml), increased AUC (61.368 ± 3.54 vs. 5.812 ± 0.49 μg/ml h), decreased clearance (36 ± 0.01 vs. 619 ± 0.005 mL/h kg), and volume of distribution (733 ± 0.092 vs. 2,064 ± 0.061 mL/kg) when compared to free DOX. The collective results of cardiac and kidney enzyme assay, antioxidant enzyme levels, hematological parameters, effect on body weight and tumor volume, tumor necrosis factor-α level, histopathological examination, and survival analysis confirmed the improved efficacy and safety profile of DOX-SLN in 7,12-dimethyl benzanthracene-induced breast cancer in SD rats.

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

Oxcarbazepine-loaded polymeric nanoparticles: development and permeability studies across in vitro models of the blood–brain barrier and human placental trophoblast

PubMed Central

Lopalco, Antonio; Ali, Hazem; Denora, Nunzio; Rytting, Erik

2015-01-01

Encapsulation of antiepileptic drugs (AEDs) into nanoparticles may offer promise for treating pregnant women with epilepsy by improving brain delivery and limiting the transplacental permeability of AEDs to avoid fetal exposure and its consequent undesirable adverse effects. Oxcarbazepine-loaded nanoparticles were prepared by a modified solvent displacement method from biocompatible polymers (poly(lactic-co-glycolic acid) [PLGA] with or without surfactant and PEGylated PLGA [Resomer® RGPd5055]). The physical properties of the developed nanoparticles were determined with subsequent evaluation of their permeability across in vitro models of the blood–brain barrier (hCMEC/D3 cells) and human placental trophoblast cells (BeWo b30 cells). Oxcarbazepine-loaded nanoparticles with encapsulation efficiency above 69% were prepared with sizes ranging from 140–170 nm, polydispersity indices below 0.3, and zeta potential values below -34 mV. Differential scanning calorimetry and X-ray diffraction studies confirmed the amorphous state of the nanoencapsulated drug. The apparent permeability (Pe) values of the free and nanoencapsulated oxcarbazepine were comparable across both cell types, likely due to rapid drug release kinetics. Transport studies using fluorescently-labeled nanoparticles (loaded with coumarin-6) demonstrated increased permeability of surfactant-coated nanoparticles. Future developments in enzyme-prodrug therapy and targeted delivery are expected to provide improved options for pregnant patients with epilepsy. PMID:25792832

Oxcarbazepine-loaded polymeric nanoparticles: development and permeability studies across in vitro models of the blood-brain barrier and human placental trophoblast.

PubMed

Lopalco, Antonio; Ali, Hazem; Denora, Nunzio; Rytting, Erik

2015-01-01

Encapsulation of antiepileptic drugs (AEDs) into nanoparticles may offer promise for treating pregnant women with epilepsy by improving brain delivery and limiting the transplacental permeability of AEDs to avoid fetal exposure and its consequent undesirable adverse effects. Oxcarbazepine-loaded nanoparticles were prepared by a modified solvent displacement method from biocompatible polymers (poly(lactic-co-glycolic acid) [PLGA] with or without surfactant and PEGylated PLGA [Resomer(®) RGPd5055]). The physical properties of the developed nanoparticles were determined with subsequent evaluation of their permeability across in vitro models of the blood-brain barrier (hCMEC/D3 cells) and human placental trophoblast cells (BeWo b30 cells). Oxcarbazepine-loaded nanoparticles with encapsulation efficiency above 69% were prepared with sizes ranging from 140-170 nm, polydispersity indices below 0.3, and zeta potential values below -34 mV. Differential scanning calorimetry and X-ray diffraction studies confirmed the amorphous state of the nanoencapsulated drug. The apparent permeability (Pe ) values of the free and nanoencapsulated oxcarbazepine were comparable across both cell types, likely due to rapid drug release kinetics. Transport studies using fluorescently-labeled nanoparticles (loaded with coumarin-6) demonstrated increased permeability of surfactant-coated nanoparticles. Future developments in enzyme-prodrug therapy and targeted delivery are expected to provide improved options for pregnant patients with epilepsy.

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

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.

Signal Amplification in Field Effect-Based Sandwich Enzyme-Linked Immunosensing by Tuned Buffer Concentration with Ionic Strength Adjuster.

PubMed

Kumar, Satyendra; Kumar, Narendra; Panda, Siddhartha

2016-04-01

Miniaturization of the sandwich enzyme-based immunosensor has several advantages but could result in lower signal strength due to lower enzyme loading. Hence, technologies for amplification of the signal are needed. Signal amplification in a field effect-based electrochemical immunosensor utilizing chip-based ELISA is presented in this work. First, the molarities of phosphate buffer saline (PBS) and concentrations of KCl as ionic strength adjuster were optimized to maximize the GOx glucose-based enzymatic reactions in a beaker for signal amplification measured by change in the voltage shift with an EIS device (using 20 μl of solution) and validated with a commercial pH meter (using 3 ml of solution). The PBS molarity of 100 μM with 25 mM KCl provided the maximum voltage shift. These optimized buffer conditions were further verified for GOx immobilized on silicon chips, and similar trends with decreased PBS molarity were obtained; however, the voltage shift values obtained on chip reaction were lower as compared to the reactions occurring in the beaker. The decreased voltage shift with immobilized enzyme on chip could be attributed to the increased Km (Michaelis-Menten constant) values in the immobilized GOx. Finally, a more than sixfold signal enhancement (from 8 to 47 mV) for the chip-based sandwich immunoassay was obtained by altering the PBS molarity from 10 to 100 μM with 25 mM KCl.

Techno-economic analysis of ethanol production from sugarcane bagasse using a Liquefaction plus Simultaneous Saccharification and co-Fermentation process.

PubMed

Gubicza, Krisztina; Nieves, Ismael U; Sagues, William J; Barta, Zsolt; Shanmugam, K T; Ingram, Lonnie O

2016-05-01

A techno-economic analysis was conducted for a simplified lignocellulosic ethanol production process developed and proven by the University of Florida at laboratory, pilot, and demonstration scales. Data obtained from all three scales of development were used with Aspen Plus to create models for an experimentally-proven base-case and 5 hypothetical scenarios. The model input parameters that differed among the hypothetical scenarios were fermentation time, enzyme loading, enzymatic conversion, solids loading, and overall process yield. The minimum ethanol selling price (MESP) varied between 50.38 and 62.72 US cents/L. The feedstock and the capital cost were the main contributors to the production cost, comprising between 23-28% and 40-49% of the MESP, respectively. A sensitivity analysis showed that overall ethanol yield had the greatest effect on the MESP. These findings suggest that future efforts to increase the economic feasibility of a cellulosic ethanol process should focus on optimization for highest ethanol yield. Copyright © 2016 Elsevier Ltd. All rights reserved.

Optimization of renewable pinene production from the conversion of macroalgae Saccharina latissima.

PubMed

Scullin, Chessa; Stavila, Vitalie; Skarstad, Anita; Keasling, Jay D; Simmons, Blake A; Singh, Seema

2015-05-01

Enzymatic hydrolysis of Saccharina latissima with laminarinase was compared to hydrolysis with different combinations of cellulase and hemicellulase enzyme mixtures. The hemicellulase mixture resulted in similar release of glucose, while the cellulase mixture released 40% more glucose than laminarinase alone. The combination of a laminarinase augmented with a cellulase mixture resulted in a 53% increase of glucose release from S. latissima than laminarinase. Increasing biomass loading above 4% (w/v) reduced the sugar yield. Resulting macroalgae hydrolysates were used as a carbon source for the production of pinene, making use of a novel two plasmid Escherichia coli system. The macroalgal hydrolysates were suitable for the novel microbial production of pinene with no further treatment and/or purification. Copyright © 2015. Published by Elsevier Ltd.

Improving the yield from fermentative hydrogen production.

PubMed

Kraemer, Jeremy T; Bagley, David M

2007-05-01

Efforts to increase H(2) yields from fermentative H(2) production include heat treatment of the inoculum, dissolved gas removal, and varying the organic loading rate. Although heat treatment kills methanogens and selects for spore-forming bacteria, the available evidence indicates H(2) yields are not maximized compared to bromoethanesulfonate, iodopropane, or perchloric acid pre-treatments and spore-forming acetogens are not killed. Operational controls (low pH, short solids retention time) can replace heat treatment. Gas sparging increases H(2) yields compared to un-sparged reactors, but no relationship exists between the sparging rate and H(2) yield. Lower sparging rates may improve the H(2) yield with less energy input and product dilution. The reasons why sparging improves H(2) yields are unknown, but recent measurements of dissolved H(2) concentrations during sparging suggest the assumption of decreased inhibition of the H(2)-producing enzymes is unlikely. Significant disagreement exists over the effect of organic loading rate (OLR); some studies show relatively higher OLRs improve H(2) yield while others show the opposite. Discovering the reasons for higher H(2) yields during dissolved gas removal and changes in OLR will help improve H(2) yields.

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.

Effects of exogenous enzymes in corn-based and Canadian pearl millet-based diets with reduced soybean meal on growth performance, intestinal nutrient digestibility, villus development, and selected microbial populations in broiler chickens.

PubMed

Baurhoo, N; Baurhoo, B; Zhao, X

2011-12-01

An experiment was conducted to compare a commercial corn-soybean meal diet with a pearl millet diet containing less soybean meal (-27%), alone or in combination with exogenous enzymes, on growth performance, jejunal villus development, ileal CP, and AA digestibility, and cecal microbial populations in broilers. One hundred sixty 1-d-old male Ross 508 broilers (5/cage) were randomly allocated to one of the following dietary treatments: 1) a standard corn-soybean meal control diet (CTL); 2) a pearl millet-soybean meal diet (PM); 3) CTL + exogenous enzymes (CE); and 4) PM + exogenous enzymes (PE) with 8 replicate cages/treatment. The PM and PE diets contained less soybean meal because of greater CP and AA contents of pearl millet. All diets were isonitrogenous and isocaloric. Body weight and feed intake were recorded weekly over 35 d. At d 21 and 35, 8 broilers per treatment were euthanized for sample collection and analyses. Gain-to-feed was greater (P < 0.01) for pearl millet- than corn-based diets. Apparent ileal digestibility (AID) of CP and most AA was similar between corn-based and pearl millet-based diets, and enzyme supplementation improved AID of CP (P < 0.01) and most AA at both d 21 and 35. However, for AID of some AA at d 21, the response to enzyme supplementation was less pronounced in broilers fed pearl millet-based diets than those fed corn-based diets (grain × enzyme, P ≤ 0.05). The villus was longer (P < 0.01) in broilers fed PM and PE than CTL and CE at d 35. Similarly, at d 35, lactobacilli loads were greater (P < 0.01) in broilers fed PM and PE than CTL and CE. It is concluded that, in comparison with corn, broiler diets formulated with pearl millet require less soybean meal and can be used to improve growth performance traits, intestinal lactobacilli populations, and villus development, whereas enzyme supplementation increases AID of CP and AA.

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.

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.

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 Hydrolysis and Simultaneous Saccharification and Fermentation of Soybean Processing Intermediates for the Production of Ethanol and Concentration of Protein and Lipids

PubMed Central

Long, Craig C.; Gibbons, William

2012-01-01

Carbohydrates in soybeans are generally undesirable due to their low digestibility and because they “dilute” more valuable components (proteins, lipids). To remove these carbohydrates and raise the titer of more valuable components, ethanol production was investigated. Commercial enzymes (Novozyme cellulase, β-glucosidase, and pectinase) were added to ground soybeans (SB), soybean meal (SBM), soybean hulls (SH), and soybean white flakes (WF) at a 10% solids loading rate to quantify hydrolyzed glucan. Saccharification resulted in glucan reductions of 28%, 45%, 76%, and 80% (SBM, SB, SH, WF, resp.). Simultaneous saccharification and fermentation (SSF) trials were conducted at 5%, 10%, 15%, and 20% solids loading with Saccharomyces cerevisiae NRRL Y-2034 and Scheffersomyces stipitis NRRL Y-7124, with protein, fiber, and lipids analyzed at SSF 10% solids and saccharification trials. S. cerevisiae and S. stipitis produced ~3–12.5 g/L ethanol and ~2.5–8.6 g/L ethanol, respectively, on SB, SBM, and WF over all solid loading rates. SH resulted in higher ethanol titers for both S. cerevisiae (~9–23 g/L) and S. stipitis (~9.5–14.5 g/L). Protein concentrations decreased by 2.5–10% for the SB, SBM, and WF, but increased by 53%–55% in SH. Oil concentrations increased by ~50% for SB; by ~500%–1300% for the others. PMID:23762751

Integrated experimental and technoeconomic evaluation of two-stage Cu-catalyzed alkaline–oxidative pretreatment of hybrid poplar

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

Bhalla, Aditya; Fasahati, Peyman; Particka, Chrislyn A.

2018-05-17

When applied to recalcitrant lignocellulosic feedstocks, multi-stage pretreatments can provide more processing flexibility to optimize or balance process outcomes such as increasing delignification, preserving hemicellulose, and maximizing enzymatic hydrolysis yields. We previously reported that adding an alkaline pre-extraction step to a copper-catalyzed alkaline hydrogen peroxide (Cu-AHP) pretreatment process resulted in improved sugar yields, but the process still utilized relatively high chemical inputs (catalyst and H2O2) and enzyme loadings. We hypothesized that by increasing the temperature of the alkaline pre-extraction step in water or ethanol, we could reduce the inputs required during Cu-AHP pretreatment and enzymatic hydrolysis without significant loss inmore » sugar yield. We also performed technoeconomic analysis to determine if ethanol or water was the more cost-effective solvent during alkaline pre-extraction and if the expense associated with increasing the temperature was economically justified.« less

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.

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.

Tocotrienol Affects Oxidative Stress, Cholesterol Homeostasis and the Amyloidogenic Pathway in Neuroblastoma Cells: Consequences for Alzheimer’s Disease

PubMed Central

Grimm, Marcus O. W.; Regner, Liesa; Mett, Janine; Stahlmann, Christoph P.; Schorr, Pascal; Nelke, Christopher; Streidenberger, Olga; Stoetzel, Hannah; Winkler, Jakob; Zaidan, Shatha R.; Thiel, Andrea; Endres, Kristina; Grimm, Heike S.; Volmer, Dietrich A.; Hartmann, Tobias

2016-01-01

One of the characteristics of Alzheimer´s disease (AD) is an increased amyloid load and an enhanced level of reactive oxidative species (ROS). Vitamin E has known beneficial neuroprotective effects, and previously, some studies suggested that vitamin E is associated with a reduced risk of AD due to its antioxidative properties. However, epidemiological studies and nutritional approaches of vitamin E treatment are controversial. Here, we investigate the effect of α-tocotrienol, which belongs to the group of vitamin E, on AD-relevant processes in neuronal cell lines. In line with the literature, α-tocotrienol reduced the ROS level in SH-SY5Y cells. In the presence of tocotrienols, cholesterol and cholesterol esters, which have been shown to be risk factors in AD, were decreased. Besides the unambiguous positive effects of tocotrienol, amyloid-β (Aβ) levels were increased accompanied by an increase in the activity of enzymes responsible for Aβ production. Proteins and gene expression of the secretases and their components remained unchanged, whereas tocotrienol accelerates enzyme activity in cell-free assays. Besides enhanced Aβ production, tocotrienols inhibited Aβ degradation in neuro 2a (N2a)-cells. Our results might help to understand the controversial findings of vitamin E studies and demonstrate that besides the known positive neuroprotective properties, tocotrienols also have negative characteristics with respect to AD. PMID:27801864

Tocotrienol Affects Oxidative Stress, Cholesterol Homeostasis and the Amyloidogenic Pathway in Neuroblastoma Cells: Consequences for Alzheimer's Disease.

PubMed

Grimm, Marcus O W; Regner, Liesa; Mett, Janine; Stahlmann, Christoph P; Schorr, Pascal; Nelke, Christopher; Streidenberger, Olga; Stoetzel, Hannah; Winkler, Jakob; Zaidan, Shatha R; Thiel, Andrea; Endres, Kristina; Grimm, Heike S; Volmer, Dietrich A; Hartmann, Tobias

2016-10-29

One of the characteristics of Alzheimer´s disease (AD) is an increased amyloid load and an enhanced level of reactive oxidative species (ROS). Vitamin E has known beneficial neuroprotective effects, and previously, some studies suggested that vitamin E is associated with a reduced risk of AD due to its antioxidative properties. However, epidemiological studies and nutritional approaches of vitamin E treatment are controversial. Here, we investigate the effect of α-tocotrienol, which belongs to the group of vitamin E, on AD-relevant processes in neuronal cell lines. In line with the literature, α-tocotrienol reduced the ROS level in SH-SY5Y cells. In the presence of tocotrienols, cholesterol and cholesterol esters, which have been shown to be risk factors in AD, were decreased. Besides the unambiguous positive effects of tocotrienol, amyloid-β (Aβ) levels were increased accompanied by an increase in the activity of enzymes responsible for Aβ production. Proteins and gene expression of the secretases and their components remained unchanged, whereas tocotrienol accelerates enzyme activity in cell-free assays. Besides enhanced Aβ production, tocotrienols inhibited Aβ degradation in neuro 2a (N2a)-cells. Our results might help to understand the controversial findings of vitamin E studies and demonstrate that besides the known positive neuroprotective properties, tocotrienols also have negative characteristics with respect to AD.

[Adaptation to hypoxia and hyperoxia improves physical endurance: the role of reactive oxygen species and redox-signaling].

PubMed

Sazontova, T G; Glazachev, O S; Bolotova, A V; Dudnik, E N; Striapko, N V; Bedareva, I V; Anchishkina, N A; Arkhipenko, Iu V

2012-06-01

We have conducted theoretical foundation, experimental analysis and a pilot study of a new method of adaptation to hypoxia and hyperoxia in the prevention of hypoxic and stress-induced disorders and improving the body's tolerance to physical stress. It has been shown in the experimental part that a combination of physical exercise with adaptation to hypoxia-hyperoxia significantly increased tolerance to acute physical load (APL) and its active phase. Analysis of lipid peroxidation processes, antioxidant enzymes and HSPs showed that short-term training for physical exercise by itself compensates the stressor, but not the hypoxic component of the APL, the combination of training with adaptation to hypoxia-hyperoxia completely normalizes the stressor and hypoxic components of APL. The pilot study has been performed to evaluate the effectiveness of hypoxic-hyperoxic training course in qualified young athletes with over-training syndrome. After completing the course of hypoxia-hyperoxia adaptation, 14 sessions, accompanied by light mode sports training, the athletes set the normalization of autonomic balance, increased resistance to acute hypoxia in hypoxic test, increased physical performance--increased PWC170, maximal oxygen consumption (VO2max) parameters, their relative values to body mass, diminished shift of rate pressure product in the load. Thus, we confirmed experimental findings that hypoxic-hyperoxic training optimizes hypoxic (increased athletes resistance to proper hypoxia) and stress (myocardium economy in acute physical stress testing) components in systemic adaptation and restoration of athletes' with over-training syndrome.

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.

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.

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.

Ascorbic Acid Prevents VEGF-induced Increases in Endothelial Barrier Permeability

PubMed Central

Ulker, Esad; Parker, William H.; Raj, Amita; Qu, Zhi-chao; May, James M.

2015-01-01

Vascular endothelial growth factor (VEGF) increases endothelial barrier permeability, an effect that may contribute to macular edema in diabetic retinopathy. Since vitamin C, or ascorbic acid, can tighten the endothelial permeability barrier, we examined whether it could prevent the increase in permeability due to VEGF in human umbilical vein endothelial cells (HUVECs). As previously observed, VEGF increased HUVEC permeability to radiolabeled inulin within 60 min in a concentration-dependent manner. Loading the cells with increasing concentrations of ascorbate progressively prevented the leakage caused by 100 ng/ml VEGF, with a significant inhibition at 13 μM and complete inhibition at 50 μM. Loading cells with 100 μM ascorbate also decreased basal generation of reactive oxygen species and prevented the increase caused by both 100 ng/ml VEGF. VEGF treatment decreased intracellular ascorbate by 25%, thus linking ascorbate oxidation to its prevention of VEGF-induced barrier leakage. The latter was blocked by treating the cells with 60 μM L-NAME (but not D-NAME) as well as by 30 μM sepiapterin, a precursor of tetrahydrobiopterin that is required for proper function of endothelial nitric oxide synthase (eNOS). These findings suggest that VEGF-induced barrier leakage uncouples eNOS. Ascorbate inhibition of the VEGF effect could thus be due either to scavenging superoxide or to peroxynitrite generated by the uncoupled eNOS, or more likely to its ability to recycle tetrahydrobiopterin, thus avoiding enzyme uncoupling in the first place. Ascorbate prevention of VEGF-induced increases in endothelial permeability opens the possibility that its repletion could benefit diabetic macular edema. PMID:26590088

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.

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.

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.

Cardioprotective effects of curcumin-loaded magnetic hydrogel nanocomposite (nanocurcumin) against doxorubicin-induced cardiac toxicity in rat cardiomyocyte cell lines.

PubMed

Namdari, Mehrdad; Eatemadi, Ali

2017-06-01

Curcumin, is a yellow substance extracted from Curcuma longa rhizomes, it is a crystalline compound that has been traditionally applied in culinary practices and medicines in India. The aim of our study is to demonstrate the efficacy of curcumin-loaded magnetic hydrogel nanocomposite in the treatment of heart hypertrophy. 10 rats weighing 150-200 g each were induced with heart failure using 2.5 mg/kg doxorubicin for 2 weeks. The test groups were treated with curcumin-loaded magnetic hydrogel nanocomposite while the control was treated with curcumin alone. malondialdehyde (MDA) levels, superoxide dismutase (SOD), and glutathione peroxidase (GPX) enzymes activities were monitored after two weeks of last the dose. In addition, the expression of three heart failure markers; atrial natriuretic peptide (ANP), B type natriuretic peptide (BNP), and beta major histocompatibility complex (β-MHC) were observed, it was found that the expression of these markers decreases with an increase in the concentration of curcumin (P < 0.05). Curcumin elevated the decreased level of GPX and SOD, and reduced the elevated level of MDA in cardiac tissue. We suggest this combination to be a potent therapy for heart failure and hypertension in the nearest future.

Effects of prolonged space flight on rat skeletal muscle.

PubMed

Nesterov, V P; Zheludkova, Z P; Kuznetsova, L A

1979-10-01

The effect of a 20-day space flight on water, Na+, K+, Mg2+, Ca2+ and glycogen contents as well as on activities of glycogen metabolism enzymes--glycogen synthetase and glycogen phosphorylase--of rat skeletal muscles was studied. This data is regarded as an integral test characterizing the state of contractile tissue of the animals at the final stage of flight aboard biosatellites. The measurements indicate that there were no significant changes of cations and glycogen contents nor of the enzymic activities in fast-twitch muscles during the 20-day spaceflight. At the same time dehydration in these muscles was observed, which disappeared on the 25th postflight day. In slow-twitch antigravitational skeletal muscle (m. soleus) there was a decrease of K+ and increase of Na+ in the tissue contents. The changes disappeared at the end of the on-earth readaptation period. From the pattern of these observations, we can conclude that the 20-day space flight leads to some reversible biochemical changes of the rat skeletal muscles. A conclusion can be drawn about necessity of creating, aboard the spaceship, an artificial load on antigravitational skeletal muscles.

Impact of ultraviolet radiation treatments on the physicochemical properties, antioxidants, enzyme activity and microbial load in freshly prepared hand pressed strawberry juice.

PubMed

Bhat, Rajeev; Stamminger, Rainer

2015-07-01

Freshly prepared, hand-pressed strawberry fruit juice was exposed to ultraviolet radiation (254 nm) at room temperature (25 ℃ ± 1 ℃) for 15, 30 and 60 min with 0 min serving as control. Results revealed decrease in pH, total soluble solids and titratable acidity, while colour parameters (L*, a* and b* values) and clarity of juice (% transmittance) increased significantly. All the results corresponded to exposure time to ultraviolet radiation. Bioactive compounds (total phenolics, ascorbic acid and anthocyanins) decreased along with a recorded reduction in polyphenol oxidase enzyme and 1,1-diphenyl-2-picryl hydrazyl radical scavenging activities, which were again dependent on exposure time. Results on the microbial studies showed significant reduction by 2-log cycles in aerobic plate count as well as in total yeast and mould counts. Though negative results were observed for certain parameters, this is the first time it was endeavoured to demonstrate the impact of ultraviolet radiation radiation on freshly prepared, hand-pressed strawberries juice. © The Author(s) 2014.

Prevention by zinc of cadmium-induced alterations in pancreatic and hepatic functions.

PubMed Central

Merali, Z; Singhal, R L

1976-01-01

Subacute cadmium treatment (CdCl2, 1 mg/kg twice daily for 7 days) in rats disturbs glucose homeostasis as shown by hyperglycemia and decreased glucose tolerance associated with suppression of insulin release, enhancement of hepatic gluconeogenic enzymes and decrease in hepatic glycogen content. 2 Exposure to cadmium increases hepatic cyclic adenosine 3',5'-monophosphate (cyclic AMP) and this is accompanied by stimulation of basal, adrenaline- as well as glucagon-stimulated form(s) of adenylate cyclase. 3 In contrast to cadmium, subacute administration of zinc (ZnCl2, 2 mg/kg twice daily for 7 days) fails to alter the activities of hepatic gluconeogenic enzymes, cyclic AMP synthesis, as well as glucose clearance and insulin release in response to a glucose load. 4 Zinc, when administered at the same time as cadmium, prevents the cadmium-induced lesions in both hepatic and pancreatic functions. 5 The results are discussed in relation to the possible mechanisms of cadmium toxicity and to the role of sulphydryl groups in the protection exercised by zinc. PMID:183849

Efficient production of ethanol from waste paper and the biochemical methane potential of stillage eluted from ethanol fermentation.

PubMed

Nishimura, Hiroto; Tan, Li; Sun, Zhao-Yong; Tang, Yue-Qin; Kida, Kenji; Morimura, Shigeru

2016-02-01

Waste paper can serve as a feedstock for ethanol production due to being rich in cellulose and not requiring energy-intensive thermophysical pretreatment. In this study, an efficient process was developed to convert waste paper to ethanol. To accelerate enzymatic saccharification, pH of waste paper slurry was adjusted to 4.5-5.0 with H2SO4. Presaccharification and simultaneous saccharification and fermentation (PSSF) with enzyme loading of 40 FPU/g waste paper achieved an ethanol yield of 91.8% and productivity of 0.53g/(Lh) with an ethanol concentration of 32g/L. Fed-batch PSSF was used to decrease enzyme loading to 13 FPU/g waste paper by feeding two separate batches of waste paper slurry. Feeding with 20% w/w waste paper slurry increased ethanol concentration to 41.8g/L while ethanol yield decreased to 83.8%. To improve the ethanol yield, presaccharification was done prior to feeding and resulted in a higher ethanol concentration of 45.3g/L, a yield of 90.8%, and productivity of 0.54g/(Lh). Ethanol fermentation recovered 33.2% of the energy in waste paper as ethanol. The biochemical methane potential of the stillage eluted from ethanol fermentation was 270.5mL/g VTS and 73.0% of the energy in the stillage was recovered as methane. Integrating ethanol fermentation with methane fermentation, recovered a total of 80.4% of the energy in waste paper as ethanol and methane. Copyright © 2015 Elsevier Ltd. 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.

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.

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.

Comparison of four functionalization methods of gold nanoparticles for enhancing the enzyme-linked immunosorbent assay (ELISA).

PubMed

Ciaurriz, Paula; Fernández, Fátima; Tellechea, Edurne; Moran, Jose F; Asensio, Aaron C

2017-01-01

The enzyme-linked immunosorbent assay (ELISA) technique is based on the specific recognition ability of the molecular structure of an antigen (epitope) by an antibody and is likely the most important diagnostic technique used today in bioscience. With this methodology, it is possible to diagnose illness, allergies, alimentary fraud, and even to detect small molecules such as toxins, pesticides, heavy metals, etc. For this reason, any procedures that improve the detection limit, sensitivity or reduce the analysis time could have an important impact in several fields. In this respect, many methods have been developed for improving the technique, ranging from fluorescence substrates to methods for increasing the number of enzyme molecules involved in the detection such as the biotin-streptavidin method. In this context, nanotechnology has offered a significant number of proposed solutions, mainly based on the functionalization of nanoparticles from gold to carbon which could be used as antibody carriers as well as reporter enzymes like peroxidase. However, few works have focused on the study of best practices for nanoparticle functionalization for ELISA enhancement. In this work, we use 20 nm gold nanoparticles (AuNPs) as a vehicle for secondary antibodies and peroxidase (HRP). The design of experiments technique (DOE) and four different methods for biomolecule loading were compared using a rabbit IgG/goat anti-rabbit IgG ELISA model (adsorption, directional, covalent and a combination thereof). As a result, AuNP probes prepared by direct adsorption were the most effective method. AuNPs probes were then used to detect gliadin, one of the main components of wheat gluten, the protein composite that causes celiac disease. With this optimized approach, our data showed a sensitivity increase of at least five times and a lower detection limit with respect to a standard ELISA of at least three times. Additionally, the assay time was remarkably decreased.

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

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...

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.

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.

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.

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.

A comparison of the autohydrolysis and ammonia fiber explosion (AFEX) pretreatments on the subsequent enzymatic hydrolysis of coastal Bermuda grass.

PubMed

Lee, Jung Myoung; Jameel, Hasan; Venditti, Richard A

2010-07-01

Two distinct pretreatment technologies, autohydrolysis and AFEX, have been applied to coastal Bermuda grass (CBG) followed by enzymatic hydrolysis in order to compare the effects of pretreatment on the subsequent sugar generation. Furthermore, the influence of structural features from each pretreatment on biomass digestibility was characterized with SEM, ATR-FTIR, and XRD. Enzymatic conversion of pretreated solids from the pretreatments increased with elevated temperature and longer residence times. AFEX pretreatment at 100 degrees C for 30 min produced a sugar yield of 94.8% of theoretical possible with 30 FPU/g enzymatic loading, the maximum achieved with AFEX. It was also shown that with autohydrolysis at 170 degrees C for 60 min that 55.4% sugar yield of the theoretical possible was produced with a 30 FPU/g enzymatic loading, the maximum with autohydrolysis. AFEX pretreatment does not change the chemical composition of CBG but autohydrolysis reduces hemicellulose content in the pretreated solids. Both pretreatments cause re-localization of lignin components. There was no observed correlation between crystallinity and enzyme digestibility of the pretreated solids. AFEX pretreatment developed more enzymatic accessibility to pretreated solids of CBG than did autohydrolysis pretreatment, leading to more sugar generation through the whole process. The total amount of sugars accounted for with autohydrolysis decreases with increasing temperature, consistent with increased byproduct generation via thermal degradation reactions. Published by Elsevier Ltd.

Endostar-loaded PEG-PLGA nanoparticles: in vitro and in vivo evaluation.

PubMed

Hu, Sanyuan; Zhang, Yangde

2010-11-24

Endostar, a novel recombinant human endostatin, which was approved by the Chinese State Food and Drug Administration in 2005, has a broad spectrum of activity against solid tumors. In this study, we aimed to determine whether the anticancer effect of Endostar is increased by using a nanocarrier system. It is expected that the prolonged circulation of endostar will improve its anticancer activity. Endostar-loaded nanoparticles were prepared to improve controlled release of the drug in mice and rabbits, as well as its anticancer effects in mice with colon cancer. A protein release system could be exploited to act as a drug carrier. Nanoparticles were formulated from poly (ethylene glycol) modified poly (DL-lactide-co-glycolide) (PEG-PLGA) by a double emulsion technique. Physical and release characteristics of endostar-loaded nanoparticles in vitro were evaluated by transmission electron microscopy (TEM), photon correlation spectroscopy (PCS), and micro bicinchoninic acid protein assay. The pharmacokinetic parameters of endostar nanoparticles in rabbit and mice plasma were measured by enzyme-linked immunosorbent assay. Western blot was used to detect endostatin in different tissues. To study the effects of endostar-loaded nanoparticles in vivo, nude mice in which tumor cells HT-29 were implanted, were subsequently treated with endostar or endostar-loaded PEG-PLGA nanoparticles. Using TEM and PCS, endostar-loaded PEG-PLGA nanoparticles were found to have a spherical core-shell structure with a diameter of 169.56 ± 35.03 nm. Drug-loading capacity was 8.22% ± 2.35% and drug encapsulation was 80.17% ± 7.83%. Compared with endostar, endostar-loaded PEG-PLGA nanoparticles had a longer elimination half-life and lower peak concentration, caused slower growth of tumor cell xenografts, and prolonged tumor doubling times. The nanoparticles changed the pharmacokinetic characteristics of endostar in mice and rabbits, thereby reinforcing anticancer activity. In conclusion, PEG-PLGA nanoparticles are a feasible carrier for endostar. Endostar-loaded PEG-PLGA nanoparticles seem to have a better anticancer effect than conventional endostar. We believe that PEG-PLGA nanoparticles are an effective carrier for protein medicines.

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.

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

Impaired small-bowel barrier integrity in the presence of lumenal pancreatic digestive enzymes leads to circulatory shock.

PubMed

Kistler, Erik B; Alsaigh, Tom; Chang, Marisol; Schmid-Schönbein, Geert W

2012-08-01

In bowel ischemia, impaired mucosal integrity may allow intestinal pancreatic enzyme products to become systemic and precipitate irreversible shock and death. This can be attenuated by pancreatic enzyme inhibition in the small-bowel lumen. It is unresolved, however, whether ischemically mediated mucosal disruption is the key event allowing pancreatic enzyme products systemic access and whether intestinal digestive enzyme activity in concert with increased mucosal permeability leads to shock in the absence of ischemia. To test this possibility, the small intestinal lumen of nonischemic rats was perfused for 2 h with either digestive enzymes, a mucin disruption strategy (i.e., mucolytics) designed to increase mucosal permeability, or both, and animals were observed for shock. Digestive enzymes perfused included trypsin, chymotrypsin, elastase, amylase, and lipase. Control (n = 6) and experimental animals perfused with pancreatic enzymes only (n = 6) or single enzymes (n = 3 for each of the five enzyme groups) maintained stable hemodynamics. After mucin disruption using a combination of enteral N-acetylcysteine, atropine, and increased flow rates, rats (n = 6) developed mild hypotension (P < 0.001 compared with groups perfused with pancreatic enzymes only after 90 min) and increased intestinal permeability to intralumenally perfused fluorescein isothiocyanate-dextran 20 kd (P < 0.05) compared with control and enzyme-only groups, but there were no deaths. All animals perfused with both digestive enzymes and subjected to mucin disruption (n = 6) developed hypotension and increased intestinal permeability (P < 0.001 after 90 min). Pancreatic enzymes were measured in the intestinal wall of both groups subjected to mucin disruption, but not in the enzyme-only or control groups. Depletion of plasma protease inhibitors was found only in animals perfused with pancreatic enzymes plus mucin disruption, implicating increased permeability and intralumenal pancreatic enzyme egress in this group. These experiments demonstrate that increased bowel permeability via mucin disruption in the presence of pancreatic enzymes can induce shock and increase systemic protease activation in the absence of ischemia, implicating bowel mucin disruption as a key event in early ischemia. Digestive enzymes and their products, if allowed to penetrate the gut wall, may trigger multiorgan failure and death.

IMPAIRED SMALL BOWEL BARRIER INTEGRITY IN THE PRESENCE OF LUMENAL PANCREATIC DIGESTIVE ENZYMES LEADS TO CIRCULATORY SHOCK

PubMed Central

Kistler, Erik B.; Alsaigh, Tom; Chang, Marisol; Schmid-Schönbein, Geert W.

2012-01-01

In bowel ischemia, impaired mucosal integrity may allow intestinal pancreatic enzyme products to become systemic and precipitate irreversible shock and death. This can be attenuated by pancreatic enzyme inhibition in the small bowel lumen. It is unresolved, however, whether ischemically-mediated mucosal disruption is the key event allowing pancreatic enzyme products systemic access, and whether intestinal digestive enzyme activity in concert with increased mucosal permeability leads to shock in the absence of ischemia. To test this possibility, the small intestinal lumen of non-ischemic rats was perfused for two hours with either digestive enzymes, a mucin disruption strategy (i.e., mucolytics) designed to increase mucosal permeability, or both, and animals were observed for shock. Digestive enzymes perfused included trypsin, chymotrypsin, elastase, amylase and lipase. Control (n=6) and experimental animals perfused with pancreatic enzymes only (n=6) or single enzymes (n=3 for each of the five enzyme groups) maintained stable hemodynamics. After mucin disruption using a combination of enteral N-acetylcysteine, atropine, and increased flow rates, rats (n=6) developed mild hypotension (p<0.001 compared to groups perfused with pancreatic enzymes only after 90 minutes) and increased intestinal permeability to intralumenally perfused FITC-dextrans-20kD (p<0.05) compared to control and enzyme-only groups, but there were no deaths. All animals perfused with both digestive enzymes and subjected to mucin disruption (n=6) developed hypotension and increased intestinal permeability (p<0.001 after 90 minutes). Pancreatic enzymes were measured in the intestinal wall of both groups subjected to mucin disruption, but not in the enzyme-only or control groups. Depletion of plasma protease inhibitors was found only in animals perfused with pancreatic enzymes plus mucin disruption, implicating increased permeability and intralumenal pancreatic enzyme egress in this group. These experiments demonstrate that increased bowel permeability via mucin disruption in the presence of pancreatic enzymes can induce shock and increase systemic protease activation in the absence of ischemia, implicating bowel mucin disruption as a key event in early ischemia. Digestive enzymes and their products, if allowed to penetrate the gut wall may trigger multiorgan failure and death. PMID:22576000

A comparative study of enzyme immobilization strategies for multi-walled carbon nanotube glucose biosensors

NASA Astrophysics Data System (ADS)

Shi, Jin; Claussen, Jonathan C.; McLamore, Eric S.; Haque, Aeraj ul; Jaroch, David; Diggs, Alfred R.; Calvo-Marzal, Percy; Rickus, Jenna L.; Porterfield, D. Marshall

2011-09-01

This work addresses the comparison of different strategies for improving biosensor performance using nanomaterials. Glucose biosensors based on commonly applied enzyme immobilization approaches, including sol-gel encapsulation approaches and glutaraldehyde cross-linking strategies, were studied in the presence and absence of multi-walled carbon nanotubes (MWNTs). Although direct comparison of design parameters such as linear range and sensitivity is intuitive, this comparison alone is not an accurate indicator of biosensor efficacy, due to the wide range of electrodes and nanomaterials available for use in current biosensor designs. We proposed a comparative protocol which considers both the active area available for transduction following nanomaterial deposition and the sensitivity. Based on the protocol, when no nanomaterials were involved, TEOS/GOx biosensors exhibited the highest efficacy, followed by BSA/GA/GOx and TMOS/GOx biosensors. A novel biosensor containing carboxylated MWNTs modified with glucose oxidase and an overlying TMOS layer demonstrated optimum efficacy in terms of enhanced current density (18.3 ± 0.5 µA mM - 1 cm - 2), linear range (0.0037-12 mM), detection limit (3.7 µM), coefficient of variation (2%), response time (less than 8 s), and stability/selectivity/reproducibility. H2O2 response tests demonstrated that the most possible reason for the performance enhancement was an increased enzyme loading. This design is an excellent platform for versatile biosensing applications.

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

Plasmonic Enzyme-Linked Immunosorbent Assay Using Nanospherical Brushes as a Catalase Container for Colorimetric Detection of Ultralow Concentrations of Listeria monocytogenes.

PubMed

Chen, Rui; Huang, Xiaolin; Xu, Hengyi; Xiong, Yonghua; Li, Yanbin

2015-12-30

Plasmonic enzyme-linked immunosorbent assay (pELISA) based on catalase (CAT)-mediated gold nanoparticle growth exhibits ultrahigh sensitivity for detecting disease-related biomarkers using sandwich formats. However, the limit of detection (LOD) of this strategy for Listeria monocytogenes is only around 10(3) CFU/mL, which considerably exceeds the amount of L. monocytogenes commonly present in food products (<100 CFU/g). Herein, we report an improved pELISA method for detection of L. monocytogenes at ultralow concentrations with the sandwich formats using silica nanoparticles carrying poly(acrylic acid) brushes as a "CAT container" to increase enzyme loading for enhancing the detection signal. Under optimal conditions, the proposed pELISA exhibits good specificity and excellent sensitivity for L. monocytogenes with a LOD of 8 × 10(1) CFU/mL in 0.01 M phosphate-buffered saline, via a reaction that can be discriminated by the naked eye. The LOD obtained by this method was 2 and 5 orders of magnitude lower than that of conventional CAT-based pELISA and horseradish peroxidase (HRP)-based conventional ELISA, respectively. Coupled with large-volume immunomagnetic separation, the LOD for L. monocytogenes-spiked lettuce samples reached 8 × 10(1) CFU/g. The improved pELISA also exhibited a great potential in detecting a single cell of L. monocytogenes in 100 μL of solution.

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.

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

Cardioprotective effect of magnetic hydrogel nanocomposite loaded N,α-L-rhamnopyranosyl vincosamide isolated from Moringa oleifera leaves against doxorubicin-induced cardiac toxicity in rats: in vitro and in vivo studies.

PubMed

Cheraghi, Mostafa; Namdari, Mehrdad; Daraee, Hadis; Negahdari, Babak

2017-06-01

Cardioprotective effect of N, α-L-rhamnopyranosyl vincosamide (VR), isolated from the leaves of Moringa oleifera plant in doxorubicin (Dox)-induced cardiac toxicity rats was evaluated. Twelve (12) rats were randomly selected into three groups; two rats received distilled water in the control group, five rats in group I received varying concentration of VR treatment, and group II containing five rats received varying concentration of VR-loaded magnetic hydrogel nanocomposite. Malondialdehyde (MDA), glutathione peroxidase (GSH) and superoxide dismutase (SOD) enzymes activities level were analysed after two weeks. In addition, the expression of three heart failure markers; beta major histocompatibility complex (β-MHC), atrial natriuretic peptide (ANP), and B type natriuretic peptide (BNP) were also evaluated. It was observed that the level of these markers expression decreases with an increase in VR concentration (p < 0.05). The reduced GSH and SOD level were increased after VR administration, this extract also reduced the initially increased MDA level in cardiac tissue. Pharmacokinetic parameters evaluation showed that nanogel treated rats possesses a significantly increased VR plasma concentration, C max , K el , t ½(a), t ½(el), K a and AUC. The result of this study indicated that VR may help to lower the dosage level, and reduces the treatment course in cardiovascular diseases (CVD). Our conclusion proposes the cardio-protective ability of the isolated VR and its beneficial effect via free radical scavenging properties.

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.

Antidiabetic effect of Sida cordata in alloxan induced diabetic rats.

PubMed

Shah, Naseer Ali; Khan, Muhammad Rashid

2014-01-01

Medicinal plants are efficient ameliorator of oxidative stress associated with diabetes mellitus. In this study, ethyl acetate fraction (SCEE) of Sida cordata was investigated for scientific validation of its folk use in diabetes. Antidiabetic effect of SCEE was confirmed by antihyperglycemic activity in normal glucose loaded and diabetic glucose loaded animals as well as normal off feed animals. Confirmation of antidiabetic activity and toxicity ameliorative role of S. cordata was investigated in a chronic multiple dose treatment study of fifteen days. A single dose of alloxan (120 mg/kg) produced a decrease in insulin level, hyperglycemia, elevated total lipids, triglycerides, and cholesterol and decreased the high-density lipoproteins. Concurrent with these changes, there was an increase in the concentration of lipid peroxidation (TBARS), H2O2, and nitrite in pancreas, liver, and testis. This oxidative stress was related to a decrease in glutathione content (GSH) and antioxidant enzymes. Administration of SCEE for 15 days after diabetes induction ameliorated hyperglycemia, restored lipid profile, blunted the increase in TBARS, H2O2, and nitrite content, and stimulated the GSH production in the organs of alloxan-treated rats. We suggested that SCEE could be used as antidiabetic component in case of diabetes mellitus. This may be related to its antioxidative properties.

Preexposure to Olive Oil Polyphenols Extract Increases Oxidative Load and Improves Liver Mass Restoration after Hepatectomy in Mice via Stress-Sensitive Genes

PubMed Central

Marinić, Jelena; Broznić, Dalibor; Milin, Čedomila

2016-01-01

Polyphenols can act as oxidants in some conditions, inducing redox-sensitive genes. We investigated the effect of preexposure to the olive oil polyphenols extract (PFE) on time-dependent changes in the hepatic oxidative state in a model of liver regeneration—a process in which oxidative stress associated with the metabolic overload accounts for the early events that contribute to the onset of liver self-repair. Liver regeneration was induced by one-third hepatectomy in mice. Prior to hepatectomy, mice were intraperitoneally given either PFE (50 mg/kg body weight) or saline for seven consecutive days, while respective controls received vehicle alone. Redox state-regulating enzymes and thiol proteins along with the mRNA levels of Nrf2 gene and its targets γ-glutamylcysteine synthetase and heme oxygenase-1 were determined at different time intervals after hepatectomy. The liver mass restoration was calculated to assess hepatic regeneration. The resulting data demonstrate the effectiveness of preexposure to PFE in stimulating liver regeneration in a model of a small tissue loss which may be ascribed to the transient increase in oxidant load during the first hours after hepatectomy and associated induction of stress response gene-profiles under the control of Nrf2. PMID:26925195

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.

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.

A population-based study of salivary lysozyme concentrations and candidal counts.

PubMed

Yeh, C K; Dodds, M W; Zuo, P; Johnson, D A

1997-01-01

The relationship between salivary lysozyme concentration and oral candida load was examined in 595 adults. Unstimulated whole saliva, and citrate-stimulated parotid and submandibular/sublingual saliva were collected from each participant. Candida colony-forming units (c.f.u.) in unstimulated whole saliva were determined. An enzyme-linked immunosorbent assay for lysozyme using commercially available antibodies was developed. This assay showed a linear relation of salivary lysozyme concentrations from 0.5 to 4.0 ng/ml. Significant negative relations were observed between lysozyme concentration and flow rate: r = -0.16 (p < 0.001) for stimulated parotid and r = -0.22 (p < 0.0001) for stimulated submandibular/sublingual saliva. The lysozyme concentration in stimulated submandibular/sublingual saliva was higher in males than in female, but no sex difference was observed for stimulated parotid saliva. The lysozyme concentration of stimulated parotid saliva was positively correlated with candida counts (r = 0.18: p < 0.005). Further study of groups according to their levels of candida in whole saliva revealed that lysozyme concentrations were higher in the high candida (> or = 1000 c.f.u./ml) group than in the zero and moderate candida categories in stimulated parotid saliva (p < 0.001): there were no concentration differences in stimulated submandibular/sublingual saliva. These results suggest that parotid lysozyme concentration increases as candida load increases.

Expression of matrix metalloproteinase-2 and metalloproteinase-9 in the skin of dogs with visceral leishmaniasis.

PubMed

Jacintho, Ana Paula Prudente; Melo, Guilherme D; Machado, Gisele F; Bertolo, Paulo Henrique Leal; Moreira, Pamela Rodrigues Reina; Momo, Claudia; Souza, Thiago A; Vasconcelos, Rosemeri de Oliveira

2018-06-01

The skin is the first organ to be infected by the parasite in canine visceral leishmaniasis. The enzyme matrix metalloproteinase (MMP) acts towards degradation of the extracellular matrix (ECM) and modulation of the inflammatory response against many kinds of injuries. The aims of this study were to evaluate the expression of MMP-2 and MMP-9 through immunohistochemistry and zymography on the skin (muzzle, ears, and abdomen) of dogs that were naturally infected by Leishmania spp. and to compare these results with immunodetection of the parasite and with alterations to the dermal ECM. Picrosirius red staining was used to differentiate collagen types I and III in three regions of the skin. The parasite load, intensity of inflammation, and production of MMP-2 (latent) and MMP-9 (active and latent) were higher in the ear and muzzle regions. MMP-9 (active) predominated in the infected group of dogs and its production was significantly different to that of the control group. Macrophages, lymphocytes, and plasma cells predominated in the dermal inflammation and formed granulomas in association with degradation of mature collagen (type I) and with discrete deposition of young collagen (type III). This dermal change was more pronounced in dogs with high parasite load in the skin. Therefore, it was concluded that the greater parasite load and intensity of inflammation in the skin led consequently to increased degradation of mature collagen, caused by increased production of MMPs, particularly active MMP-9, in dogs with visceral leishmaniasis. This host response profile possibly favors systemic dissemination of the parasite.

Alkaline hydrogen peroxide pretreatment of cashew apple bagasse for ethanol production: study of parameters.

PubMed

Correia, Jessyca Aline da Costa; Júnior, José Edvan Marques; Gonçalves, Luciana Rocha B; Rocha, Maria Valderez Ponte

2013-07-01

The alkaline hydrogen peroxide (AHP) pretreatment of cashew apple bagasse (CAB) was evaluated based on the conversion of the resultant cellulose into glucose. The effects of the concentration of hydrogen peroxide at pH 11.5, the biomass loading and the pretreatment duration performed at 35°C and 250 rpm were evaluated after the subsequent enzymatic saccharification of the pretreated biomass using a commercial cellulase enzyme. The CAB used in this study contained 20.56 ± 2.19% cellulose, 10.17 ± 0.89% hemicellulose and 35.26 ± 0.90% lignin. The pretreatment resulted in a reduced lignin content in the residual solids. Increasing the H2O2 concentration (0-4.3% v/v) resulted in a higher rate of enzymatic hydrolysis. Lower biomass loadings gave higher glucose yields. In addition, no measurable furfural and hydroxymethyl furfural were produced in the liquid fraction during the pretreatment. The results show that alkaline hydrogen peroxide is effective for the pretreatment of CAB. Crown Copyright © 2013. Published by 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.

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.

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.

Cellular Basis of Secondary Infections and Impaired Desquamation in Certain Inherited Ichthyoses

PubMed Central

Chan, Aegean; Godoy-Gijon, Elena; Nuno-Gonzalez, Almudena; Crumrine, Debra; Hupe, Melanie; Choi, Eung-Ho; Gruber, Robert; Williams, Mary L.; Choate, Keith; Fleckman, Philip H.; Elias, Peter M.

2015-01-01

IMPORTANCE Secondary infections and impaired desquamation complicate certain inherited ichthyoses, but their cellular basis remains unknown. In healthy human epidermis, the antimicrobial peptides cathelicidin (LL-37) and human β-defensin 2 (HBD2), as well as the desquamatory protease kallikrein-related peptidase 7 (KLK7), are delivered to the stratum corneum (SC) interstices by lamellar body (LB) exocytosis. OBJECTIVE To assess whether abnormalities in the LB secretory system could account for increased risk of infections and impaired desquamation in inherited ichthyoses with known abnormalities in LB assembly (Harlequin ichthyosis [HI]), secretion (epidermolytic ichthyosis [EI]), or postsecretory proteolysis (Netherton syndrome [NS]). DESIGN, SETTING, AND PARTICIPANTS Samples from library material were taken from patients with HI, EI, NS, and other ichthyoses, but with a normal LB secretory system, and in healthy controls and were evaluated by electron microscopy and immunohistochemical analysis from July 1, 2010, through March 31, 2013. MAIN OUTCOME AND MEASURES Changes in LB secretion and in the fate of LB-derived enzymes and antimicrobial peptides in ichthyotic patients vs healthy controls. RESULTS In healthy controls and patients with X-linked ichthyosis, neutral lipid storage disease with ichthyosis, and Gaucher disease, LB secretion is normal, and delivery of LB-derived proteins and LL-37 immunostaining persists high into the SC. In contrast, proteins loaded into nascent LBs and their delivery to the SC interstices decrease markedly in patients with HI, paralleled by reduced immunostaining for LL-37, HBD2, and KLK7 in the SC. In patients with EI, the cytoskeletal abnormality impairs the exocytosis of LB contents and thus results in decreased LL-37, HBD2, and KLK7 secretion, causing substantial entombment of these proteins within the corneocyte cytosol. Finally, in patients with NS, although abundant enzyme proteins loaded in parallel with accelerated LB production, LL-37 disappears, whereas KLK7 levels increase markedly in the SC. CONCLUSIONS AND RELEVANCE Together, these results suggest that diverse abnormalities in the LB secretory system account for the increased risk of secondary infections and impaired desquamation in patients with HI, EI, and NS. PMID:25565224

Ultrasound assisted enzymatic depolymerization of aqueous guar gum solution.

PubMed

Prajapat, Amrutlal L; Subhedar, Preeti B; Gogate, Parag R

2016-03-01

The present work investigates the effectiveness of application of low intensity ultrasonic irradiation for the intensification of enzymatic depolymerization of aqueous guar gum solution. The extent of depolymerization of guar gum has been analyzed in terms of intrinsic viscosity reduction. The effect of ultrasonic irradiation on the kinetic and thermodynamic parameters related to the enzyme activity as well as the intrinsic viscosity reduction of guar gum using enzymatic approach has been evaluated. The kinetic rate constant has been found to increase with an increase in the temperature and cellulase loading. It has been observed that application of ultrasound not only enhances the extent of depolymerization but also reduces the time of depolymerization as compared to conventional enzymatic degradation technique. In the presence of cellulase enzyme, the maximum extent of depolymerization of guar gum has been observed at 60 W of ultrasonic rated power and ultrasonic treatment time of 30 min. The effect of ultrasound on the kinetic and thermodynamic parameters as well as the molecular structure of cellulase enzyme was evaluated with the help of the chemical reaction kinetics model and fluorescence spectroscopy. Application of ultrasound resulted in a reduction in the thermodynamic parameters of activation energy (Ea), enthalpy (ΔH), entropy (ΔS) and free energy (ΔG) by 47%, 50%, 65% and 1.97%, respectively. The changes in the chemical structure of guar gum treated using ultrasound assisted enzymatic approach in comparison to the native guar gum were also characterized by FTIR. The results revealed that enzymatic depolymerization of guar gum resulted in a polysaccharide with low degree of polymerization, viscosity and consistency index without any change in the core chemical structure which could make it useful for incorporation in food products. Copyright © 2015 Elsevier B.V. All rights reserved.

Mechanism of product inhibition for cellobiohydrolase Cel7A during hydrolysis of insoluble cellulose.

PubMed

Olsen, Johan P; Alasepp, Kadri; Kari, Jeppe; Cruys-Bagger, Nicolaj; Borch, Kim; Westh, Peter

2016-06-01

The cellobiohydrolase cellulase Cel7A is extensively utilized in industrial treatment of lignocellulosic biomass under conditions of high product concentrations, and better understanding of inhibition mechanisms appears central in attempts to improve the efficiency of this process. We have implemented an electrochemical biosensor assay for product inhibition studies of cellulases acting on their natural substrate, cellulose. Using this method we measured the hydrolytic rate of Cel7A as a function of both product (inhibitor) concentration and substrate load. This data enabled analyses along the lines of conventional enzyme kinetic theory. We found that the product cellobiose lowered the maximal rate without affecting the Michaelis constant, and this kinetic pattern could be rationalized by two fundamentally distinct molecular mechanisms. One was simple reversibility, that is, an increasing rate of the reverse reaction, lowering the net hydrolytic velocity as product concentrations increase. Strictly this is not a case of inhibition, as no catalytically inactive is formed. The other mechanism that matched the kinetic data was noncompetitive inhibition with an inhibition constant of 490 ± 40 μM. Noncompetitive inhibition implies that the inhibitor binds with comparable strength to either free enzyme or an enzymesubstrate complex, that is, that association between enzyme and substrate has no effect on the binding of the inhibitor. This mechanism is rarely observed, but we argue, that the special architecture of Cel7A with numerous subsites for binding of both substrate and product could give rise to a true noncompetitive inhibition mechanism. Biotechnol. Bioeng. 2016;113: 1178-1186. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

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.

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-...

Template synthesis of test tube nanoparticles using non-destructive replication

PubMed Central

Wagner, Jonathan; Yao, Jingyuan; Rodgers, David; Hinds, Bruce

2013-01-01

Nano test tubes are a promising delivery vehicle for a range of therapeutics including small molecule drugs and biologics. However, current template synthesis methods of producing nano test tubes are prohibitively expensive and time consuming. Here, non-destructive template replication was used to increase nano test tube yield from porous alumina by more than a hundredfold. We demonstrate how to produce nano test tubes of several sizes and compositions including hybrid tubes with different inner and outer surfaces for targeted surface chemistry. Nano test tubes were readily suspended and stored in aqueous solutions without the need for chemical treatment. These nano test tubes should find application as delivery vehicles for therapeutics, particularly for processive “bionanoreactors” loaded with enzymes. PMID:23376956

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.

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.

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

Greater Mechanical Loading During Walking Is Associated With Less Collagen Turnover in Individuals With Anterior Cruciate Ligament Reconstruction.

PubMed

Pietrosimone, Brian; Blackburn, J Troy; Harkey, Matthew S; Luc, Brittney A; Hackney, Anthony C; Padua, Darin A; Driban, Jeffrey B; Spang, Jeffrey T; Jordan, Joanne M

2016-02-01

Individuals who have sustained an anterior cruciate ligament (ACL) injury and undergo ACL reconstruction (ACLR) are at higher risk of developing knee osteoarthritis. It is hypothesized that altered knee loading may influence the underlying joint metabolism and hasten development of posttraumatic knee osteoarthritis. To explore the associations between serum biomarkers of cartilage metabolism and peak vertical ground-reaction force (vGRF) and vGRF loading rate in the injured and uninjured limbs of individuals with ACLR. Descriptive laboratory study. Patients with a history of a primary unilateral ACLR who had returned to unrestricted physical activity (N = 19) participated in the study. Resting blood was collected from each participant before completing 5 walking gait trials at a self-selected comfortable speed. Peak vGRF was extracted for both limbs during the first 50% of the stance phase of gait, and the linear vGRF loading rate was determined between heel strike and peak vGRF. Sera were assessed for collagen breakdown (collagen type II cleavage product [C2C]) and synthesis (collagen type II C-propeptide [CPII]), as well as aggrecan concentrations, via commercially available specific enzyme-linked immunosorbent assays. Pearson product-moment correlations (r) and Spearman rank-order correlations (ρ) were used to evaluate associations between loading characteristics and biomarkers of cartilage metabolism. Lower C2C:CPII ratios were associated with higher peak vGRF in the injured limb (ρ = -0.59, uncorrected P = .007). There were no significant associations between peak vGRF or linear vGRF loading rate and CPII, C2C, or aggrecan serum concentrations. Lower C2C:CPII ratios were associated with higher peak vGRF in the ACLR limb during gait, suggesting that higher peak loading in the ACLR limb is related to lower type II collagen breakdown relative to type II collagen synthesis. These data suggest that type II collagen synthesis may be higher relative to the amount of type II collagen breakdown in the ACLR limb with higher lower extremity loading. Future study should determine if metabolic compensations to increase collagen synthesis may affect the risk of developing osteoarthritis after ACLR. © 2015 The Author(s).

Nitrogen fixation in boreal peatlands: the effects of increased N deposition on N2-fixation

NASA Astrophysics Data System (ADS)

Popma, J. M.; Wieder, R.; Lamers, L.; Vile, M. A.

2013-12-01

Boreal peatlands are of great importance to global carbon and nitrogen cycling. While covering only 3-4 % of the terrestrial surface, they account for 25-30 % of the world's soil C and 9-15 % of the world's soil N. In Western Canada atmospheric dry deposition rates are extremely low: approximately 1 kg N ha-1 yr-1. Though these systems have been functioning as net sinks over the past 11,000 years, natural and anthropogenic disturbances might compromise the historical balance of C and N. Biological N2-fixation has recently been shown to represent a very significant input of N into these systems, contributing to 62% of total N in Western Canada. Interactions between N deposition and biological N2-fixation are as yet, unknown, but the impact of elevated deposition of N-compounds from increased industrial expansion of oil sands mining to peatlands, is concerning. Given that nitrogenase, the enzyme responsible for catalyzing N2-fixation, is energetically costly when active, enhanced inputs of atmospheric N deposition could be a major determinant for enzyme activity and rates of biological N input to these bogs. Understanding interactions between N deposition and N2 fixation in boreal peatlands can aid in predicting the consequences of increased N deposition and setting critical loads. We conducted a field-fertilization experiment in a poor fen in Alberta, Canada, to determine the effects of enhanced N deposition on a dominant fen species Sphagnum angustifolium. The experiment consisted of seven N treatments: Control, 0, 5, 10, 15, 20 and 25 kg N ha-1 y1, n=3. N2-fixation was measured during summer 2012 and 2013 using the acetylene reduction assay (ARA). ARA rates were converted to rates of N2-fixation by calibrating ARA with paired 15N2-incubations. In both 2012 and 2013, with increasing N deposition from 0 kg N ha-1 yr-1 to 25 kg N ha-1 yr-1, rates of N2 fixation decreased, with highest rates in the 0 kg N ha-1 yr-1 treatment mosses (54.2 × 1.40; 48.58 × 7.12 kg N ha-1 yr-1, mean × std err for 2012 and 2013, respectively) followed by progressively lower rates with a low of 5.02 × 0.87 in 2012 and 8.94 × 3.09 in 2013 (mean × std err). As biological N2-fixation is an energetically costly process, up-regulating enzyme activity when N availability is low and down-regulating activity when N deposition is enhanced makes thermodynamic and evolutionary sense. N2-fixation shows to be one of the most early-warning indicators to the early response of boreal peatlands to increased N deposition, and can aid in setting critical loads to protect these historically pristine ecosystems.

Vitamin C activity of dehydroascorbic acid in humans--association between changes in the blood vitamin C concentration or urinary excretion after oral loading.

PubMed

Tsujimura, Masaru; Higasa, Shizu; Nakayama, Kazuhiro; Yanagisawa, Yoshiko; Iwamoto, Sadahiko; Kagawa, Yasuo

2008-08-01

We performed oral loading of AsA or DAsA (1 mmol) in subjects who had consumed a diet low in vitamin C (C) (C< or =5 mg/d) for 3 d before loading, and measured urinary and blood vitamin C. Since the crossover method was used, the same experiment was repeated after an interval of about 1 mo in each subject. The results of the experiment including a total of 17 subjects for 2005 and 2006, were as follows. (1) There were marked individual differences in urinary C excretion. (2) The C level in 24-h urine after C loading did not differ between the two orally administered C forms (AsA and DAsA). (3) C excretion between 0 and 3 h after C loading was significantly higher (p<0.05) for the DAsA group, while those between 3 and 6, 6 and 9, 9 and 12, and 12 and 24 h after C loading were significantly higher (p<0.05 or p<0.01) for the AsA group. (4) The blood C concentration and the increase in C 1 h after C loading were significantly higher (p<0.05 and p<0.01, respectively) in the DAsA than in the AsA group. (5) Evaluation of the association between C metabolism and the single nucleotide polymorphisms of glutathione S-transferase P (GSTP) 1-1 showed a lower urinary C excretion and a significantly lower C level in 24-h urine (p<0.05) after AsA loading, and a significantly lower urinary C excretion between 0 and 3 h after DAsA loading (p<0.05) for the GA heterozygotes than for the AA homozygotes. Considering the activity of C as DAsA in humans, based on urinary and blood C levels after a single loading of C, the utilization of DAsA is equivalent to that of AsA, although the metabolic turnover time is different. The involvement of polymorphisms in the xenobiotic metabolizing enzyme, GSTP1-1, in C metabolism, particularly urinary C excretion, was also clarified. This demonstrates the necessity of considering gene polymorphisms in determining individual C requirements. An abstract of this paper was reported by the Vitamin C Research Committee (Ochanomizu University) in 2007.

Cell recruiting chemokine-loaded sprayable gelatin hydrogel dressings for diabetic wound healing.

PubMed

Yoon, Dong Suk; Lee, Yunki; Ryu, Hyun Aae; Jang, Yeonsue; Lee, Kyoung-Mi; Choi, Yoorim; Choi, Woo Jin; Lee, Moses; Park, Kyung Min; Park, Ki Dong; Lee, Jin Woo

2016-07-01

In this study, we developed horseradish peroxidase (HRP)-catalyzed sprayable gelatin hydrogels (GH) as a bioactive wound dressing that can deliver cell-attracting chemotactic cytokines to the injured tissues for diabetic wound healing. We hypothesized that topical administration of chemokines using GH hydrogels might improve wound healing by inducing recruitment of the endogenous cells. Two types of chemokines (interleukin-8; IL-8, macrophage inflammatory protein-3α; MIP-3α) were simply loaded into GH hydrogels during in situ cross-linking, and then their wound-healing effects were evaluated in streptozotocin-induced diabetic mice. The incorporation of chemokines did not affect hydrogels properties including swelling ratio and mechanical stiffness, and the bioactivities of IL-8 and MIP-3α released from hydrogel matrices were stably maintained. In vivo transplantation of chemokine-loaded GH hydrogels facilitated cell infiltration into the wound area, and promoted wound healing with enhanced re-epithelialization/neovascularization and increased collagen deposition, compared with no treatment or the GH hydrogel alone. Based on our results, we suggest that cell-recruiting chemokine-loaded GH hydrogel dressing can serve as a delivery platform of various therapeutic proteins for wound healing applications. Despite development of materials combined with therapeutic agents for diabetic wound treatment, impaired wound healing by insufficient chemotactic responses still remain as a significant problem. In this study, we have developed enzyme-catalyzed gelatin (GH) hydrogels as a sprayable dressing material that can deliver cell-attracting chemokines for diabetic wound healing. The chemotactic cytokines (IL-8 and MIP-3α) were simply loaded within hydrogel during in situ gelling, and wound healing efficacy of chemokine-loaded GH hydrogels was investigated in STZ-induced diabetic mouse model. These hydrogels significantly promoted wound-healing efficacy with faster wound closure, neovascularization, and thicker granulation. Therefore, we expect that HRP-catalyzed in situ forming GH hydrogels can serve as an injectable/sprayable carrier of various therapeutic agents for wound healing applications. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Binding affinity and adhesion force of organophosphate hydrolase enzyme with soil particles related to the isoelectric point of the enzyme.

PubMed

Islam, Shah Md Asraful; Yeasmin, Shabina; Islam, Md Saiful; Islam, Md Shariful

2017-07-01

The binding affinity of organophosphate hydrolase enzyme (OphB) with soil particles in relation to the isoelectric point (pI) was studied. Immobilization of OphB with soil particles was observed by confocal microscopy, Fourier transform infrared spectroscopy (FT-IR), and Atomic force microscopy (AFM). The calculated pI of OphB enzyme was increased from 8.69 to 8.89, 9.04 and 9.16 by the single, double and triple mutant of OphB enzyme, respectively through the replacement of negatively charged aspartate with positively charged histidine. Practically, the binding affinity was increased to 5.30%, 11.50%, and 16.80% for single, double and triple mutants, respectively. In contrast, enzyme activity of OphB did not change by the mutation of the enzyme. On the other hand, adhesion forces were gradually increased for wild type OphB enzyme (90 pN) to 96, 100 and 104 pN for single, double and triple mutants of OphB enzyme, respectively. There was an increasing trend of binding affinity and adhesion force by the increase of isoelectric point (pI) of OphB enzyme. Copyright © 2017 Elsevier Inc. All rights reserved.

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.

Ocular Inserts for Sustained Release of the Angiotensin-Converting Enzyme 2 Activator, Diminazene Aceturate, to Treat Glaucoma in Rats

PubMed Central

Nogueira, José Carlos; Fulgêncio, Gustavo de Oliveira; Ribeiro, Tatiana Gomes; Castilho, Rachel Oliveira; Yoshida, Maria Irene; Fuscaldi, Leonardo Lima; Fernandes, Simone Odília Antunes; Cardoso, Valbert Nascimento; Cronemberger, Sebastião; Faraco, André Augusto Gomes; Ferreira, Anderson José

2015-01-01

The aim of this study was to develop and evaluate the effects of chitosan inserts for sustained release of the angiotensin-converting enzyme 2 (ACE2) activator, diminazene aceturate (DIZE), in experimental glaucoma. Monolayer DIZE loaded inserts (D+I) were prepared and characterized through swelling, attenuated total reflectance Fourier transformed infrared spectroscopy (ATR-FTIR), differential scanning calorimetry (DSC) and in vitro drug release. Functionally, the effects of D+I were tested in glaucomatous rats. Glaucoma was induced by weekly injections of hyaluronic acid (HA) into the anterior chamber and intraocular pressure (IOP) measurements were performed. Retinal ganglion cells (RGC) and optic nerve head cupping were evaluated in histological sections. Biodistribution of the drug was accessed by scintigraphic images and ex vivo radiation counting. We found that DIZE increased the swelling index of the inserts. Also, it was molecularly dispersed and interspersed in the polymeric matrix as a freebase. DIZE did not lose its chemical integrity and activity when loaded in the inserts. The functional evaluation demonstrated that D+I decreased the IOP and maintained the IOP lowered for up to one month (last week: 11.0±0.7 mmHg). This effect of D+I prevented the loss of RGC and degeneration of the optic nerve. No toxic effects in the eyes related to application of the inserts were observed. Moreover, biodistribution studies showed that D+I prolonged the retention of DIZE in the corneal site. We concluded that D+I provided sustained DIZE delivery in vivo, thereby evidencing the potential application of polymeric-based DIZE inserts for glaucoma management. PMID:26204514

Dual-functionalized graphene oxide for enhanced siRNA delivery to breast cancer cells.

PubMed

Imani, Rana; Shao, Wei; Taherkhani, Samira; Emami, Shahriar Hojjati; Prakash, Satya; Faghihi, Shahab

2016-11-01

The aim of this study is to improve hydrocolloid stability and siRNA transfection ability of a reduced graphene oxide (rGO) based nano-carrier using a phospholipid-based amphiphilic polymer (PL-PEG) and cell penetrating peptide (CPPs). The dual functionalized nano-carrier is comprehensively characterized for its chemical structure, size, surface charge and morphology as well as thermal stability. The nano-carrier cytocompatibility, siRNA condensation ability both in the presence and absence of enzyme, endosomal buffering capacity, cellular uptake and intracellular localization are also assessed. The siRNA loaded nano-carrier is used for internalization to MCF-7 cells and its gene silencing ability is compared with AllStars Hs Cell Death siRNA as a model gene. The nano-carrier remains stable in biological solution, exhibits excellent cytocompatibility, retards the siRNA migration and protects it against enzyme degradation. The buffering capacity analysis shows that incorporation of the peptide in nano-carrier structure would increase the resistance to endo/lysosomal like acidic condition (pH 6-4) The functionalized nano-carrier which is loaded with siRNA in an optimal N:P ratio presents superior internalization efficiency (82±5.1% compared to HiPerFect(®)), endosomal escape quality and capable of inducing cell death in MCF-7 cancer cells (51±3.1% compared to non-treated cells). The success of siRNA-based therapy is largely dependent on the safe and efficient delivery system, therefore; the dual functionalized rGO introduced here could have a great potential to be used as a carrier for siRNA delivery with relevancy in therapeutics and clinical applications. Copyright © 2016 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.

Risk factors for vertical transmission of hepatitis C virus: a single center experience with 710 HCV-infected mothers.

PubMed

Garcia-Tejedor, Amparo; Maiques-Montesinos, Vicente; Diago-Almela, Vicente José; Pereda-Perez, Antonio; Alberola-Cuñat, Vicente; López-Hontangas, José Luís; Perales-Puchalt, Alfredo; Perales, Alfredo

2015-11-01

The aim of this study was to analyze the risk factors on the perinatal transmission of hepatitis C virus (HCV). A retrospective cohort study with 711 infants born to 710 HCV-infected mothers was conducted at the Hospital La Fe, in Valencia, Spain, from 1986 to 2011. As potential risk factors for transmission we analyzed: maternal age, mode of acquisition of HCV infection, HIV co-infection, antiretroviral treatment against HIV, CD4 cell count, HIV and HCV viral load, liver enzyme levels during pregnancy, smoking habit, gestational age, intrapartum invasive procedures, length of rupture of membranes, length of labor, mode of delivery, episiotomy, birth weight, newborn gender and type of feeding. Overall perinatal HCV transmission rate was 2.4%. The significant risk factors related with HCV transmission were maternal virus load >615copies/mL (OR 9.3 [95% CI 1.11-78.72]), intrapartum invasive procedures (OR 10.1 [95% CI 2.6-39.02]) and episiotomy (OR 4.2 [95% CI 1.2-14.16]). HIV co-infection and newborn female were near significance (p=0.081 and 0.075, respectively). Invasive procedures as fetal scalp blood sampling or internal electrode and episiotomy increase vertical transmission of HCV, especially in patients with positive HCV RNA virus load at delivery. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

β-Lapachone and Paclitaxel Combination Micelles with Improved Drug Encapsulation and Therapeutic Synergy as Novel Nanotherapeutics for NQO1-Targeted Cancer Therapy.

PubMed

Zhang, Ling; Chen, Zhen; Yang, Kuan; Liu, Chun; Gao, Jinming; Qian, Feng

2015-11-02

β-Lapachone (LPC) is a novel cytotoxic agent that is bioactivated by NADP(H): quinone oxidoreductase 1 (NQO1), an enzyme elevated in a variety of tumors, such as non-small cell lung cancer (NSCLC), pancreatic cancer, liver cancer, and breast cancer. Despite its unique mechanism of action, its clinical evaluation has been largely hindered by low water solubility, short blood half-life, and narrow therapeutic window. Although encapsulation into poly(ethylene glycol)-b-poly(D,L-lactic acid) (PEG-PLA) micelles could modestly improve its solubility and prolong its half-life, the extremely fast intrinsic crystallization tendency of LPC prevents drug loading higher than ∼2 wt %. The physical stability of the LPC-loaded micelles is also far from satisfactory for further development. In this study, we demonstrate that paclitaxel (PTX), a front-line drug for many cancers, can provide two functions when coencapsulated together with LPC in the PEG-PLA micelles; first, as a strong crystallization inhibitor for LPC, thus to significantly increase the LPC encapsulation efficiency in the micelle from 11.7 ± 2.4% to 100.7 ± 2.2%. The total drug loading efficiency of both PTX and LPC in the combination polymeric micelle reached 100.3 ± 3.0%, and the drug loading density reached 33.2 ± 1.0%. Second, the combination of LPC/PTX demonstrates strong synergistic cytotoxicity effect against the NQO1 overexpressing cancer cells, including A549 NSCLC cells, and several pancreatic cancer cells (combination index <1). In vitro drug release study showed that LPC was released faster than PTX either in phosphate-buffered saline (PH = 7.4) or in 1 M sodium salicylate, which agrees with the desired dosing sequence of the two drugs to exert synergistic pharmacologic effect at different cell checkpoints. The PEG-PLA micelles coloaded with LPC and PTX offer a novel nanotherapeutic, with high drug loading, sufficient physical stability, and biological synergy to increase drug delivery efficiency and optimize the therapeutic window for NOQ1-targeted therapy of cancer.

Plant, cell, and molecular mechanisms of abscisic-acid regulation of stomatal apertures. In vivo phosphorylation of phosphoenolpyruvate carboxylase in guard cells of Vicia faba L. is enhanced by fusicoccin and suppressed by abscisic acid

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

Du, Z.; Aghoram, K.; Outlaw, W.H. Jr.

Plants regulate water loss and CO{sub 2} gain by modulating the aperture sizes of stomata that penetrate the epidermis. Aperture size itself is increased by osmolyte accumulation and consequent turgor increase in the pair of guard cells that flank each stoma. Guard-cell phosphoenolpyruvate carboxylase, which catalyzes the regulated step leading to malate synthesis, is crucial for charge and pH maintenance during osmolyte accumulation. Regulation of this cytosolic enzyme by effectors is well documented, but additional regulation by posttranslational modification is predicted by the alteration of PEPC kinetics during stomatal opening. In this study, the authors have investigated whether this alterationmore » is associated with the phosphorylation status of this enzyme. Using sonicated epidermal peels (isolated guard cells) pre-loaded with {sub 32}PO{sub 4}, the authors induced stomatal opening and guard-cell malate accumulation by incubation with 5 {micro}M fusicoccin (FC). In corroboratory experiments, guard cells were incubated with 5 {micro}M fusicoccin (FC). In corroboratory experiments, guard cells were incubated with the FC antagonist, 10 {micro}M abscisic acid (ABA). The phosphorylation status of PEPC was assessed by immunoprecipitation, electrophoresis, immunoblotting, and autoradiography. PEPC was phosphorylated when stomata were stimulated to open, and phosphorylation was lessened by incubation with ABA.« less

Glycogen phosphorylase isoenzyme BB plasma kinetics is not related to myocardial ischemia induced by exercise stress echo test.

PubMed

Dobric, Milan; Giga, Vojislav; Beleslin, Branko; Ignjatovic, Svetlana; Paunovic, Ivana; Stepanovic, Jelena; Djordjevic-Dikic, Ana; Kostic, Jelena; Nedeljkovic, Ivana; Nedeljkovic, Milan; Tesic, Milorad; Dajak, Marijana; Ostojic, Miodrag

2013-10-01

Glycogen phosphorylase BB (GPBB) is released from cardiac cells during myocyte damage. Previous studies have shown contradictory results regarding the relation of enzyme release and reversible myocardial ischemia. The aim of this study was to determine the plasma kinetics of GPBB as a response to the exercise stress echocardiographic test (ESET), and to define the relationship between myocardial ischemia and enzyme plasma concentrations. We studied 46 consecutive patients undergoing ESET, with recent coronary angiography. In all patients, a submaximal stress echo test according to Bruce protocol was performed. Concentration of GPBB was measured in peripheral blood that was sampled 5 min before and 10, 30 and 60 min after ESET. There was significant increase of GPBB concentration after the test (p=0.021). Significant increase was detected 30 min (34.9% increase, p=0.021) and 60 min (34.5% increase, p=0.016) after ESET. There was no significant effect of myocardial ischemia on GPBB concentrations (p=0.126), and no significant interaction between sampling intervals and myocardial ischemia, suggesting a similar release profile of GPBB in ischemic and non-ischemic conditions (p=0.558). Patients in whom ESET was terminated later (stages 4 or 5 of standard Bruce protocol; n=13) had higher GPBB concentrations than patients who terminated ESET earlier (stages 1, 2 or 3; n=33) (p=0.049). Baseline GPBB concentration was not correlated to any of the patients' demographic, clinical and hemodynamic characteristics. GPBB plasma concentration increases after ESET, and it is not related to inducible myocardial ischemia. However, it seems that GPBB release during ESET might be related to exercise load/duration.

Effects of Two Types of Melatonin-Loaded Nanocapsules with Distinct Supramolecular Structures: Polymeric (NC) and Lipid-Core Nanocapsules (LNC) on Bovine Embryo Culture Model.

PubMed

Komninou, Eliza Rossi; Remião, Mariana Härter; Lucas, Caroline Gomes; Domingues, William Borges; Basso, Andrea Cristina; Jornada, Denise Soledade; Deschamps, João Carlos; Beck, Ruy Carlos Ruver; Pohlmann, Adriana Raffin; Bordignon, Vilceu; Seixas, Fabiana Kömmling; Campos, Vinicius Farias; Guterres, Silvia Stanisçuaski; Collares, Tiago

2016-01-01

Melatonin has been used as a supplement in culture medium to improve the efficiency of in vitro produced mammalian embryos. Through its ability to scavenge toxic oxygen derivatives and regulate cellular mRNA levels for antioxidant enzymes, this molecule has been shown to play a protective role against damage by free radicals, to which in vitro cultured embryos are exposed during early development. In vivo and in vitro studies have been performed showing that the use of nanocapsules as active substances carriers increases stability, bioavailability and biodistribution of drugs, such as melatonin, to the cells and tissues, improving their antioxidant properties. These properties can be modulated through the manipulation of formula composition, especially in relation to the supramolecular structures of the nanocapsule core and the surface area that greatly influences drug release mechanisms in biological environments. This study aimed to evaluate the effects of two types of melatonin-loaded nanocapsules with distinct supramolecular structures, polymeric (NC) and lipid-core (LNC) nanocapsules, on in vitro cultured bovine embryos. Embryonic development, apoptosis, reactive oxygen species (ROS) production, and mRNA levels of genes involved in cell apoptosis, ROS and cell pluripotency were evaluated after supplementation of culture medium with non-encapsulated melatonin (Mel), melatonin-loaded polymeric nanocapsules (Mel-NC) and melatonin-loaded lipid-core nanocapsules (Mel-LNC) at 10-6, 10-9, and 10-12 M drug concentrations. The highest hatching rate was observed in embryos treated with 10-9 M Mel-LNC. When compared to Mel and Mel-NC treatments at the same concentration (10-9 M), Mel-LNC increased embryo cell number, decreased cell apoptosis and ROS levels, down-regulated mRNA levels of BAX, CASP3, and SHC1 genes, and up-regulated mRNA levels of CAT and SOD2 genes. These findings indicate that nanoencapsulation with LNC increases the protective effects of melatonin against oxidative stress and cell apoptosis during in vitro embryo culture in bovine species.

Hypertrophic response to hemodynamic overload: role of load vs. renin-angiotensin system activation

NASA Technical Reports Server (NTRS)

Koide, M.; Carabello, B. A.; Conrad, C. C.; Buckley, J. M.; DeFreyte, G.; Barnes, M.; Tomanek, R. J.; Wei, C. C.; Dell'Italia, L. J.; Cooper, G. 4th;

Enhancement of Penicillium echinulatum glycoside hydrolase enzyme complex.

PubMed

dos Santos Costa, Patrícia; Büchli, Fernanda; Robl, Diogo; Delabona, Priscila da Silva; Rabelo, Sarita Candida; Pradella, José Geraldo da Cruz

2016-05-01

The enhancement of enzyme complex produced by Penicillium echinulatum grown in several culture media components (bagasse sugarcane pretreated by various methods, soybean meal, wheat bran, sucrose, and yeast extract) was studied to increment FPase, xylanase, pectinase, and β-glucosidase enzyme activities. The present results indicated that culture media composed with 10 g/L of the various bagasse pretreatment methods did not have any substantial influence with respect to the FPase, xylanase, and β-glucosidase attained maximum values of, respectively, 2.68 FPU/mL, 2.04, and 115.4 IU/mL. On the other hand, proposed culture media to enhance β-glucosidase production composed of 10 g/L steam-exploded bagasse supplemented with soybean flour 5.0 g/L, yeast extract 1.0 g/L, and sucrose 10.0 g/L attained, respectively, 3.19 FPU/mL and 3.06 IU/mL while xylanase was maintained at the same level. The proteomes obtained from the optimized culture media for enhanced FPase, xylanase, pectinase, and β-glucosidase production were analyzed using mass spectrometry and a panel of GH enzyme activities against 16 different substrates. Culture medium designed to enhance β-glucosidase activity achieved higher enzymatic activities values (13 measured activities), compared to the culture media for FPase/pectinase (9 measured activities) and xylanase (7 measured activities), when tested against the 16 substrates. Mass spectrometry analyses of secretome showed a consistent result and the greatest number of spectral counts of Cazy family enzymes was found in designed β-glucosidase culture medium, followed by FPase/pectinase and xylanase. Most of the Cazy identified protein was cellobiohydrolase (GH6 and GH7), endoglucanase (GH5), and endo-1,4-β-xylanase (GH10). Enzymatic hydrolysis of hydrothermally pretreated sugarcane bagasse performed with β-glucosidase enhanced cocktail achieved 51.4 % glucose yield with 10 % w/v insoluble solids at enzyme load of 15 FPU/g material. Collectively the results demonstrated that it was possible to rationally modulate the GH activity of the enzymatic complex secreted by P. echinulatum using adjustment of the culture medium composition. The proposed strategy may contribute to increase enzymatic hydrolysis of lignocellulosic materials.

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 ...

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...

Enhancing of Osseointegration with Propolis-Loaded TiO2 Nanotubes in Rat Mandible for Dental Implants

PubMed Central

Somsanith, Nithideth; Jang, Young-Seok; Lee, Young-Hee; Yi, Ho-Keun; Kim, Kyoung-A; Bae, Tae-Sung; Lee, Min-Ho

2018-01-01

TiO2 nanotubes (TNT) formation is beneficial for improving bone cell–material interaction and drug delivery for Ti dental implants. Among the natural drugs to be installed in TNT, selected propolis has antibacterial and anti-inflammatory properties. It is a resinous natural product which is collected by the honeybees from the various types of plants with their salivary enzymes. This study concludes that TNT loaded with a propolis (PL-TNT-Ti) dental implant has the ability to improve osseointegration. The propolis particles were embedded within the TNT or adhered to the top. In a cytotoxicity test using osteoblast, PL-TNT-Ti group exhibited an increased cell proliferation and differentiation. A Sprague Dawley rat mandibular model was used to evaluate the osseointegration and bone bonding of TNT or PL-TNT-Ti. From the µ-CT and hematoxylin and eosin (HE) histological results after implantation at 1 and 4 weeks to rat mandibular, an increase in the extent of new bone formation and mineral density around the PL-TNT-Ti implant was confirmed. The Masson’s trichrome staining showed the expression of well-formed collagenous for bone formation on the PL-TNT-Ti. Immunohistochemistry staining indicate that bone morphogenetic proteins (BMP-2 and BMP-7) around the PL-TNT-Ti increased the expression of collagen fibers and of osteogenic differentiation whereas the expression of inflammatory cytokine such as interleukin-1 beta (IL-1ß) and tumor necrosis factor-alpha (TNF-α) is decreased. PMID:29301269

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.

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.

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.

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).

Phosphoric acid based pretreatment of switchgrass and fermentation of entire slurry to ethanol using a simplified process.

PubMed

Wu, Wei; Rondon, Vanessa; Weeks, Kalvin; Pullammanappallil, Pratap; Ingram, Lonnie O; Shanmugam, K T

2018-03-01

Switchgrass (Alamo) was pretreated with phosphoric acid (0.75 and 1%, w/w) at three temperatures (160, 175 and 190 °C) and time (5, 7.5 and 10 min) using a steam gun. The slurry after pretreatment was liquefied by enzymes and the released sugars were fermented in a simultaneous saccharification and co-fermentation process to ethanol using ethanologenic Escherichia coli strain SL100. Among the three variables in pretreatment, temperature and time were critical in supporting ethanol titer and yield. Enzyme hydrolysis significantly increased the concentration of furans in slurries, apparently due to release of furans bound to the solids. The highest ethanol titer of 21.2 ± 0.3 g/L ethanol obtained at the pretreatment condition of 190-1-7.5 (temperature-acid concentration-time) and 10% solids loading accounted for 190 ± 2.9 g ethanol/kg of raw switch grass. This converts to 61.7 gallons of ethanol per ton of dry switchgrass, a value that is comparable to other published pretreatment conditions. Copyright © 2017 Elsevier Ltd. All rights reserved.

Recovery and purification of chitosanase produced by Bacillus cereus using expanded bed adsorption and central composite design.

PubMed

de Araújo, Nathália Kelly; Pimentel, Vanessa Carvalho; da Silva, Nayane Macedo Portela; de Araújo Padilha, Carlos Eduardo; de Macedo, Gorete Ribeiro; Dos Santos, Everaldo Silvino

2016-02-01

This study presents a system for expanded bed adsorption for the purification of chitosanase from broth extract in a single step. A chitosanase-producing strain was isolated and identified as Bacillus cereus C-01 and used to produce chitosanases. The expanded bed adsorption conditions for chitosanase purification were optimized statistically using STREAMLINE(TM) DEAE and a homemade column (2.6 × 30.0 cm). Dependent variables were defined by the quality criteria purification factor (P) and enzyme yield to optimize the chromatographic process. Statistical analyses showed that the optimum conditions for the maximum P were 150 cm/h load flow velocity, 6.0 cm settled bed height, and 7.36 cm distributor height. Distributor height had a strong influence on the process, considerably affecting both the P and enzyme yield. Optimizing the purification variables resulted in an approximately 3.66-fold increase in the P compared with the value under nonoptimized conditions. This system is promising for the recovery of chitosanase from B. cereus C-01 and is economically viable because it promotes the reduction steps. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Hydrologic connectivity to streams increases nitrogen and phosphorus inputs and cycling in soils of created and natural floodplain wetlands

USGS Publications Warehouse

Wolf, Kristin L.; Noe, Gregory B.; Ahn, Changwoo

2013-01-01

Greater connectivity to stream surface water may result in greater inputs of allochthonous nutrients that could stimulate internal nitrogen (N) and phosphorus (P) cycling in natural, restored, and created riparian wetlands. This study investigated the effects of hydrologic connectivity to stream water on soil nutrient fluxes in plots (n = 20) located among four created and two natural freshwater wetlands of varying hydrology in the Piedmont physiographic province of Virginia. Surface water was slightly deeper; hydrologic inputs of sediment, sediment-N, and ammonium were greater; and soil net ammonification, N mineralization, and N turnover were greater in plots with stream water classified as their primary water source compared with plots with precipitation or groundwater as their primary water source. Soil water-filled pore space, inputs of nitrate, and soil net nitrification, P mineralization, and denitrification enzyme activity (DEA) were similar among plots. Soil ammonification, N mineralization, and N turnover rates increased with the loading rate of ammonium to the soil surface. Phosphorus mineralization and ammonification also increased with sedimentation and sediment-N loading rate. Nitrification flux and DEA were positively associated in these wetlands. In conclusion, hydrologic connectivity to stream water increased allochthonous inputs that stimulated soil N and P cycling and that likely led to greater retention of sediment and nutrients in created and natural wetlands. Our findings suggest that wetland creation and restoration projects should be designed to allow connectivity with stream water if the goal is to optimize the function of water quality improvement in a watershed.

Development of polymer-biomolecule core-shell particles for biomedical applications

NASA Astrophysics Data System (ADS)

Suthiwangcharoen, Nisaraporn

Developing efficient strategies to introduce biomolecules around polymeric nanoparticles (NPs) is critical for targeted delivery of therapeutic or diagnostic agents. Although polymeric NPs have been well established, problems such as toxicity, stability, and immunoresistance remain potential concerns. The first part of this dissertation focuses on the development of nanosized targeted drug delivery vehicle in cancer chemotherapy. The vehicle was created by the self-assembly of folate-grafted filamentous bacteriophage M13 with poly(caprolactone- b-2-vinylpyridine) while doxorubicin, the antitumor drugs, was successfully loaded in the interior of the vehicles. These particles offer unique properties of being able to selectively target tumor cells while appearing to be safe and non-toxic to normal cells. Although they have shown great prospects in many biomedical applications, less is known about the interactions between biomolecules and polymers. The next part of the dissertation focuses on the self-assembly of proteins and polymers to create polymer-protein core-shell nanoparticles (PPCS-NPs). Several proteins with different isoelectric points and molecular weights were employed to demonstrate a versatility of our assembly method while a series of esterified derivatives of poly(2-hydroxyethyl methacrylate) (pHEMA) were synthesized to evaluate the interaction between proteins and polymers. Our data indicated that the polymers containing pyridine residues can successfully assemble with proteins, and the mechanism is mainly governed by hydrogen bonding and the hydrophobic/hydrophilic interactions. This in turn helps retaining proteins' folding conformation and functionality, which are also demonstrated in the in vitro/in vivo cellular uptake of the PPCS-NPs in endothelial cells. The last part of the dissertation focuses on the self-assembly of the bienzyme-polymer NPs. Glucose oxidase (GOX) together with horseradish peroxidase (HRP) were employed to construct bienzyme-NPs. GOX was initially used as a model enzyme to assemble with pyridine-modified pHEMA, and its enzymatic activity was shown to increase after self-assembly. When both GOX and HRP were assembled with the polymers, the activity was shown to be even greater than that of the single enzyme-polymer NPs or a free enzyme. The results indicated that NPs enhance enzymatic activity, likely due to an increase in enzyme localization, and the assembly between GOX and polymers might prevent conformational transitions of these enzymes.

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

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.

Effect of enzyme concentration, addition of water and incubation time on increase in yield of starch from potato.

PubMed

Sit, Nandan; Agrawal, U S; Deka, Sankar C

2014-05-01

Enzymatic treatment process for starch extraction from potato was investigated using cellulase enzyme and compared with conventional process. The effects of three parameters, cellulase enzyme concentration, incubation time and addition of water were evaluated for increase in starch yield as compared to the conventional process i.e., without using enzyme. A two-level full factorial design was used to study the process. The results indicated that all the main parameters and their interactions are statistically significant. Enzyme concentration and incubation time had a positive effect on the increase in starch yield while addition of water had a negative effect. The increase in starch yield ranged from 1.9% at low enzyme concentration and incubation time and high addition of water to a maximum of 70% increase from conventional process in starch yield was achieved when enzyme concentration and incubation time were high and addition of water was low suggesting water present in the ground potato meal is sufficient for access to the enzyme with in the slurry ensuring adequate contact with the substrate.

Effects of space environment on biological characters of cultured rose seedlings

NASA Astrophysics Data System (ADS)

Min, L.; Huai, X.; Jinying, L.; Yi, P.; Chunhua, Z.

Cultured rose seedlings were carried into space by SHENZHOU-4 spacecraft and then used as the experimental material to investigate effects of the space environmental conditions on morphology cytology physiology and molecular biology of the seedlings After loaded on the space flight the plant s height number of leaves and fresh weight per seedling were all increased significantly compared to the ground controls The content of chlorophyll was basically unchanged In some cells the ultrastructural changes involved twist contraction and deformation of cell wall curvature and loose arrangement of lamellae of some chloroplasts and a significant increase in number of starch grains per chloroplast In addition the number of mitochondria increased but some mitochondrial outer membrane broke and some mitochondrial cristae disappeared The activities of the defense enzymes such as superoxide dismutase peroxidase and catalyse in rose leaves increased and the content of malondialdehyde decreased In the RAPD analysis with 40 10-mer primers 36 primers generated 148 DNA bands from both of the space flight treated seedlings and the ground controls and five primers amplified polymorphic products The rate of DNA variation was 6 34

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

Soil microbial communities and enzyme activities under various poultry litter application rates.

PubMed

Acosta-Martínez, V; Harmel, R Daren

2006-01-01

The potential excessive nutrient and/or microbial loading from mismanaged land application of organic fertilizers is forcing changes in animal waste management. Currently, it is not clear to what extent different rates of poultry litter impact soil microbial communities, which control nutrient availability, organic matter quality and quantity, and soil degradation potential. From 2002 to 2004, we investigated the microbial community and several enzyme activities in a Vertisol soil (fine, smectitic, thermic, Udic Haplustert) at 0 to 15 cm as affected by different rates of poultry litter application to pasture (0, 6.7, and 13.4 Mg ha(-1)) and cultivated sites (0, 4.5, 6.7, 9.0, 11.2, and 13.4 Mg ha(-1)) in Texas, USA. No differences in soil pH (average: 7.9), total N (pasture: 2.01-3.53, cultivated: 1.09-1.98 g kg(-1) soil) or organic C (pasture average: 25-26.7, cultivated average: 13.9-16.1 g kg(-1) soil) were observed following the first four years of litter application. Microbial biomass carbon (MBC) and nitrogen (MBN) increased at litter rates greater than 6.7 Mg ha(-1) (pasture: MBC = >863, MBN = >88 mg kg(-1) soil) compared to sites with no applied litter (MBC = 722, MBN = 69 mg kg(-1) soil). Enzyme activities of C (beta-glucosidase, alpha-galactosidase, beta-glucosaminidase) or N cycling (beta-glucosaminidase) were increased at litter rates greater than 6.7 Mg ha(-1). Enzyme activities of P (alkaline phosphatase) and S (arylsulfatase) mineralization showed the same response in pasture, but they were only increased at the highest (9.0, 11.2, and 13.4 Mg ha(-1)) litter application rates in cultivated sites. According to fatty acid methyl ester (FAME) analysis, the pasture soils experienced shifts to higher bacterial populations at litter rates of 6.7 Mg ha(-1), and shifts to higher fungal populations at the highest litter application rates in cultivated sites. While rates greater than 6.7 Mg ha(-1) provided rapid enhancement of the soil microbial populations and enzymatic activities, they result in P application in excess of crop needs. Thus, studies will continue to investigate whether litter application at rates below 6.7 Mg ha(-1), previously recommended to maintain water quality, will result in similar improved soil microbial and biochemical functioning with continued annual litter application.

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.

Microbial responses to multi-factor climate change: effects on soil enzymes.

PubMed

Steinweg, J Megan; Dukes, Jeffrey S; Paul, Eldor A; Wallenstein, Matthew D

2013-01-01

The activities of extracellular enzymes, the proximate agents of decomposition in soils, are known to depend strongly on temperature, but less is known about how they respond to changes in precipitation patterns, and the interaction of these two components of climate change. Both enzyme production and turnover can be affected by changes in temperature and soil moisture, thus it is difficult to predict how enzyme pool size may respond to altered climate. Soils from the Boston-Area Climate Experiment (BACE), which is located in an old field (on abandoned farmland), were used to examine how climate variables affect enzyme activities and microbial biomass carbon (MBC) in different seasons and in soils exposed to a combination of three levels of precipitation treatments (ambient, 150% of ambient during growing season, and 50% of ambient year-round) and four levels of warming treatments (unwarmed to ~4°C above ambient) over the course of a year. Warming, precipitation and season had very little effect on potential enzyme activity. Most models assume that enzyme dynamics follow microbial biomass, because enzyme production should be directly controlled by the size and activity of microbial biomass. We observed differences among seasons and treatments in mass-specific potential enzyme activity, suggesting that this assumption is invalid. In June 2009, mass-specific potential enzyme activity, using chloroform fumigation-extraction MBC, increased with temperature, peaking under medium warming and then declining under the highest warming. This finding suggests that either enzyme production increased with temperature or turnover rates decreased. Increased maintenance costs associated with warming may have resulted in increased mass-specific enzyme activities due to increased nutrient demand. Our research suggests that allocation of resources to enzyme production could be affected by climate-induced changes in microbial efficiency and maintenance costs.

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.

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.

The efficacy of using exogenous enzymes cocktail on production, egg quality, egg nutrients and blood metabolites of laying hens fed distiller's dried grains with solubles.

PubMed

Abd El-Hack, M E; Chaudhry, M T; Mahrose, K M; Noreldin, A; Emam, M; Alagawany, M

2018-04-01

An experiment was performed using 120 Hisex Brown laying hens for evaluating the effects of different inclusion levels of corn distiller's dried grains with solubles (DDGS) as a replacement of soybean meal (SBM) with or without enzyme cocktail on performance, egg quality, egg nutrients and blood metabolites in laying hens through 22-42 weeks of age. A 4 × 2 factorial design experiment was performed including four substitution levels of DDGS (0, 250, 500 and 750 g/kg respectively) and two enzyme cocktail levels (0 and 250 mg/kg diet). The used enzyme in this study "Gallazyme" composed of xylanase, Trichoderma longibrachiatum (600 units/g), protease, Bacillus subtilis (8,000 units/g) and amylase and Bacillus amyloliquofaciens (800 units/g). The control diet showed the best feed efficiency followed by the intermediate levels of DDGS. The lowest value of feed efficiency was found in the group fed the highest level of DDGS. Enzyme addition improved feed efficiency and decreased laying rate. Increasing DDGS levels was associated with albumin and shell thickness increases. Dietary DDGS depressed all egg components except the organic matter which maximised in enzyme-treated groups. Increasing DDGS level was accompanied by increase in yolk cholesterol and total lipids. No significant impacts were detected with enzymes supplementation on yolk lipids profile. Excepting serum calcium and phosphorous, all serum constituents increased with increasing level of DDGS. Using enzyme markedly depressed serum ammonia by 15.02% and increased calcium by 6.44% compared with enzyme-free diets. Interaction between DDGS and enzyme was significant on most of studied parameters. It could be concluded that using enzyme cocktail in DDGS-based diets may improve feed efficiency and egg quality, in addition to lowering blood ammonia and increasing blood calcium. It is recommended to substitute SBM by DDGS up to 500 g/kg diet. © 2017 Blackwell Verlag GmbH.

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

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...

Exploring the Potential Role of Chemopreventive Agent, Hesperetin Conjugated Pegylated Gold Nanoparticles in Diethylnitrosamine-Induced Hepatocellular Carcinoma in Male Wistar Albino Rats.

PubMed

Gokuladhas, Krishnan; Jayakumar, Subramaniyan; Rajan, Balan; Elamaran, Ramasamy; Pramila, Chengalvarayan Subramani; Gopikrishnan, Mani; Tamilarasi, Sasivarman; Devaki, Thiruvengadam

2016-04-01

Liver cancer is the fifth most common cancer and is still one of the leading causes of death world wide, due to food additives, alcohol, fungal toxins, air, toxic industrial chemicals, and water pollutants. Chemopreventive drugs play a potential role in liver cancer treatment. Obviously in the production of anticancer drugs, the factors like poor solubility, bioavailability, biocompatibility, limited chemical stability, large amount of dose etc., plays a major role. Against this backdrop, the idea of designing the chemopreventive nature of bio flavanoid hesperetin (HP) drug conjugated with pegylated gold nanoparticles to increasing the solubility, improve bioavailability and enhance the targeting capabilities of the drug during diethylnitrosamine (DEN) induced liver cancer in male wistar albino rats. The dose fixation studies and the toxicity of pure HP and HP conjugated gold nanoparticles (Au-mPEG(5000)-S-HP) were analysed. After concluded the dose fixation and toxicity studies the experimental design were segregated in six groups for the anticancer analysis of DEN induced HCC for 16 weeks. After the experimental period the body weight, relative liver weight, number of nodules and size of nodules, the levels of tumor markers like CEA, AFP and the level of lipid peroxidation, lipid hydroperoxides and the activities of antioxidant enzymes were assessed. The administration of DEN to rats resulted in increased relative liver weight and serum marker enzymes aspartate transaminase, alanine transaminase, alkaline phosphatase, lactate dehydrogenase, and gamma glutamyl transpeptidase. The levels of lipid peroxides elevated (in both serum and tissue) with subsequent decrease in the final body weight and tissue antioxidants like superoxide dismutase, catalase, reduced glutathione, glutathione peroxidise, and glutathione reductase. HP supplementation (20 mg/kg b.wt) significantly attenuated these alterations, thereby showing potent anticancer effect in liver cancer and the HP loaded gold nanoparticels (Au-mPEG(5000)-S-HP) treated animals shows the better treatment than the pure HP due to the solubility of drug, bioavailability and the target drug delivery of the biodegradable polymer. Histological observations were also carried out, which added supports to the chemopreventive action of the pure HP and HP loaded gold nanoparticles (Au-mPEG(5000)-S-HP) against DEN induction during liver cancer progression. These findings suggest that HP loaded gold nanoparticels (Au-mPEG(5000)-S-HP) shows better efficacy than the pure HP against lipid peroxidation, hepatic cell damage and protects the antioxidant system in DEN induced hepatocellular carcinogenesis.

Effect of low frequency, low amplitude magnetic fields on the permeability of cationic liposomes entrapping carbonic anhydrase: II. No evidence for surface enzyme involvement.

PubMed

Ramundo-Orlando, A; Mattia, F; Palombo, A; D'Inzeo, G

2000-10-01

Observations recently reported by our group indicate that combined 7 Hz sinusoidal (B(acpeak) = 50 mu T) and parallel static (B(dc) = 50 mu T) magnetic fields can induce a significant increase in diffusion rate of substrate across carbonic anhydrase (CA)-loaded liposomes (DPPC:Chol:SA). A direct involvement of charges of stearylamine (SA) on the lipid membrane surface was also demonstrated. Kinetic studies showed that CA was mainly entrapped in liposomes at 5:3:2 molar ratio, although a small amount (17%) of enzyme was also located on the external surface of these cationic liposomes. In this paper we report steady state kinetic studies on this latter CA after ELF-EMFs exposure. No difference in the apparent K(m) between exposed and sham samples was observed. On the contrary the apparent V(max) was increased by approximately a factor of 2 after field exposure. In spite of the proteolytic digestion of this external CA, a significant increase of enzymatic activity, as a function of increase in the diffusion rate of substrate across the lipid bilayer, was observed in the exposed samples. Based on these results, a conformational change induced by the field on the CA located on the external surface of 5:3:2 liposomes is excluded as an explanation for our previous observations, supporting the primary role of bilayer SA in the interaction with ELF. A model of ELF interaction, based on the Larmor precession theory, explaining the physical phenomenon induced on the dipole of SA has been developed. Copyright 2000 Wiley-Liss, Inc.

Investigation of Pleurotus ostreatus pretreatment on switchgrass for ethanol production

NASA Astrophysics Data System (ADS)

Slavens, Shelyn Gehle

Fungal pretreatment using the white-rot fungus Pleurotus ostreatus on switchgrass for ethanol production was studied. In a small-scale storage study, small switchgrass bales were inoculated with fungal spawn and automatically watered to maintain moisture. Sampled at 25, 53, and 81 d, the switchgrass composition was determined and liquid hot water (LHW) pretreatment was conducted. Fungal pretreatment significantly decreased the xylan and lignin content; glucan was not significantly affected by fungal loading. The glucan, xylan, and lignin contents significantly decreased with increased fungal pretreatment time. The effects of the fungal pretreatment were not highly evident after the LHW pretreatment, showing only changes based on sampling time. Although other biological activity within the bales increased cellulose degradation, the fungal pretreatment successfully reduced the switchgrass lignin and hemicellulose contents. In a laboratory-scale nutrient supplementation study, copper, manganese, glucose, or water was added to switchgrass to induce production of ligninolytic enzymes by P. ostreatus. After 40 d, ligninolytic enzyme activities and biomass composition were determined and simultaneous saccharification and fermentation (SSF) was conducted to determine ethanol yield. Laccase activity was similar for all supplements and manganese peroxidase (MnP) activity was significantly less in copper-treated samples than in the other fungal-inoculated samples. The fungal pretreatment reduced glucan, xylan, and lignin content, while increasing extractable sugars content. The lowest lignin contents occurred in the water-fungal treated samples and produced the greatest ethanol yields. The greatest lignin contents occurred in the copper-fungal treated samples and produced the lowest ethanol yields. Manganese-fungal and glucose-fungal treated samples had similar, intermediate lignin contents and produced similar, intermediate ethanol yields. Ethanol yields from switchgrass were increased significantly by fungal pretreatment.

Improving functional properties of pea protein isolate for microencapsulation of flaxseed oil.

PubMed

Bajaj, Poonam R; Bhunia, Kanishka; Kleiner, Leslie; Joyner Melito, Helen S; Smith, Denise; Ganjyal, Girish; Sablani, Shyam S

2017-03-01

Unhydrolysed pea protein (UN) forms very viscous emulsions when used at higher concentrations. To overcome this, UN was hydrolysed using enzymes alcalase, flavourzyme, neutrase, alcalase-flavourzyme, and neutrase-flavourzyme at 50 °C for 0 min, 30 min, 60 min, and 120 min to form hydrolysed proteins A, F, N, AF, and NF, respectively. All hydrolysed proteins had lower apparent viscosity and higher solubility than UN. Foaming capacity of A was the highest, followed by NF, N, and AF. Hydrolysed proteins N60, A60, NF60, and AF60 were prepared by hydrolysing UN for 60 min and used further for microencapsulation. At 20% oil loading (on a total solid basis), the encapsulated powder N60 had the highest microencapsulation efficiency (ME = 56.2). A decrease in ME occurred as oil loading increased to 40%. To improve the ME of N60, >90%, UN and maltodextrin were added. Flowability and particle size distribution of microencapsulated powders with >90% microencapsulation efficiency and morphology of all powders were investigated. This study identified a new way to improve pea protein functionality in emulsions, as well as a new application of hydrolysed pea protein as wall material for microencapsulation.

Insolubilization process increases enzyme stability

NASA Technical Reports Server (NTRS)

Billingham, J.; Lyn, J.

1971-01-01

Enzymes complexed with polymeric matrices contain properties suggesting application to enzyme-controlled reactions. Stability of insolubilized enzyme derivatives is markedly greater than that of soluble enzymes and physical form of insolubilized enzymes is useful in column and batch processes.

Metabolic plasticity and the energy economizing effect of ibogaine, the principal alkaloid of Tabernanthe iboga.

PubMed

Paškulin, Roman; Jamnik, Polona; Danevčič, Tjaša; Koželj, Gordana; Krašovec, Rok; Krstić-Milošević, Dijana; Blagojević, Duško; Strukelj, Borut

2012-08-30

The root bark of iboga plant-Tabernanthe iboga has been used traditionally in Central Africa as a psychoactive substance in religious rituals, while in smaller doses it is appreciated due to its stimulant properties. The iboga root bark, iboga extract or pure ibogaine are being recognized in the West as an anti-addiction remedy and their use is increasing. Our previous studies have demonstrated a transient ATP pool reduction under ibogaine accompanied by the induction of energy metabolism related enzymes. The present study aimed to find the cause of this energy deprivation and to foresee its immediate and long-term impact on metabolism. The overall project is designed to disclose the common mechanism of action at these seemingly diverse indications for iboga use, to predict eventual adverse effects and to build the grounds for its safe and beneficial utilization. The rate of carbon dioxide (CO(2)) as a marker of energy metabolism in stationary yeast model under aerobic conditions in the presence of ibogaine at concentration of 1, 4 and 20mg/l was measured for 5h by gas chromatography. The overall oxidative load was determined fluorimetrically by 2',7'-dichlorofluorescein diacetate (H(2)DCFDA) and in vitro antioxidant properties of ibogaine were defined by 1,1-diphenyl-2-picrylhydrazyl (DPPH) test. The CO(2) production under ibogaine was temporarily increased in a dose dependent manner. The increased energy consumption as an early effect of ibogaine was proven by the fact that in spite of energy mobilization, the ATP pool has been simultaneously decreased. Although increased cellular respiration co-produces reactive oxygen species (ROS), the overall oxidative load was significantly lowered by ibogaine. Since ibogaine does not show any significant in vitro antioxidant properties, the results indicate its stimulating influence on physiological oxidative stress defence system. Ibogaine triggers remodeling of the housekeeping metabolism. Under the initial energy cost it results in increased efficacy of physiological antioxidative systems, which reduce oxidative damage and lowers basal metabolic needs. Together with induced catabolic enzymes they set a new metabolic equilibrium that saves energy and makes it easily available in case of extra needs. While healthy organism profits from improved fitness and mental performance and can withstand higher stress without risking a disease, due to the same principle ibogaine provides beneficial support at the recovery after diseases including addiction syndrome. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Opposite effects of capacity load and resolution load on distractor processing.

PubMed

Zhang, Weiwei; Luck, Steven J

2015-02-01

According to the load theory of attention, an increased perceptual load reduces distractor processing whereas an increased working memory load facilitates distractor processing. Here we raise the possibility that the critical distinction may instead be between an emphasis on resolution and an emphasis on capacity. That is, perceptual load manipulations typically emphasize resolution (fine-grained discriminations), whereas working memory load manipulations typically emphasize capacity (simultaneous processing of multiple relevant stimuli). To test the plausibility of this hypothesis, we used a visual working memory task that emphasized either the number of items to be stored (capacity load, retaining 2 vs. 4 colors) or the precision of the representations (resolution load, detecting small vs. large color changes). We found that an increased capacity load led to increased flanker interference (a measure of distractor processing), whereas an increased resolution load led to reduced flanker interference. These opposite effects of capacity load and resolution load on distractor processing mirror the previously described opposite effects of perceptual load and working memory load.

Opposite Effects of Capacity Load and Resolution Load on Distractor Processing

PubMed Central

Zhang, Weiwei; Luck, Steven J.

2014-01-01

According to the load theory of attention, an increased perceptual load reduces distractor processing whereas an increased working memory load facilitates distractor processing. Here we raise the possibility that the critical distinction may instead be between an emphasis on resolution and an emphasis on capacity. That is, perceptual load manipulations typically emphasize resolution (fine-grained discriminations), whereas working memory load manipulations typically emphasize capacity (simultaneous processing of multiple relevant stimuli). To test the plausibility of this hypothesis, we used a visual working memory task that emphasized either the number of items to be stored (capacity load, retaining two versus four colors) or the precision of the representations (resolution load, detecting small versus large color changes). We found that an increased capacity load led to increased flanker interference (a measure of distractor processing), whereas an increased resolution load led to reduced flanker interference. These opposite effects of capacity load and resolution load on distractor processing mirror the previously described opposite effects of perceptual load and working memory load. PMID:25365573

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.

Menisci of the rabbit knee require mechanical loading to maintain homeostasis: cyclic hydrostatic compression in vitro prevents derepression of catabolic genes.

PubMed

Natsu-Ume, Takashi; Majima, Tokifumi; Reno, Carol; Shrive, Nigel G; Frank, Cyril B; Hart, David A

2005-07-01

The purpose of this study was to examine the influence of removing menisci from their in vivo loading environment on gene expression patterns and to determine whether in vitro loading can maintain the tissues in their in vivo phenotype. Lateral and medial rabbit meniscal explants from one leg were cultured in vitro and subjected to intermittent cyclic hydrostatic pressure (CHP) of 1 MPa at 0.5 Hz for 1 min and a rest period of 14 min (4 h of culture). The contralateral menisci were incubated at atmospheric pressure for 4 h. Menisci from both legs of another set of rabbits were frozen immediately to yield time zero values reflective of in vivo mRNA levels. Total RNA was isolated from all groups and processed for reverse transcription-polymerase chain reaction analysis for a subset of relevant genes (matrix molecules, cytokines, proteinases and inhibitors, enzymes). It was found that mRNA levels for MMP-1, MMP-3, TIMPs, iNOS, COX-2, interleukin-1beta in both menisci, and interleukin-6 in medial menisci were significantly elevated in tissues cultured under nonloading conditions compared to the time zero controls. Subjecting menisci to CHP significantly prevented these increases in mRNA levels for nearly all of the indicated molecules. In contrast, there were no significant differences in mRNA levels for collagens, biglycan, MMP-13, or TIMP-4 between the time zero values and those cultured under either nonloading or loading conditions. These studies demonstrate that removing rabbit menisci from their normal in vivo mechanical environment leads to an apparent up-regulation of a subset of potent effector molecules that could mediate catabolic activities, and that in vitro CHP can largely prevent this apparent up-regulation.

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

Aβ-degrading enzymes: potential for treatment of Alzheimer disease.

PubMed

Miners, James Scott; Barua, Neil; Kehoe, Patrick Gavin; Gill, Steven; Love, Seth

2011-11-01

There is increasing evidence that deficient clearance of β-amyloid (Aβ) contributes to its accumulation in late-onset Alzheimer disease (AD). Several Aβ-degrading enzymes, including neprilysin (NEP), insulin-degrading enzyme, and endothelin-converting enzyme reduce Aβ levels and protect against cognitive impairment in mouse models of AD. The activity of several Aβ-degrading enzymes rises with age and increases still further in AD, perhaps as a physiological response to minimize the buildup of Aβ. The age- and disease-related changes in expression of more recently recognized Aβ-degrading enzymes (e.g. NEP-2 and cathepsin B) remain to be investigated, and there is strong evidence that reduced NEP activity contributes to the development of cerebral amyloid angiopathy. Regardless of the role of Aβ-degrading enzymes in the development of AD, experimental data indicate that increasing the activity of these enzymes (NEP in particular) has therapeutic potential in AD, although targeting their delivery to the brain remains a major challenge. The most promising current approaches include the peripheral administration of agents that enhance the activity of Aβ-degrading enzymes and the direct intracerebral delivery of NEP by convection-enhanced delivery. In the longer term, genetic approaches to increasing the intracerebral expression of NEP or other Aβ-degrading enzymes may offer advantages.

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.

Antioxidant functionalized polymer capsules to prevent oxidative stress.

PubMed

Larrañaga, Aitor; Isa, Isma Liza Mohd; Patil, Vaibhav; Thamboo, Sagana; Lomora, Mihai; Fernández-Yague, Marc A; Sarasua, Jose-Ramon; Palivan, Cornelia G; Pandit, Abhay

2018-02-01

Polymeric capsules exhibit significant potential for therapeutic applications as microreactors, where the bio-chemical reactions of interest are efficiently performed in a spatial and time defined manner due to the encapsulation of an active biomolecule (e.g., enzyme) and control over the transfer of reagents and products through the capsular membrane. In this work, catalase loaded polymer capsules functionalized with an external layer of tannic acid (TA) are fabricated via a layer-by-layer approach using calcium carbonate as a sacrificial template. The capsules functionalised with TA exhibit a higher scavenging capacity for hydrogen peroxide and hydroxyl radicals, suggesting that the external layer of TA shows intrinsic antioxidant properties, and represents a valid strategy to increase the overall antioxidant potential of the developed capsules. Additionally, the hydrogen peroxide scavenging capacity of the capsules is enhanced in the presence of the encapsulated catalase. The capsules prevent oxidative stress in an in vitro inflammation model of degenerative disc disease. Moreover, the expression of matrix metalloproteinase-3 (MMP-3), and disintegrin and metalloproteinase with thrombospondin motif-5 (ADAMTS-5), which represents the major proteolytic enzymes in intervertebral disc, are attenuated in the presence of the polymer capsules. This platform technology exhibits potential to reduce oxidative stress, a key modulator in the pathology of a broad range of inflammatory diseases. Oxidative stress damages important cell structures leading to cellular apoptosis and senescence, for numerous disease pathologies including cancer, neurodegeneration or osteoarthritis. Thus, the development of biomaterials-based systems to control oxidative stress has gained an increasing interest. Herein, polymer capsules loaded with catalase and functionalized with an external layer of tannic acid are fabricated, which can efficiently scavenge important reactive oxygen species (i.e., hydroxyl radicals and hydrogen peroxide) and modulate extracellular matrix activity in an in vitro inflammation model of nucleus pulposus. The present work represents accordingly, an important advance in the development and application of polymer capsules with antioxidant properties for the treatment of oxidative stress, which is applicable for multiple inflammatory disease targets. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Chestnut astringent skin extract, an alpha-amylase inhibitor, retards carbohydrate absorption in rats and humans.

PubMed

Tsujita, Takahiro; Takaku, Takeshi; Suzuki, Tsuneo

2008-02-01

Inhibitors of carbohydrate-hydrolyzing enzyme play an important role to control postprandial blood glucose levels. In this paper, we investigated the effect of an ethanol extract from chestnut astringent skin (CAS) on alpha-amylase. Chestnut astringent skin extract strongly inhibited human and porcine pancreatic alpha-amylase. We also investigated the effect of CAS extract on carbohydrate absorption in rats and humans. Oral administration of CAS extract to normal rats fed corn starch (2 g/kg body weight), significantly suppressed the increase of blood glucose levels after starch loading in a dose-dependent manner. The effective dose of CAS extract required to achieve 20 and 40% suppression of the rise in blood glucose level was estimated to be 40 and 155 mg/kg body weight, respectively. Chestnut astringent skin extract also suppressed the rise in plasma insulin level and the fall in plasma non-esterified fatty acid level. In the type 2 diabetic rat model, CAS extract significantly suppressed the rise in blood glucose level after starch loading in a dose-dependent manner. Chestnut astringent skin extract also suppressed the rise in plasma glucose level after boiled rice loading in a dose-dependent manner in humans. The amount of CAS extract required to achieve 11 and 23% suppression in the rise in plasma glucose level was 300 and 600 mg/person, respectively. These results suggest that CAS extract retards absorption of carbohydrate and reduces post-prandial hyperglycemia.

Stimulation by Erwinia carotovora of the synthesis of ethylene in cauliflower tissue

PubMed Central

Lund, Barbara M.; Mapson, L. W.

1970-01-01

The synthesis of ethylene by cauliflower floret tissue was increased when the tissue was inoculated with the soft-rot bacterium Erwinia carotovora. This effect was clearly associated with the production of pectic enzymes by the micro-organism. These enzymes, acting together with the plant enzymes, stimulated the production of ethylene from methionine. The increased synthetic activity was due to the release and increased activity of a glucose oxidase enzyme apparently attached to plant cell-wall material and liberated by the action of pectic enzymes of the bacterium. ImagesPLATE 1 PMID:5488914

Formation and oral administration of alginate microspheres loaded with pDNA coding for lymphocystis disease virus (LCDV) to Japanese flounder.

PubMed

Tian, Ji-Yuan; Sun, Xiu-Qin; Chen, Xi-Guang

2008-05-01

Oral delivery of plasmid DNA (pDNA) is a desirable approach for fish immunization in intensive culture. However, its effectiveness is limited because of possible degradation of pDNA in the fish's digestive system. In this report, alginate microspheres loaded with pDNA coding for fish lymphocystis disease virus (LCDV) and green fluorescent protein were prepared with a modified oil containing water (W/O) emulsification method. Yield, loading percent and encapsulation efficiency of alginate microspheres were 90.5%, 1.8% and 92.7%, respectively. The alginate microspheres had diameters of less than 10 microm, and their shape was spherical. As compared to sodium alginate, a remarkable increase of DNA-phosphodiester and DNA-phosphomonoester bonds was observed for alginate microspheres loaded with pDNA by Fourier transform infrared (FTIR) spectroscopic analysis. Agarose gel electrophoresis showed a little supercoiled pDNA was transformed to open circular and linear pDNA during encapsulation. The cumulative release of pDNA in alginate microspheres was or=0.3) for anti-LCDV antibody from week 3 to week 16 for fish orally vaccinated with alginate microspheres loaded with pDNA, in comparison with fish orally vaccinated with naked pDNA. Our results display that alginate microspheres obtained by W/O emulsification are promising carriers for oral delivery of pDNA. This encapsulation technique has the potential for DNA vaccine delivery applications due to its ease of operation, low cost and significant immune effect.

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.

Production of tannase by the immobilized cells of Bacillus licheniformis KBR6 in Ca-alginate beads.

PubMed

Mohapatra, P K D; Mondal, K C; Pati, B R

2007-06-01

The present study was aimed at finding the optimal conditions for immobilization of Bacillus licheniformis KBR6 cells in calcium-alginate (Ca-alginate) beads and determining the operational stability during the production of tannin-acyl-hydrolase (tannase) under semicontinous cultivation. The active cells of B. licheniformis KBR6 were immobilized in Ca-alginate and used for the production of tannase. The influence of alginate concentration (5, 10, 20 and 30 g l(-1)) and initial cell loading on enzyme production were studied. The production of tannase increased significantly with increasing alginate concentration and reached a maximum enzyme yield of 0.56 +/- 0.03 U ml(-1) at 20 g l(-1). This was about 1.70-fold higher than that obtained by free cells. The immobilized cells produced tannase consistently over 13 repeated cycles and reached a maximum level at the third cycle. Scanning electron microscope study indicated that the cells in Ca-alginate beads remain in normal shape. The Ca-alginate entrapment is a promising immobilization method of B. licheniformis KBR6 for repeated tannase production. Tannase production by immobilized cells is superior to that of free cells because it leads to higher volumetric activities within the same period of fermentation. This is the first report of tannase production from immobilized bacterial cells. The bacterium under study can produce higher amounts of tannase with respect to other fungal strains within a short cultivation period.

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

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.

Enzymatic synthesis of 6-O-glucosyl-poly(3-hydroxyalkanoate) in organic solvents and their binary mixture.

PubMed

Gumel, A M; Annuar, M S M; Heidelberg, T

2013-04-01

The effects of organic solvents and their binary mixture in the glucose functionalization of bacterial poly-3-hydroxyalkanoates catalyzed by Lecitase™ Ultra were studied. Equal volume binary mixture of DMSO and chloroform with moderate polarity was more effective for the enzyme catalyzed synthesis of the carbohydrate polymer at ≈38.2 (±0.8)% reactant conversion as compared to the mono-phasic and other binary solvents studied. The apparent reaction rate constant as a function of medium water activity (aw) was observed to increase with increasing solvent polarity, with optimum aw of 0.2, 0.4 and 0.7 (±0.1) observed in hydrophilic DMSO, binary mixture DMSO:isooctane and hydrophobic isooctane, respectively. Molecular sieve loading between 13 to 15gL(-1) (±0.2) and reaction temperature between 40 to 50°C were found optimal. Functionalized PHA polymer showed potential characteristics and biodegradability. Copyright © 2012 Elsevier B.V. All rights reserved.

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.

Abnormal Mechanical Loading Induces Cartilage Degeneration by Accelerating Meniscus Hypertrophy and Mineralization After ACL Injuries In Vivo.

PubMed

Du, Guoqing; Zhan, Hongsheng; Ding, Daofang; Wang, Shaowei; Wei, Xiaochun; Wei, Fangyuan; Zhang, Jianzhong; Bilgen, Bahar; Reginato, Anthony M; Fleming, Braden C; Deng, Jin; Wei, Lei

2016-03-01

Although patients with an anterior cruciate ligament (ACL) injury have a high risk of developing posttraumatic osteoarthritis (PTOA), the role of meniscus hypertrophy and mineralization in PTOA after an ACL injury remains unknown. The purpose of this study was to determine if menisci respond to abnormal loading and if an ACL injury results in meniscus hypertrophy and calcification. The hypotheses were that (1) abnormal mechanical loading after an ACL injury induces meniscus hypertrophy and mineralization, which correlates to articular cartilage damage in vivo, and (2) abnormal mechanical loading on bovine meniscus explants induces the overexpression of hypertrophic and mineralization markers in vitro. Controlled laboratory study. In vivo guinea pig study (hypothesis 1): Three-month-old male Hartley guinea pigs (n = 9) underwent ACL transection (ACLT) on the right knee; the left knee served as the control. Calcification in the menisci was evaluated by calcein labeling 1 and 5 days before knee harvesting at 5.5 months. Cartilage and meniscus damage and mineralization were quantified by the Osteoarthritis Research Society International score and meniscus grade, respectively. Indian hedgehog (Ihh), matrix metalloproteinase-13 (MMP-13), collagen type X (Col X), progressive ankylosis homolog (ANKH), ectonucleotide pyrophosphatase/phosphodiesterase-1 (ENPP1), alkaline phosphatase (ALP), inorganic pyrophosphate (PPi), and inorganic phosphate (Pi) concentrations were evaluated by immunohistochemistry and enzyme-linked immunosorbent assay. In vitro bovine meniscus explant study (hypothesis 2): Bovine meniscus explants were subjected to 25% strain at 0.3 Hz for 1, 2, and 3 hours. Cell viability was determined using live/dead staining. The levels of mRNA expression and protein levels were measured using real-time quantitative reverse transcription polymerase chain reaction and Western blot after 24, 48, and 72 hours in culture. The conditioned medium was collected for sulfated glycosaminoglycan (GAG) release and Pi/PPi assay. In vivo guinea pig study: Meniscus size and area as well as intensity of meniscus calcification were significantly increased in the ACLT group compared with the control group. Both calcified area and intensity were correlated with cartilage damage in the ACLT group (meniscus calcified area: r = 0.925, P < .0001; meniscus calcified intensity: r = 0.944, P < .0001). Ihh, MMP-13, Col X, ANKH, ENPP1, and ALP expression were increased in the ACLT group compared with the control group. The Pi level and Pi/PPi ratio increased by 63% and 42%, respectively, in the ACLT group compared with the control group. In vitro bovine meniscus explant study: Cell death was found in the superficial zone of the bovine meniscus explants after loading for 3 hours. The mRNA expression and protein levels of MMP-13, ANKH, ENPP1, and ALP were up-regulated in all 3-hour loaded samples. The Pi/PPi ratio and sulfated GAG content in the culture medium were increased in the 3-hour loaded group. Meniscus hypertrophy and mineralization correlated to cartilage degeneration after ACL injuries. The study data suggest that the suppression of meniscus hypertrophy and calcification may decrease the risk of PTOA after ACL injuries. © 2016 The Author(s).

Abnormal Mechanical Loading Induces Cartilage Degeneration by Accelerating Meniscus Hypertrophy and Mineralization After ACL Injuries In Vivo

PubMed Central

Du, Guoqing; Zhan, Hongsheng; Ding, Daofang; Wang, Shaowei; Wei, Xiaochun; Wei, Fangyuan; Zhang, Jianzhong; Bilgen, Bahar; Reginato, Anthony M.; Fleming, Braden C.; Deng, Jin; Wei, Lei

2016-01-01

Background Although patients with an anterior cruciate ligament (ACL) injury have a high risk of developing posttraumatic osteoarthritis (PTOA), the role of meniscus hypertrophy and mineralization in PTOA after an ACL injury remains unknown. Purpose/Hypothesis The purpose of this study was to determine if menisci respond to abnormal loading and if an ACL injury results in meniscus hypertrophy and calcification. The hypotheses were that (1) abnormal mechanical loading after an ACL injury induces meniscus hypertrophy and mineralization, which correlates to articular cartilage damage in vivo, and (2) abnormal mechanical loading on bovine meniscus explants induces the overexpression of hypertrophic and mineralization markers in vitro. Study Design Controlled laboratory study. Methods In vivo guinea pig study (hypothesis 1): Three-month-old male Hartley guinea pigs (n = 9) underwent ACL transection (ACLT) on the right knee; the left knee served as the control. Calcification in the menisci was evaluated by calcein labeling 1 and 5 days before knee harvesting at 5.5 months. Cartilage and meniscus damage and mineralization were quantified by the Osteoarthritis Research Society International score and meniscus grade, respectively. Indian hedgehog (Ihh), matrix metalloproteinase–13 (MMP-13), collagen type X (Col X), progressive ankylosis homolog (ANKH), ectonucleotide pyrophosphatase/phosphodiesterase–1 (ENPP1), alkaline phosphatase (ALP), inorganic pyrophosphate (PPi), and inorganic phosphate (Pi) concentrations were evaluated by immunohistochemistry and enzyme-linked immunosorbent assay. In vitro bovine meniscus explant study (hypothesis 2): Bovine meniscus explants were subjected to 25% strain at 0.3 Hz for 1, 2, and 3 hours. Cell viability was determined using live/dead staining. The levels of mRNA expression and protein levels were measured using real-time quantitative reverse transcription polymerase chain reaction and Western blot after 24, 48, and 72 hours in culture. The conditioned medium was collected for sulfated glycosaminoglycan (GAG) release and Pi/PPi assay. Results In vivo guinea pig study: Meniscus size and area as well as intensity of meniscus calcification were significantly increased in the ACLT group compared with the control group. Both calcified area and intensity were correlated with cartilage damage in the ACLT group (meniscus calcified area: r = 0.925, P < .0001; meniscus calcified intensity: r = 0.944, P < .0001). Ihh, MMP-13, Col X, ANKH, ENPP1, and ALP expression were increased in the ACLT group compared with the control group. The Pi level and Pi/PPi ratio increased by 63% and 42%, respectively, in the ACLT group compared with the control group. In vitro bovine meniscus explant study: Cell death was found in the superficial zone of the bovine meniscus explants after loading for 3 hours. The mRNA expression and protein levels of MMP-13, ANKH, ENPP1, and ALP were up-regulated in all 3-hour loaded samples. The Pi/PPi ratio and sulfated GAG content in the culture medium were increased in the 3-hour loaded group. Conclusion Meniscus hypertrophy and mineralization correlated to cartilage degeneration after ACL injuries. Clinical Relevance The study data suggest that the suppression of meniscus hypertrophy and calcification may decrease the risk of PTOA after ACL injuries. PMID:26792705

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...

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

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.

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.

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.

Dihydroquercetin Does Not Affect Age-Dependent Increase in Blood Pressure and Angiotensin-Converting Enzyme Activity in the Aorta of Hypertensive Rats.

PubMed

Slashcheva, G A; Rykov, V A; Lobanov, A V; Murashev, A N; Kim, Yu A; Arutyunyan, T V; Korystova, A F; Kublik, L N; Levitman, M Kh; Shaposhnikona, V V; Korystov, Yu N

2016-09-01

We analyzed changes in angiotensin-converting enzyme activity in the aorta of hypertensive SHR rats against the background of age-related BP increase (from week 7 to 14) and the effect of dihydroquercetin on BP rise and angiotensin-converting enzyme activity. Normotensive WKY rats of the same age were used as the control. BP and activity of angiotensin-converting enzyme in the aorta of SHR rats increased with age. Dihydroquercetin in doses of 100 and 300 μg/kg per day had no effect on the increase of these parameters; dihydroquercetin administered to 14-week-old WKY rats in a dose of 300 μg/kg reduced activity of the angiotensin-converting enzyme. Thus, the early (7-14 weeks) increase in BP and angiotensin-converting enzyme activity in the aorta of SHR rats was not modified by flavonoids (dihydroquercetin) in contrast to other rat strains and humans, which is indicative of specificity of hypertension mechanism in SHR rats.

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.

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.

Optimization of parameters for enhanced oil recovery from enzyme treated wild apricot kernels.

PubMed

Rajaram, Mahatre R; Kumbhar, Baburao K; Singh, Anupama; Lohani, Umesh Chandra; Shahi, Navin C

2012-08-01

Present investigation was undertaken with the overall objective of optimizing the enzymatic parameters i.e. moisture content during hydrolysis, enzyme concentration, enzyme ratio and incubation period on wild apricot kernel processing for better oil extractability and increased oil recovery. Response surface methodology was adopted in the experimental design. A central composite rotatable design of four variables at five levels was chosen. The parameters and their range for the experiments were moisture content during hydrolysis (20-32%, w.b.), enzyme concentration (12-16% v/w of sample), combination of pectolytic and cellulolytic enzyme i.e. enzyme ratio (30:70-70:30) and incubation period (12-16 h). Aspergillus foetidus and Trichoderma viride was used for production of crude enzyme i.e. pectolytic and cellulolytic enzyme respectively. A complete second order model for increased oil recovery as the function of enzymatic parameters fitted the data well. The best fit model for oil recovery was also developed. The effect of various parameters on increased oil recovery was determined at linear, quadric and interaction level. The increased oil recovery ranged from 0.14 to 2.53%. The corresponding conditions for maximum oil recovery were 23% (w.b.), 15 v/w of the sample, 60:40 (pectolytic:cellulolytic), 13 h. Results of the study indicated that incubation period during enzymatic hydrolysis is the most important factor affecting oil yield followed by enzyme ratio, moisture content and enzyme concentration in the decreasing order. Enzyme ratio, incubation period and moisture content had insignificant effect on oil recovery. Second order model for increased oil recovery as a function of enzymatic hydrolysis parameters predicted the data adequately.

Inhibition of nitric oxide production and the effects of arginine and Lactobacillus administration in an acute liver injury model.

PubMed

Adawi, D; Molin, G; Jeppsson, B

1998-12-01

To study the effect of inhibiting nitric oxide production and the effects of arginine and lactobacilli administration in an acute liver injury (LI) model. Infectious complications caused by enteric bacteria are common in patients with liver diseases and those who have undergone liver surgery. Increased bacterial translocation has been proposed as one underlying mechanism. Lactobacilli constitute an integral part of the normal gastrointestinal microecology; they are involved in host metabolism and have many beneficial properties. Arginine has numerous roles in cellular metabolism and may be metabolized by lactobacilli in some cases. We have previously shown that rectal administration of Lactobacillus plantarum DSM 9843 (strain 299v), with and without arginine, in an acute LI model significantly reduces the extent of the LI and reduces bacterial translocation. To clarify the pathogenetic mechanisms, we studied the role of nitric oxide in the effects of L. plantarum and arginine in acute LI, as determined by bacterial translocation, ileal, cecal, and colonic nucleotides, RNA, and DNA. Male Sprague-Dawley rats were used. L. plantarum, 2% arginine, and/or N-nitro-L-arginine methyl ester (L-NAME), as appropriate, were administered rectally once daily for 8 days. Acute LI was induced on the eighth day by intraperitoneal injection of D-galactosamine (1.1 g/kg body weight), and samples were collected after 24 hours. Bacterial translocation was evaluated by culture of portal and arterial blood, mesenteric lymph nodes, and liver tissue. Liver enzymes and bilirubin were assayed in the serum. The bacterial load in the cecum and colon was determined. Ileal, cecal, and colonic mucosal nucleotides, RNA, and DNA were evaluated. The levels of liver enzymes and bilirubin were lower in liver-injured rats supplemented with arginine and Lactobacillus, and this effect was abolished by the addition of L-NAME. Inhibition of nitric oxide production (by L-NAME) increased bacterial translocation in many groups. L-NAME administration increased the cecal and colonic bacterial count and decreased the levels of mucosal nucleotides, RNA, and DNA. Inhibition of nitric oxide production modulated the effects of arginine and L. plantarum in this acute LI model. L-NAME potentiated the LI, as indicated by elevation of liver enzymes and bilirubin, and it also increased bacterial translocation and the cecal and colonic bacterial count. Increased bacterial translocation could be one of the mechanisms by which LI is potentiated.

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.

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

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.

Synergism between ultrasonic pretreatment and white rot fungal enzymes on biodegradation of wheat chaff.

PubMed

Sabarez, Henry; Oliver, Christine Maree; Mawson, Raymond; Dumsday, Geoff; Singh, Tanoj; Bitto, Natalie; McSweeney, Chris; Augustin, Mary Ann

2014-11-01

Lignocellulosic biomass samples (wheat chaff) were pretreated by ultrasound (US) (40kHz/0.5Wcm(-2)/10min and 400kHz/0.5Wcm(-2)/10min applied sequentially) prior to digestion by enzyme extracts obtained from fermentation of the biomass with white rot fungi (Phanerochaete chrysosporium or Trametes sp.). The accessibility of the cellulosic components in wheat chaff was increased, as demonstrated by the increased concentration of sugars produced by exposure to the ultrasound treatment prior to enzyme addition. Pretreatment with ultrasound increased the concentration of lignin degradation products (guaiacol and syringol) obtained from wheat chaff after enzyme addition. In vitro digestibility of wheat chaff was also enhanced by the ultrasonics pretreatment in combination with treatment with enzyme extracts. Degradation was enhanced with the use of a mixture of the enzyme extracts compared to that for a single enzyme extract. Copyright © 2014. Published by Elsevier B.V.

Regulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity in murine epidermis. Modulation of enzyme content and activation state by barrier requirements.

PubMed Central

Proksch, E; Elias, P M; Feingold, K R

1990-01-01

Epidermal cholesterol biosynthesis is regulated by barrier function. We quantitated the amount and activation state (phosphorylation-dephosphorylation) of the rate-limiting enzyme, 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, in epidermis before and after barrier disruption. In murine epidermis we found high enzyme activity (1.75 +/- 0.02 nmol/min per mg protein). After acute barrier disruption, enzyme activity began to increase after 1.5 h, reaching a maximum increase by 2.5 h, and returned to normal by 15 h. Chronic barrier disruption increased total enzyme activity by 83%. In normal epidermis, measurement of HMG CoA reductase activity in microsomes isolated in NaF- vs. NaCl-containing buffers demonstrated that 46 +/- 2% of the enzyme was in the active form. After acute or chronic barrier disruption, a marked increase in the percentage of HMG CoA reductase in the active form was observed. Acute disruption increased enzyme activation state as early as 15 min, reaching a maximum after 2.5 h, with an increase still present at 15 h, indicating that changes in activation state had a close temporal relationship with barrier function. Increases in total HMG CoA reductase activity occurred only after profound barrier disruption, whereas changes in activation state occur with lesser degrees of barrier disruption. Artificial correction of barrier function prevented the increase in total HMG CoA reductase activity, and partially prevented the increase in enzyme activation. These results show that barrier requirements regulate epidermal cholesterol synthesis by modulating both the HMG CoA reductase amount and activation state. Images PMID:2312730

ENZYME ACTIVITIES DURING THE ASEXUAL CYCLE OF NEUROSPORA CRASSA

PubMed Central

Stine, G. J.

1968-01-01

Three enzymes, (a) nicotinamide adenine diphosphate-dependent glutamic dehydrogenase (NAD enzyme), (b) nictoinamide adenine triphosphate-dependent glutamic dehydrogenase (NADP enzyme), and (c) nicotinamide-adenine dinucleotidase (NADase), were measured in separate extracts of Neurospora crassa grown in Vogel's medium N and medium N + glutamate. Specific activities and total units per culture of each enzyme were determined at nine separate intervals phased throughout the asexual cycle. The separate dehydrogenases were lowest in the conidia, increased slowly during germination, and increased rapidly during logarithmic mycelial growth. The amounts of these enzymes present during germination were small when compared with those found later during the production of the conidiophores. The NAD enzyme may be necessary for pregermination synthesis. The NADP-enzyme synthesis was associated with the appearance of the germ tube. Although higher levels of the dehydrogenases in the conidiophores resulted in more enzyme being found in the differentiated conidia, the rate of germination was uneffected. The greatest activity for the NADase enzyme was associated with the conidia, early phases of germination, and later production of new conidia. NADase decreased significantly with the onset of logarithmic growth, remained low during the differentiation of conidiophores, and increased considerably as the conidiophores aged. PMID:4384627

Effect of enzyme induction on nephrotoxicity of halothane-related compounds.

PubMed Central

Hitt, B A; Mazze, R I

1977-01-01

Nephrotoxicity following administration of methoxyflurane has been shown to be directly related to anesthetic metabolism to inorganic fluoride. Enzyme induction should increase metabolic rate and the amount of inorganic fluoride that is released. In vivo studies in Fischer 344 rats show that enzyme induction with phenobarbital or phenytoin increases defluorination following methoxyflurane anesthesia but not after enflurane or isoflurane. In vitro, methoxyflurane defluorinase activity was increased far more than that of any of the other anesthetics. These data suggest that treatment with enzyme inducing drugs increases the risk of nephrotoxocity only if methoxyflurane is the anesthetic agent. PMID:612443

Integration of Enzymes in Polyaniline-Sensitized 3D Inverse Opal TiO2 Architectures for Light-Driven Biocatalysis and Light-to-Current Conversion.

PubMed

Riedel, Marc; Lisdat, Fred

2018-01-10

Inspired by natural photosynthesis, coupling of artificial light-sensitive entities with biocatalysts in a biohybrid format can result in advanced photobioelectronic systems. Herein, we report on the integration of sulfonated polyanilines (PMSA1) and PQQ-dependent glucose dehydrogenase (PQQ-GDH) into inverse opal TiO 2 (IO-TiO 2 ) electrodes. While PMSA1 introduces sensitivity for visible light into the biohybrid architecture and ensures the efficient wiring between the IO-TiO 2 electrode and the biocatalytic entity, PQQ-GDH provides the catalytic activity for the glucose oxidation and therefore feeds the light-driven reaction with electrons for an enhanced light-to-current conversion. Here, the IO-TiO 2 electrodes with pores of around 650 nm provide a suitable interface and morphology needed for the stable and functional assembly of polymer and enzyme. The IO-TiO 2 electrodes have been prepared by a template approach applying spin coating, allowing an easy scalability of the electrode height and surface area. The successful integration of the polymer and the enzyme is confirmed by the generation of an anodic photocurrent, showing an enhanced magnitude with increasing glucose concentrations. Compared to flat and nanostructured TiO 2 electrodes, the three-layered IO-TiO 2 electrodes give access to a 24-fold and 29-fold higher glucose-dependent photocurrent due to the higher polymer and enzyme loading in IO films. The three-dimensional IO-TiO 2 |PMSA1|PQQ-GDH architecture reaches maximum photocurrent densities of 44.7 ± 6.5 μA cm -2 at low potentials in the presence of glucose (for a three TiO 2 layer arrangement). The onset potential for the light-driven substrate oxidation is found to be at -0.315 V vs Ag/AgCl (1 M KCl) under illumination with 100 mW cm -2 , which is more negative than the redox potential of the enzyme. The results demonstrate the advantageous properties of IO-TiO 2 |PMSA1|PQQ-GDH biohybrid architectures for the light-driven glucose conversion with improved performance.

Advances in Targeted Pesticides with Environmentally Responsive Controlled Release by Nanotechnology

PubMed Central

Huang, Bingna; Chen, Feifei; Shen, Yue; Wang, Yan; Sun, Changjiao; Zhao, Xiang; Cui, Bo; Gao, Fei; Zeng, Zhanghua; Cui, Haixin

2018-01-01

Pesticides are the basis for defending against major biological disasters and important for ensuring national food security. Biocompatible, biodegradable, intelligent, and responsive materials are currently an emerging area of interest in the field of efficient, safe, and green pesticide formulation. Using nanotechnology to design and prepare targeted pesticides with environmentally responsive controlled release via compound and chemical modifications has also shown great potential in creating novel formulations. In this review, special attention has been paid to intelligent pesticides with precise controlled release modes that can respond to micro-ecological environment changes such as light-sensitivity, thermo-sensitivity, humidity sensitivity, soil pH, and enzyme activity. Moreover, establishing intelligent and controlled pesticide release technologies using nanomaterials are reported. These technologies could increase pesticide-loading, improve the dispersibility and stability of active ingredients, and promote target ability. PMID:29439498

An enzyme complex increases in vitro dry matter digestibility of corn and wheat in pigs.

PubMed

Park, Kyu Ree; Park, Chan Sol; Kim, Beob Gyun

2016-01-01

Two experiments were conducted to determine the effects of enzyme complex on in vitro dry matter (DM) digestibility for feed ingredients. The objective of experiment 1 was to screen feed ingredients that can be effective substrates for an enzyme complex, mainly consisted of β-pentosanase, β-glucanase and α-amylase, using in vitro digestibility methods. In experiment 1, the test ingredients were three grain sources (barley, corn and wheat) and six protein supplements (canola meal, copra expellers, cottonseed meal, distillers dried grains with solubles, palm kernel expellers and soybean meal). In vitro ileal and total tract digestibility (IVID and IVTTD, respectively) of DM for test ingredients were determined. In vitro digestibility methods consisted of two- or three-step procedure simulating in vivo digestion in the pig gastrointestinal tracts with or without enzyme complex. As the enzyme complex added, the IVID of DM for corn and wheat increased (p < 0.05) by 5.0 and 2.6 percentage unit, respectively. The IVTTD of DM for corn increased (p < 0.05) by 3.1 percentage unit with enzyme complex addition. As the effect of enzyme complex was the greatest in corn digestibility, corn grains were selected to determine the in vitro digestibility of the fractions (starch, germ, hull and gluten) that maximally respond to the enzyme complex in experiment 2. The IVID of DM for corn starch, germ and hull increased (p < 0.05) by 16.0, 2.8 and 1.2 percentage unit, respectively. The IVTTD of DM for corn starch and hull also increased (p < 0.05) by 8.6 and 0.9 percentage unit, respectively, with enzyme complex addition. In conclusion, the enzyme complex increases in vitro DM digestibility of corn and wheat, and the digestibility increments of corn are mainly attributed to the increased digestibility of corn starch.

Fermentation of dilute acid pretreated Populus by Clostridium thermocellum, Caldicellulosiruptor bescii, and Caldicellulosiruptor obsidiansis

DOE PAGES

Yee, Kelsey L.; Rodriguez, Jr., Miguel; Hamilton, Choo Yieng; ...

2015-07-25

Consolidated bioprocessing (CBP), which merges enzyme production, biomass hydrolysis, and fermentation into a single step, has the potential to become an efficient and economic strategy for the bioconversion of lignocellulosic feedstocks to transportation fuels or chemicals. In this study, we evaluated Clostridium thermocellum, Caldicellulosiruptor bescii, and Caldicellulosiruptor obsidiansis, three , thermophilic,cellulolytic, mixed-acid fermenting candidate CBP microorganisms, for their fermentation capabilities using dilute acid pretreated Populus as a model biomass feedstock. Under pH controlled, anaerobic fermentation conditions, each candidate successfully digested a minimum of 75% of the cellulose from dilute acid pretreated Populus, as indicated by an increase in planktonic cellsmore » and end-product metabolites and a concurrent decrease in glucan content. C. thermocellum, which employs a cellulosomal approach to biomass degradation, required 120 hours to achieve 75% cellulose utilization. In contrast, the non-cellulosomal, secreted hydrolytic enzyme system of the Caldicellulosiruptor sp. required 300 hours to achieve similar results. End-point fermentation conversions for C. thermocellum, C. bescii, and C. obsidiansis were determined to be 0.29, 0.34, and 0.38 grams of total metabolites per gram of loaded glucan, respectively. This data provide a starting point for future strain engineering efforts that can serve to improve the biomass fermentation capabilities of these three promising candidate CBP platforms.« less

Fermentation of dilute acid pretreated Populus by Clostridium thermocellum, Caldicellulosiruptor bescii, and Caldicellulosiruptor obsidiansis

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

Yee, Kelsey L.; Rodriguez, Jr., Miguel; Hamilton, Choo Yieng

Consolidated bioprocessing (CBP), which merges enzyme production, biomass hydrolysis, and fermentation into a single step, has the potential to become an efficient and economic strategy for the bioconversion of lignocellulosic feedstocks to transportation fuels or chemicals. In this study, we evaluated Clostridium thermocellum, Caldicellulosiruptor bescii, and Caldicellulosiruptor obsidiansis, three , thermophilic,cellulolytic, mixed-acid fermenting candidate CBP microorganisms, for their fermentation capabilities using dilute acid pretreated Populus as a model biomass feedstock. Under pH controlled, anaerobic fermentation conditions, each candidate successfully digested a minimum of 75% of the cellulose from dilute acid pretreated Populus, as indicated by an increase in planktonic cellsmore » and end-product metabolites and a concurrent decrease in glucan content. C. thermocellum, which employs a cellulosomal approach to biomass degradation, required 120 hours to achieve 75% cellulose utilization. In contrast, the non-cellulosomal, secreted hydrolytic enzyme system of the Caldicellulosiruptor sp. required 300 hours to achieve similar results. End-point fermentation conversions for C. thermocellum, C. bescii, and C. obsidiansis were determined to be 0.29, 0.34, and 0.38 grams of total metabolites per gram of loaded glucan, respectively. This data provide a starting point for future strain engineering efforts that can serve to improve the biomass fermentation capabilities of these three promising candidate CBP platforms.« less

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.

Methyl trypsin loaded poly(D,L-lactide-coglycolide) nanoparticles for contact lens care.

PubMed

Jimenez, N; Galan, J; Vallet, A; Egea, M A; Garcia, M L

2010-03-01

The need of an enzymatic cleaner for soft contact lens care with an improved ocular safety and stability profile led us to evaluate the use of nanoparticles (NPs) of poly(D,L-lactide-coglycolide) (PLGA) and methyl trypsin (MT). NPs were prepared by double emulsion-solvent evaporation technique. A factorial design was performed to select the lactic acid proportion in the copolymer and conditions of the second sonication. The increment in proportion of lactic acid provided higher particle size results. When the time of second sonication was decreased, the entrapment efficiency (EE) increased. PLGA 50:50 NPs were chosen for further development since PLGA 50:50H NPs settled fast with different particle size in the sediment and PLGA 75:25 NPs led to form aggregates. The addition of glycerol to the NPs provided the highest EE of MT (>90%) while the addition of Tetronic 1304 promoted the fast release of enzyme initially and decreased the zeta potential (zeta) up to neutral values after gamma irradiation. NPs are expected to be effective as a lens care cleaner after 3 days or even longer with a very low quantity of enzyme released. Formulations showed an acceptable irritation ocular tolerance after in vitro HET-CAM test and in vivo Draize test. 2009 Wiley-Liss, Inc. and the American Pharmacists Association

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.

Effects of protease and non-starch polysaccharide enzyme on performance, digestive function, activity and gene expression of endogenous enzyme of broilers.

PubMed

Yuan, Lin; Wang, Mingfa; Zhang, Xiaotu; Wang, Zhixiang

2017-01-01

Three hundred one-day-old male broiler chickens (Ross-308) were fed corn-soybean basal diets containing non-starch polysaccharide (NSP) enzyme and different levels of acid protease from 1 to 42 days of age to investigate the effects of exogenous enzymes on growth performance, digestive function, activity of endogenous digestive enzymes in the pancreas and mRNA expression of pancreatic digestive enzymes. For days 1-42, compared to the control chickens, average daily feed intake (ADFI) and average daily gain (ADG) were significantly enhanced by the addition of NSP enzyme in combination with protease supplementation at 40 or 80 mg/kg (p<0.05). Feed-to-gain ratio (FGR) was significantly improved by supplementation with NSP enzymes or NSP enzyme combined with 40 or 80 mg/kg protease compared to the control diet (p<0.05). Apparent digestibility of crude protein (ADCP) was significantly enhanced by the addition of NSP enzyme or NSP enzyme combined with 40 or 80 mg/kg protease (p<0.05). Cholecystokinin (CCK) level in serum was reduced by 31.39% with NSP enzyme combined with protease supplementation at 160 mg/kg (p<0.05), but the CCK level in serum was increased by 26.51% with NSP enzyme supplementation alone. After 21 days, supplementation with NSP enzyme and NSP enzyme combined with 40 or 80 mg/kg protease increased the activity of pancreatic trypsin by 74.13%, 70.66% and 42.59% (p<0.05), respectively. After 42 days, supplementation with NSP enzyme and NSP enzyme combined with 40 mg/kg protease increased the activity of pancreatic trypsin by 32.45% and 27.41%, respectively (p<0.05). However, supplementation with NSP enzyme and 80 or 160 mg/kg protease decreased the activity of pancreatic trypsin by 10.75% and 25.88%, respectively (p<0.05). The activities of pancreatic lipase and amylase were significantly higher in treated animals than they were in the control group (p<0.05). Supplementation with NSP enzyme, NSP enzyme combined with 40 or 80 mg/kg protease increased pancreatic trypsin mRNA levels by 40%, 44% and 28%, respectively. Supplementation with NSP enzyme and 160 mg/kg protease decreased pancreatic trypsin mRNA levels by 13%. Pancreatic lipase and amylase mRNA expression were significantly elevated in treated animals compared to the control group (p<0.05). These results suggest that the amount of NSP enzyme and acid protease in the diet significantly affects digestive function, endogenous digestive-enzyme activity and mRNA expression in broilers.

Aβ degradation or cerebral perfusion? Divergent effects of multifunctional enzymes.

PubMed

Miners, J Scott; Palmer, Jennifer C; Tayler, Hannah; Palmer, Laura E; Ashby, Emma; Kehoe, Patrick G; Love, Seth

2014-01-01

There is increasing evidence that deficient clearance of β-amyloid (Aβ) contributes to its accumulation in late-onset Alzheimer disease (AD). Several Aβ-degrading enzymes, including neprilysin (NEP), endothelin-converting enzyme (ECE), and angiotensin-converting enzyme (ACE) reduce Aβ levels and protect against cognitive impairment in mouse models of AD. In post-mortem human brain tissue we have found that the activity of these Aβ-degrading enzymes rise with age and increases still further in AD, perhaps as a physiological response that helps to minimize the build-up of Aβ. ECE-1/-2 and ACE are also rate-limiting enzymes in the production of endothelin-1 (ET-1) and angiotensin II (Ang II), two potent vasoconstrictors, increases in the levels of which are likely to contribute to reduced blood flow in AD. This review considers the possible interdependence between Aβ-degrading enzymes, ischemia and Aβ in AD: ischemia has been shown to increase Aβ production both in vitro and in vivo, whereas increased Aβ probably enhances ischemia by vasoconstriction, mediated at least in part by increased ECE and ACE activity. In contrast, NEP activity may help to maintain cerebral perfusion, by reducing the accumulation of Aβ in cerebral blood vessels and lessening its toxicity to vascular smooth muscle cells. In assessing the role of Aβ-degrading proteases in the pathogenesis of AD and, particularly, their potential as therapeutic agents, it is important to bear in mind the multifunctional nature of these enzymes and to consider their effects on other substrates and pathways.

Experiment K-6-21. Effect of microgravity on 1) metabolic enzymes of type 1 and type 2 muscle fibers and on 2) metabolic enzymes, neutransmitter amino acids, and neurotransmitter associated enzymes in motor and somatosensory cerebral cortex. Part 1: Metabolic enzymes of individual muscle fibers; part 2: metabolic enzymes of hippocampus and spinal cord

NASA Technical Reports Server (NTRS)

Lowry, O.; Mcdougal, D., Jr.; Nemeth, Patti M.; Maggie, M.-Y. Chi; Pusateri, M.; Carter, J.; Manchester, J.; Norris, Beverly; Krasnov, I.

1990-01-01

The individual fibers of any individual muscle vary greatly in enzyme composition, a fact which is obscured when enzyme levels of a whole muscle are measured. The purpose of this study was therefore to assess the changes due to weightless on the enzyme patterns composed by the individual fibers within the flight muscles. In spite of the limitation in numbers of muscles examined, it is apparent that: (1) that the size of individual fibers (i.e., their dry weight) was reduced about a third, (2) that this loss in dry mass was accompanied by changes in the eight enzymes studied, and (3) that these changes were different for the two muscles, and different for the two enzyme groups. In the soleus muscle the absolute amounts of the three enzymes of oxidative metabolism decreased about in proportion to the dry weight loss, so that their concentration in the atrophic fibers was almost unchanged. In contrast, there was little loss among the four enzymes of glycogenolysis - glycolysis so that their concentrations were substantially increased in the atrophic fibers. In the TA muscle, these seven enzymes were affected in just the opposite direction. There appeared to be no absolute loss among the oxidative enzymes, whereas the glycogenolytic enzymes were reduced by nearly half, so that the concentrations of the first metabolic group were increased within the atrophic fibers and the concentrations of the second group were only marginally decreased. The behavior of hexokinase was exceptional in that it did not decrease in absolute terms in either type of muscle and probably increased as much as 50 percent in soleus. Thus, their was a large increase in concentration of this enzyme in the atrophied fibers of both muscles. Another clear-cut finding was the large increase in the range of activities of the glycolytic enzymes among individual fibers of TA muscles. This was due to the emergence of TA fibers with activities for enzymes of this group extending down to levels as low as those found in control soleus muscles. It would be interesting to know if this represents a transition stage, and whether with prolonged weightlessness most of the fibers would be transformed into a low glycogenolytic type.

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.

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.

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.

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.