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

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.

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.

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.

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

PubMed

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

2015-01-19

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

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

NASA Astrophysics Data System (ADS)

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

2015-01-01

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

Enzymatic saccharification of brown seaweed for production of fermentable sugars.

PubMed

Sharma, Sandeep; Horn, Svein Jarle

2016-08-01

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Continuous production of ethanol from starch using glucoamylase and yeast co-immobilized in pectin gel.

PubMed

Giordano, Raquel L C; Trovati, Joubert; Schmidell, Willibaldo

2008-03-01

This work presents a continuous simultaneous saccharification and fermentation (SSF) process to produce ethanol from starch using glucoamylase and Saccharomyces cerevisiae co-immobilized in pectin gel. The enzyme was immobilized on macroporous silica, after silanization and activation of the support with glutaraldehyde. The silica-enzyme derivative was co-immobilized with yeast in pectin gel. This biocatalyst was used to produce ethanol from liquefied manioc root flour syrup, in three fixed bed reactors. The initial reactor yeast load was 0.05 g wet yeast/ml of reactor (0.1 g wet yeast/g gel), used in all SSF experiments. The enzyme concentration in the reactor was defined by running SSF batch assays, using different amount of silica-enzyme derivative, co-immobilized with yeast in pectin gel. The chosen reactor enzyme concentration, 3.77 U/ml, allowed fermentation to be the rate-limiting step in the batch experiment. In this condition, using initial substrate concentration of 166.0 g/l of total reducing sugars (TRS), 1 ml gel/1 ml of medium, ethanol productivity of 8.3 g/l/h was achieved, for total conversion of starch to ethanol and 91% of the theoretical yield. In the continuous runs, feeding 163.0 g/l of TRS and using the same enzyme and yeast concentrations used in the batch run, ethanol productivity was 5.9 g ethanol/l/h, with 97% of substrate conversion and 81% of the ethanol theoretical yield. Diffusion effects in the extra-biocatalyst film seemed to be reduced when operating at superficial velocities above 3.7 x 10(-4) cm/s.

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.

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.

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.

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.

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.

Atrazine concentrations in stream water and streambed sediment pore water in the St. Joseph and Galien River basins, Michigan and Indiana, May 2001-September 2003

USGS Publications Warehouse

Duris, Joseph W.; Reeves, Howard W.; Kiesler, James L.

2005-01-01

The U.S. Geological Survey (USGS) sampled multiple stream sites across the St. Joseph and Galien River Basins to detect and quantify the herbicide atrazine using a field enzyme-linked immunosorbent assay (ELISA) triazine test. In May 2001, July 2001, April 2002, August 2002, August 2003 and September 2003, composite samples were collected across streams at USGS streamflow-gaging stations. Concentrations and instantaneous loading for atrazine sampled in stream water throughout the St. Joseph River and Galien River Basins in Michigan and Indiana ranged from nondetection (< 0.05 part per billion (ppb)) with an associated load less than 0.001 kilogram per day (kg/d) to 6 ppb and a maximum load of 10 kg/d. Atrazine concentrations were highest in May 2001 just after the planting season. The lowest concentration was found in April 2002 just before planting. Atrazine concentrations in streambed-sediment pore water were not spatially connected with atrazine concentrations in stream-water samples. This study showed that atrazine concentrations were elevated from May to July in the St. Joseph and Galien River Basins. At many sites, concentrations exceeded the level that has been shown to feminize frog populations (0.2 ppb). There were 8 sites where concentrations exceeded 0.2 ppb atrazine in May 2001 and July 2001.

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.

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.

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.

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.

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

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.

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.

Simultaneous saccharification and fermentation of fungal pretreated cornstalk for hydrogen production using Thermoanaerobacterium thermosaccharolyticum W16.

PubMed

Zhao, Lei; Cao, Guang-Li; Wang, Ai-Jie; Guo, Wan-Qian; Ren, Hong-Yu; Ren, Nan-Qi

2013-10-01

In this research, environmentally friendly fungal pretreatment was first adopted for deconstruction of cornstalk. Then the fungal-pretreated cornstalk was employed to produce hydrogen in simultaneous saccharification and fermentation (SSF) using crude enzyme from Trichoderma viride and Thermoanaerobacterium thermosaccharolyticum W16. The influence of various factors including substrate concentration, initial pH, and enzyme loading on hydrogen production were evaluated. The highest hydrogen yield of 89.3 ml/g-cornstalk was obtained with an initial pH 6.5, 0.75% substrate concentration, and 34 FPU/g cellulose. Compared the result with SSF of physical or chemical pretreated lignocellulosic materials, this research suggested an economic and efficient way for hydrogen production from lignocellulosic biomass. Copyright © 2013 Elsevier Ltd. All rights reserved.

Continuous Production of Ethanol from Starch Using Glucoamylase and Yeast Co-Immobilized in Pectin Gel

NASA Astrophysics Data System (ADS)

Giordano, Raquel L. C.; Trovati, Joubert; Schmidell, Willibaldo

This work presents a continuous simultaneous saccharification and fermentation (SSF) process to produce ethanol from starch using glucoamylase and Saccharomyces cerevisiae co-immobilized in pectin gel. The enzyme was immobilized on macroporous silica, after silanization and activation of the support with glutaraldehyde. The silicaenzyme derivative was co-immobilized with yeast in pectin gel. This biocatalyst was used to produce ethanol from liquefied manioc root flour syrup, in three fixed bed reactors. The initial reactor yeast load was 0.05 g wet yeast/ml of reactor (0.1 g wet yeast/g gel), used in all SSF experiments. The enzyme concentration in the reactor was defined by running SSF batch assays, using different amount of silica-enzyme derivative, co-immobilized with yeast in pectin gel. The chosen reactor enzyme concentration, 3.77 U/ml, allowed fermentation to be the rate-limiting step in the batch experiment. In this condition, using initial substrate concentration of 166.0 g/1 of total reducing sugars (TRS), 1 ml gel/1 ml of medium, ethanol productivity of 8.3 g/l/h was achieved, for total conversion of starch to ethanol and 91% of the theoretical yield. In the continuous runs, feeding 163.0 g/1 of TRS and using the same enzyme and yeast concentrations used in the batch run, ethanol productivity was 5.9 g ethanol/1/h, with 97% of substrate conversion and 81% of the ethanol theoretical yield. Diffusion effects in the extra-biocatalyst film seemed to be reduced when operating at superficial velocities above 3.7 × 10-4 cm/s.

Effect of protein load on stability of immobilized enzymes.

PubMed

Fernandez-Lopez, Laura; Pedrero, Sara G; Lopez-Carrobles, Nerea; Gorines, Beatriz C; Virgen-Ortíz, Jose J; Fernandez-Lafuente, Roberto

2017-03-01

Different lipases have been immobilized on octyl agarose beads at 1mg/g and at maximum loading, via physical interfacial activation versus the octyl layer on the support. The stability of the preparations was analyzed. Most biocatalysts had the expected result: the apparent stability increased using the highly loaded preparations, due to the diffusional limitations that reduced the initial observed activity. However, lipase B from Candida antarctica (CALB) was significantly more stable using the lowly loaded preparation than the maximum loaded one. This negative effect of the enzyme crowding on enzyme stability was found in inactivations at pH 5, 7 or 9, but not in inactivations in the presence of organic solvents. The immobilization using ethanol to reduce the immobilization rate had no effect on the stability of the lowly loaded preparation, while the highly loaded enzyme biocatalysts increased their stabilities, becoming very similar to that of the lowly loaded preparation. Results suggested that CALB molecules immobilized on octyl agarose may be closely packed together due to the high immobilization rate and this produced some negative interactions between immobilized enzyme molecules during enzyme thermal inactivation. Slowing-down the immobilization rate may be a solution for this unexpected problem. Copyright © 2016 Elsevier Inc. 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.

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.

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.

[Comparative analysis of viral load by bDNA HCV RNA-2.0 and amplicor HCV monitor in patients with hepatitis C infection].

PubMed

Córdoba, J; Olaso, V; Molina, J M; López Viedma, B; Argüello, L; Ortiz, V; Esteban, R J; Garijo, R; Pastor, M; Gobernado, M

2000-01-01

Two standardized techniques, Quantiplex (bDNA-2.0) and Amplicor Monitor have been evaluated for the quantification of virus load of HCV with these objectives: a) determinate the relationship between virus load and genotype, and b) evaluate the virus load in serial serum samples and in patients with normal or slightly increased liver enzymes in an area with a high prevalence of genotype 1. A significant correlation of 0.7 (p < 0.0001) in virus load has been observed by both methods, but the virus load is smaller by Monitor than by Quantiplex and does not depend on genotype. The relationship Monitor/Quantiplex is smaller in patients with non-1 genotype than in patients with genotype 1a (p = 0.01) and 1b (p = 0.005). Virus characteristics are similar in patients with normal or slightly increased enzymes than in patients with high enzymes. Virus load by both methods is not related to the age, sex, know duration of the infection, transmission manner of the infection neither to the histologic activity index. The virus load not depends on genotype. The determination of virus load in a single serum sample adequately reflects the virus load are in several serum samples in patients with chronic HCV infection. The genotype and the virus load are similar in patients with normal enzymes than in patients with high enzymes.

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.

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.

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

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

NASA Astrophysics Data System (ADS)

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

2017-11-01

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

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.

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.

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.

Production of propyl gallate in nonaqueous medium using cell-associated tannase of Bacillus massiliensis: effect of various parameters and statistical optimization.

PubMed

Aithal, Mahesh; Belur, Prasanna D

2013-01-01

Enzymatic synthesis of propyl gallate in an organic solvent was studied using cell-associated tannase (E.C. 3.1.1.20) of Bacillus massiliensis. Lyophilized biomass showing tannase activity was used as a biocatalyst. The influence of buffer pH and strength, water activity, temperature, biocatalyst loading, gallic acid concentration, and 1-propanol concentration was studied by the one-factor-at-a-time method. Subsequently, response surface methodology was applied based on a central composite design to determine the effects of three independent variables (biocatalyst loading, gallic acid concentration, and 1-propanol concentration) and their mutual interactions. A total of 20 experiments were conducted, and a statistical model was developed, which predicted the maximum propyl gallate yield of 20.28 μg/mL in the reaction mixture comprising 40.4 mg biocatalyst, 0.4 mM gallic acid, and 6.52 % (v/v) 1-propanol in 9.5 mL benzene at 30°C. The subsequent verification experiments established the validity of the model. Under optimal conditions, 25% conversion of gallic acid to propyl gallate was achieved on a molar basis. The absence of the need for enzyme purification and subsequent immobilization steps and good conversion efficiency makes this enzyme system an interesting one. Reports on the applications of bacterial whole cell systems for synthetic reactions in organic solvents are scarce, and perhaps this is the first report on bacterial cell-associated tannase-mediated esterification in a nonaqueous medium. © 2013 International Union of Biochemistry and Molecular Biology, Inc.

Plasmonic ELISA for the ultrasensitive detection of disease biomarkers with the naked eye

NASA Astrophysics Data System (ADS)

de La Rica, Roberto; Stevens, Molly M.

2012-12-01

In resource-constrained countries, affordable methodologies for the detection of disease biomarkers at ultralow concentrations can potentially improve the standard of living. However, current strategies for ultrasensitive detection often require sophisticated instruments that may not be available in laboratories with fewer resources. Here, we circumvent this problem by introducing a signal generation mechanism for biosensing that enables the detection of a few molecules of analyte with the naked eye. The enzyme label of an enzyme-linked immunosorbent assay (ELISA) controls the growth of gold nanoparticles and generates coloured solutions with distinct tonality when the analyte is present. Prostate specific antigen (PSA) and HIV-1 capsid antigen p24 were detected in whole serum at the ultralow concentration of 1 × 10-18 g ml-1. p24 was also detected with the naked eye in the sera of HIV-infected patients showing viral loads undetectable by a gold standard nucleic acid-based test.

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

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.

Fabrication and evaluation of SDF-1 loaded galactosylated chitosan nanoparticles for liver targeting

NASA Astrophysics Data System (ADS)

Xue-Hui, Chu; Zhang-Qi, Feng; Qian, Xu; Jiang-Qiang, Xiao; Xian-Wen, Yuan; Xi-Tai, Sun

2017-03-01

Objective. SDF-1 loaded galactosylated chitosan (GC) nanoparticles for liver targeting were synthesized by electrospraying technique, and its biocompatibility and liver targeting effect were evaluated. Method. The SDF-1 loaded GC nanoparticles were constructed and its morphology was observed by the scanning electron microscopy (SEM). Hepatocytes were harvested and cocultured with the nanoparticles, and the albumin secretion and urea synthesis were detected by enzyme-linked immunosorbent assay assay, the concentration of lactate dehydrogenase (LDH) and tumor necrosis factor-α (TNF-α) was also measured. Finally, the nanoparticles were injected intravenously through the caudal vein of rat, and its liver targeting effect was evaluated. Result. SEM showed the nanoparticles distributed uniformly, with an average diameter of 100 nm and a regular spherical shape. There was no significant difference in urea synthesis, albumin secretion, concentration of LDH and TNF-α between two groups (p > 0.05). The nanoparticles were significantly accumulated in the liver tissue after its injection, but seldom fluorescence signals were observed in the lung, spleen, heart and kidney. Conclusion. The SDF-1 loaded GC nanoparticles showed uniform distribution, good biocompatibility and liver targeting effect, and suggested its potential application as a liver targeting delivery system.

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.

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.

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.

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.

Formulation, Characterization and Stability Assessment of a Food-Derived Tripeptide, Leucine-Lysine-Proline Loaded Chitosan Nanoparticles.

PubMed

Danish, Minna K; Vozza, Giuliana; Byrne, Hugh J; Frias, Jesus M; Ryan, Sinéad M

2017-09-01

The chicken- or fish-derived tripeptide, leucine-lysine-proline (LKP), inhibits the angiotensin converting enzyme and may be used as an alternative treatment for prehypertension. However, it has low permeation across the small intestine. The formulation of LKP into a nanoparticle (NP) has the potential to address this issue. LKP-loaded NPs were produced using an ionotropic gelation technique, using chitosan (CL113). Following optimization of unloaded NPs, a mixture amount design was constructed using variable concentration of CL113 and tripolyphosphate at a fixed LKP concentration. Resultant particle sizes ranged from 120 to 271 nm, zeta potential values from 29 to 37 mV, and polydispersity values from 0.3 to 0.6. A ratio of 6:1 (CL113:TPP) produced the best encapsulation of approximately 65%. Accelerated studies of the loaded NPs indicated stability under normal storage conditions (room temperature). Cytotoxicity assessment showed no significant loss of cell viability and in vitro release studies indicated an initial burst followed by a slower and sustained release. © 2017 Institute of Food Technologists®.

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

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.

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.

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.

Purification of pectinase from mango (Mangifera indica L. cv. Chokanan) waste using an aqueous organic phase system: a potential low cost source of the enzyme.

PubMed

Amid, Mehrnoush; Abdul Manap, Mohd Yazid; Mustafa, Shuhaimi

2013-07-15

As a novel method of purification, an aqueous organic phase system (AOPS) was employed to purify pectinase from mango waste. The effect of different parameters, such as the alcohol concentration (ethanol, 1-propanol, and 2-propanol), the salt type and concentration (ammonium sulfate, potassium phosphate and sodium citrate), the feed stock crude load, the aqueous phase pH and NaCl concentration, were investigated in the recovery of pectinase from mango peel. The partition coefficient (K), selectivity (S), purification factor (PF) and yield (Y, %) were investigated in this study as important parameters for the evaluation of enzyme recovery. The desirable partition efficiency for pectinase purification was achieved in an AOPS of 19% (w/w) ethanol and 22% (w/w) potassium phosphate in the presence of 5% (w/w) NaCl at pH 7.0. Based on the system, the purification factor of pectinase was enhanced 11.7, with a high yield of 97.1%. Copyright © 2013 Elsevier B.V. All rights reserved.

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.

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.

A comparison between PLGA-PEG and NIPAAm-MAA nanocarriers in curcumin delivery for hTERT silencing in lung cancer cell line.

PubMed

Roointan, A; Sharifi-Rad, M; Badrzadeh, F; Sharifi-Rad, J

2016-08-29

Lung cancer is the most common cancer among men. Since the main reason of cancer cells immortality is telomerase activity, targeting of such enzyme can be a promising approach in cancer therapy. Curcumin is a safe and efficient anticancer agent in this context, but its applications in cancer therapy are limited because of its hydrophobic structure and low solubility in water. Today, using nanocarriers for delivery of such anticancer agents is a well performed method. Here, we developed and compared the efficiency of two nanocarriers (PLGA-PEG and NIPAAm-MAA) in delivery of curcumin and also in levels of hTERT silencing in lung cancer cell line (calu-6). Scanning electron microscopy, MTT assays and real-time PCR were used for imaging, cytotoxicity testing and measuring the expression levels of hTERT after treatment of cells with different concentrations of free curcumin and curcumin loaded nanocarriers. The MTT results demonstrated that the IC50 values of curcumin loaded nanocarriers were in lower concentrations than free curcumin. The hTERT expression levels were decreased by curcumin loaded PLGA-PEG more than curcumin loaded NIPAAm-MAA and free curcumin. Our results showed that the curcumin loaded PLGA-PEG can be a useful nano based carrier for delivery of anti-cancer agents such as curcumin to fight lung cancer.

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.

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.

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

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

Xue, Saisi; Uppugundla, Nirmal; Bowman, Michael J.

Accumulation of recalcitrant oligosaccharides during high-solids loading enzymatic hydrolysis of cellulosic biomass reduces biofuel yields and increases processing costs for a cellulosic biorefinery. Recalcitrant oligosaccharides in AFEX-pretreated corn stover hydrolysate accumulate to the extent of about 18–25 % of the total soluble sugars in the hydrolysate and 12–18 % of the total polysaccharides in the inlet biomass (untreated), equivalent to a yield loss of about 7–9 kg of monomeric sugars per 100 kg of inlet dry biomass (untreated). These oligosaccharides represent a yield loss and also inhibit commercial hydrolytic enzymes, with both being serious bottlenecks for economical biofuel production frommore » cellulosic biomass. Very little is understood about the nature of these oligomers and why they are recalcitrant to commercial enzymes. This work presents a robust method for separating recalcitrant oligosaccharides from high solid loading hydrolysate in gramme quantities. Composition analysis, recalcitrance study and enzyme inhibition study were performed to understand their chemical nature. Results indicate that, 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, and ammonia fibre expansion: AFEX). The methodology for large-scale separation of recalcitrant oligosaccharides from 25 % solids-loading AFEXcorn stover hydrolysate using charcoal fractionation and size exclusion chromatography is reported for the first time. Oligosaccharides with higher degree of polymerization (DP) were recalcitrant towards commercial enzyme mixtures [Ctec2, Htec2 and Multifect pectinase (MP)] compared to lower DP oligosaccharides. Enzyme inhibition studies using processed substrates (Avicel and xylan) showed that low DP oligosaccharides also inhibit commercial enzymes. Addition of monomeric sugars to oligosaccharides increases the inhibitory effects of oligosaccharides on commercial enzymes. In conclusion, the carbohydrate composition of the recalcitrant oligosaccharides, ratios of different DP oligomers and their distribution profiles were determined. Recalcitrance and enzyme inhibition studies help determine whether the commercial enzyme mixtures lack the enzyme activities required to completely de-polymerize the plant cell wall. Such studies clarify the reasons for oligosaccharide accumulation and contribute to strategies by which oligosaccharides can be converted into fermentable sugars and provide higher biofuel yields with less enzyme.« less

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

DOE PAGES

Xue, Saisi; Uppugundla, Nirmal; Bowman, Michael J.; ...

2015-11-26

Accumulation of recalcitrant oligosaccharides during high-solids loading enzymatic hydrolysis of cellulosic biomass reduces biofuel yields and increases processing costs for a cellulosic biorefinery. Recalcitrant oligosaccharides in AFEX-pretreated corn stover hydrolysate accumulate to the extent of about 18–25 % of the total soluble sugars in the hydrolysate and 12–18 % of the total polysaccharides in the inlet biomass (untreated), equivalent to a yield loss of about 7–9 kg of monomeric sugars per 100 kg of inlet dry biomass (untreated). These oligosaccharides represent a yield loss and also inhibit commercial hydrolytic enzymes, with both being serious bottlenecks for economical biofuel production frommore » cellulosic biomass. Very little is understood about the nature of these oligomers and why they are recalcitrant to commercial enzymes. This work presents a robust method for separating recalcitrant oligosaccharides from high solid loading hydrolysate in gramme quantities. Composition analysis, recalcitrance study and enzyme inhibition study were performed to understand their chemical nature. Results indicate that, 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, and ammonia fibre expansion: AFEX). The methodology for large-scale separation of recalcitrant oligosaccharides from 25 % solids-loading AFEXcorn stover hydrolysate using charcoal fractionation and size exclusion chromatography is reported for the first time. Oligosaccharides with higher degree of polymerization (DP) were recalcitrant towards commercial enzyme mixtures [Ctec2, Htec2 and Multifect pectinase (MP)] compared to lower DP oligosaccharides. Enzyme inhibition studies using processed substrates (Avicel and xylan) showed that low DP oligosaccharides also inhibit commercial enzymes. Addition of monomeric sugars to oligosaccharides increases the inhibitory effects of oligosaccharides on commercial enzymes. In conclusion, the carbohydrate composition of the recalcitrant oligosaccharides, ratios of different DP oligomers and their distribution profiles were determined. Recalcitrance and enzyme inhibition studies help determine whether the commercial enzyme mixtures lack the enzyme activities required to completely de-polymerize the plant cell wall. Such studies clarify the reasons for oligosaccharide accumulation and contribute to strategies by which oligosaccharides can be converted into fermentable sugars and provide higher biofuel yields with less enzyme.« less

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.

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.

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

PubMed

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

2017-01-01

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

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

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

PubMed Central

2013-01-01

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

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

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

PubMed

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

2015-01-01

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

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

PubMed Central

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

2015-01-01

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

Enzymatic hydrolysis of cellulose pretreated with ionic liquids and N-methyl Morpholine N-Oxide

NASA Astrophysics Data System (ADS)

Yau Li, Elizabeth

The effect of N-methyl Morpholine N-Oxide (NMMO), 1-ethyl-3-methyl-imidazolium acetate ([Emim]Ac) and 1-ethyl-3-methyl-imidazolium diethyl phosphate ([Emim]DEP) on pretreatment and enzymatic hydrolysis of dissolving pulp was studied. X-ray diffraction measurements of regenerated cellulose from these solvents showed that solvent pretreatment reduces the crystallinity of cellulose. However, crystallinity might not be a major factor affecting the in-situ enzymatic hydrolysis of cellulose in these solvents. Although regenerated cellulose from [Emim]DEP showed the lowest crystallinity index (˜15%), in-situ enzymatic hydrolysis of cellulose dissolved in NMMO showed the highest cellulose conversion (68% compared to 65% for [Emim]Ac and 37% for [Emim]DEP at enzyme loading of 122 FPU/g). Moreover, results showed that enzymes could tolerate up to NMMO concentration of 100 g/L and still yield full conversion of cellulose. Since it is not necessary to remove all the NMMO, less amount of water will be required for the washing step and thus the process will be more economical. The HCH-1 model was used in an attempt to model the enzymatic hydrolysis of cellulose in NMMO. With the incorporation of NMMO inhibition and a factor to account for unreacted cellulose, the model was able to correlate the experimental data of the enzymatic hydrolysis of cellulose (6.68 g/L) at various NMMO concentrations (0, 50, 100, 150 and 250 g/L). However, the experimental results also suggest that NMMO might be deactivating the enzymes rather than inhibiting them. More studies need to be done at varying cellulose, NMMO and enzyme concentrations to find the exact nature of this deactivation of NMMO.

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.

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

PubMed Central

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

2010-01-01

Antioxidant enzymes have shown promise as a therapy for pathological conditions involving increased production of reactive oxygen species (ROS). However the efficiency of their use for combating oxidative stress is dependent on the ability to achieve therapeutically adequate levels of active enzymes at the site of ROS-mediated injury. Thus, the implementation of antioxidant enzyme therapy requires a strategy enabling both guided delivery to the target site and effective protection of the protein in its active form. To address these requirements we developed magnetically responsive nanoparticles (MNP) formed by precipitation of calcium oleate in the presence of magnetite-based ferrofluid (controlled aggregation/precipitation) as a carrier for magnetically guided delivery of therapeutic proteins. We hypothesized that antioxidant enzymes, catalase and superoxide dismutase, can be protected from proteolytic inactivation by encapsulation in MNP. We also hypothesized that catalase-loaded MNP applied with a high-gradient magnetic field can rescue endothelial cells from hydrogen peroxide toxicity in culture. To test these hypotheses, a family of enzyme-loaded MNP formulations were prepared and characterized with respect to their magnetic properties, enzyme entrapment yields and protection capacity. SOD- and catalase-loaded MNP were formed with average sizes ranging from 300 to 400 nm, and a protein loading efficiency of 20–33%. MNP were strongly magnetically responsive (magnetic moment at saturation of 14.3 emu/g) in the absence of magnetic remanence, and exhibited a protracted release of their cargo protein in plasma. Catalase stably associated with MNP was protected from proteolysis and retained 20% of its initial enzymatic activity after 24 hr of exposure to pronase. Under magnetic guidance catalase-loaded MNP were rapidly taken up by cultured endothelial cells providing increased resistance to oxidative stress (62±12% cells rescued from hydrogen peroxide induced cell death vs. 10±4% under non-magnetic conditions). We conclude that non-polymeric MNP formed using the controlled aggregation/precipitation strategy are a promising carrier for targeted antioxidant enzyme therapy, and in combination with magnetic guidance can be applied to protect endothelial cells from oxidative stress mediated damage. This protective effect of magnetically targeted MNP impregnated with antioxidant enzymes can be highly relevant for the treatment of cardiovascular disease and should be further investigated in animal models. PMID:20483366

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

PubMed

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

2010-08-17

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

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

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.

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.

Effect of salt acclimatization on 3 beta-hydroxysteroid dehydrogenase/isomerase activity in the interrenal of Bufo arenarum.

PubMed

Pozzi, Andrea G; Lantos, Carlos P; Ceballos, Nora R

2002-03-01

In amphibians, aldosterone (Aldo) is particularly important in the regulation of Na(+) exchange by skin and urinary bladder. In previous works we studied a key enzyme in Aldo biosynthesis, the 3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta HSD/I), in the interrenals of Bufo arenarum. In those works a dual localization of the 3 beta HSD/I in both microsomes and mitochondria was described. The mitochondrial, but not the microsomal, enzyme prefers the immediate Aldo precursor, 3 beta-analogue of aldosterone, as substrate. In this order, the enzyme 3 beta HSD/I would be not only a key enzyme for the synthesis of Aldo but additionally, due to its microsomal and mitochondrial localization, a possible target for the regulation of Aldo biosynthesis. With this rationale in mind, we have used in vivo and in vitro approaches to study Aldo regulation. In the present investigation the levels of Aldo were determined in plasma of winter (W) and summer (S) toads subjected to different saline concentrations (0.125 and 0.15 M) or kept on wet land. Saline hyperosmotically treated toads had significantly lower levels than isoosmotically treated toads. These results are consistent with the response in mammals, in which salt loading provokes a reduction in Aldo secretion. In W toads, plasmatic corticosterone (B) concentration was higher than Aldo concentration, whereas in S toads, B/Aldo ratio approached unity. The reduction of Aldo levels after saline dehydration was due to a decline in its biosynthesis. K(m) and V(max) values for 3 beta HSD/I were calculated for mitochondrial and microsomal fractions obtained from animals acclimated to 0.15 M NaCl or kept on land. As previously described, V(max) differs between W and S toads. However, only mitochondrial V(max) changed as a consequence of saline adaptation, suggesting that the mitochondrial enzyme could be involved in the regulation of Aldo biosynthesis.

Enzymatic Conversion of CO2 to Bicarbonate in Functionalized Mesoporous Silica

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

Yu, Yuehua; Chen, Baowei; Qi, Wen N.

2012-05-01

We report here that carbonic anhydrase (CA), the fastest enzyme that can covert carbon dioxide to bicarbonate, can be spontaneously entrapped in functionalized mesoporous silica (FMS) with super-high loading density (up to 0.5 mg of protein/mg of FMS) due to the dominant electrostatic interaction. The binding of CA to HOOC-FMS can result in the protein’s conformational change comparing to the enzyme free in solution, but can be overcome with increased protein loading density. The higher the protein loading density, the less conformational change, hence the higher enzymatic activity and the higher enzyme immobilization efficiency. The electrostatically bound CA can bemore » released by changing pH. The released enzyme still displayed the native conformational structure and the same high enzymatic activity as that prior to the enzyme entrapment. This work opens up a new approach converting carbon dioxide to biocarbonate in a biomimetic nanoconfiguration that can be integrated with the other part of biosynthesis process for the assimilation of carbon dioxide.« less

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.

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.

Nanoarmoring of Enzymes by Interlocking in Cellulose Fibers With Poly(Acrylic Acid).

PubMed

Riccardi, Caterina M; Kasi, Rajeswari M; Kumar, Challa V

2017-01-01

A simple method for interlocking glucose oxidase (GOx) and horseradish peroxidase (HRP) in cellulose fibers using poly(acrylic acid) (PAA) as an armor around the enzyme, without any need for activation of the cellulose support, is reported here. The resulting enzyme paper is an inexpensive, stable, simple, wearable, and washable biosensor. PAA functions as a multifunctional tether to interlock the enzyme molecules around the paper fibers so that the enzymes are protected against thermal/chemical denaturation and not released from the paper when washed with a detergent. The decreased conformational entropy of the interlocked enzyme protected by the nanoarmor is likely responsible for increased enzyme stability to heat and chemical denaturants (retained ≥70 percent enzyme activity after washing with urea or SDS for 30min), and the polymer protects the enzyme against inactivation by proteases, bacteria, inhibitors, etc. The kinetics of the interlocked enzyme were similar to that of the enzyme in solution. The V max was 6(±0.5)mM per minute before washing, then increased slightly to 9(±1.4)mM per minute after washing with water. The K m was 22(±6.4mM), which was slightly higher compared to GOx in solution (25-27mM). Because the surface area of the paper does not limit the enzyme loading, about 20% of enzyme was successfully loaded onto the paper (0.2g enzyme per gram of paper), and ≥95% of the enzyme was retained after washing. Interlocking works with other enzymes such as laccase, where ≥60% of the enzyme activity is retained. This novel methodology provides a low cost, simple, modular approach of achieving high enzyme loadings in ordinary filter paper, not limited by cellulose surface area, and there has been no need for complex methods of enzyme engineering or toxic methods of activation of the solid support to prepare highly active biocatalysts. © 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.

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.

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.

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

PubMed

Kumar, Linoj; Chandra, Richard; Saddler, Jack

2011-10-01

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

Optimization of liquid fermentation of microbial consortium WSD-5 followed by saccharification and acidification of wheat straw.

PubMed

Wen, Boting; Yuan, Xufeng; Cao, Yanzhuan; Liu, Yan; Wang, Xiaofen; Cui, Zongjun

2012-08-01

The microbial consortium WSD-5 is composed of bacteria and fungi, and the cooperation and symbiosis of the contained microbes enhance the degradation ability of WSD-5. Experiment results showed that the highest cellulase and hemicellulase were obtained when ventilation volume was 4 L/min, stirring rate was 0 rpm, and substrate loading rate was 3%. After 6 days of cultivation, a 67.60% loss in wheat straw dry weight was observed. The crude enzyme secreted from WSD-5 after optimization was evaluated by experiments of saccharification and acidification. The maximum concentration of reducing sugars was 3254 mg/L after 48 h saccharification. The concentration of sCOD peaked on day 2 with a value of 4345 mg/L during acidification, and the biogas yield and methane yield were 22.3% and 32.3% higher than un-acidified samples. This study is the first attempt to explore both the saccharification and the acidification ability of crude enzymes secreted by microbial consortium. Copyright © 2012 Elsevier Ltd. All rights reserved.

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

PubMed Central

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

2012-01-01

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

The relationship between 25-hydroxyvitamin D concentration and liver enzymes in overweight or obese adults: Cross-sectional and interventional outcomes.

PubMed

Naderpoor, Negar; Mousa, Aya; de Courten, Maximilian; Scragg, Robert; de Courten, Barbora

2018-03-01

Vitamin D deficiency is prevalent in individuals with non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis. However, there is limited and inconsistent data on the effect of vitamin D supplementation on liver function. Hepatic enzymes have been used as surrogate markers for NAFLD and have been associated with metabolic syndrome. We examined the relationships between 25-hydroxyvitamin D (25(OH)D) and γ-glutamyl transferase (GGT), alanine aminotransferase (ALT), alkaline phosphatase (ALP) in 120 drug-naïve individuals with no history of liver disease. In addition, the effect of vitamin D supplementation (100,000 loading dose of cholecalciferol followed by 4000IU daily for 16 weeks) on hepatic enzymes was investigated in a subgroup of 54 vitamin D-deficient overweight or obese individuals (28 randomised to cholecalciferol and 26 to placebo). Hepatic enzymes, anthropometric parameters, lipid profile, insulin sensitivity (hyperinsulinaemic-euglycaemic clamp, M value) and high sensitivity C-reactive protein (hs-CRP) were measured before and after the intervention. In the cross-sectional study, levels of GGT and ALT were higher in men compared to women (both p=0.001). There were no significant differences in GGT, ALT and ALP between vitamin D categories (25(OH)D<25nmol/L, 25-50nmol/L, and >50nmol/L) and no relationships were found between the three enzymes and 25(OH)D before and after adjustment for age, sex, BMI, WHR, and insulin sensitivity (all p>0.5). In the randomised trial, 25(OH)D concentrations increased in the vitamin D group (mean change 57.0±21.3nmol/L) compared to the placebo group (mean change 1.9±15.1nmol/L). Mean changes in GGT, ALT and ALP were not significantly different between vitamin D and placebo groups (all p>0.2). Change in 25(OH)D concentration was not correlated with changes in GGT, ALT and ALP before and after adjustments for age and sex (all p>0.1). In summary, 25(OH)D concentrations were not related to hepatic enzymes in drug-naive adults with no history of liver disease, and vitamin D supplementation had no effect on the serum levels of hepatic enzymes in vitamin D-deficient and overweight or obese, otherwise healthy individuals. Hence, vitamin D supplementation is unlikely to prevent incident NAFLD. Copyright © 2017 Elsevier Ltd. All rights reserved.

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.

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

A Novel Aqueous Two Phase System Composed of Surfactant and Xylitol for the Purification of Lipase from Pumpkin (Cucurbita moschata) Seeds and Recycling of Phase Components.

PubMed

Amid, Mehrnoush; Manap, Mohd Yazid; Hussin, Muhaini; Mustafa, Shuhaimi

2015-06-17

Lipase is one of the more important enzymes used in various industries such as the food, detergent, pharmaceutical, textile, and pulp and paper sectors. A novel aqueous two-phase system composed of surfactant and xylitol was employed for the first time to purify lipase from Cucurbita moschata. The influence of different parameters such as type and concentration of surfactants, and the composition of the surfactant/xylitol mixtures on the partitioning behavior and recovery of lipase was investigated. Moreover, the effect of system pH and crude load on the degree of purification and yield of the purified lipase were studied. The results indicated that the lipase was partitioned into the top surfactant rich phase while the impurities partitioned into the bottom xylitol-rich phase using an aqueous two phase system composed of 24% (w/w) Triton X-100 and 20% (w/w) xylitol, at 56.2% of tie line length (TLL), (TTL is one of the important parameters in this study and it is determined from a bimodal curve in which the tie-line connects two nodes on the bimodal, that represent concentration of phase components in the top and bottom phases) and a crude load of 25% (w/w) at pH 8.0. Recovery and recycling of components was also measured in each successive step process. The enzyme was successfully recovered by the proposed method with a high purification factor of 16.4 and yield of 97.4% while over 97% of the phase components were also recovered and recycled. This study demonstrated that the proposed novel aqueous two phase system method is more efficient and economical than the traditional aqueous two phase system method for the purification and recovery of the valuable enzyme lipase.

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.

Phloem loading--not metaphysical, only complex: towards a unified model of phloem loading.

PubMed

Komor, E; Orlich, G; Weig, A; Köckenberger, W

1996-08-01

Phloem loading comprises the entire pathway of phloem-mobile solutes from their place of generation (or delivery) to the sieve tubes in a sequence of transport steps across or passing by several different cell types. Each of these steps can be classified as symplastic or apoplastic. The detailed anatomical-cytological work in the past ten years made clear that the symplastic continuity from mesophyll to sieve tubes may be very different for different plant species or even in different vein orders. Therefore data from one species are not transferable to another species and a well-rounded picture involving different experimental methods has to be aimed at for each species separately. The information obtained with the Ricinus seedling, where phloem loading and sieve tube sap analysis can be achieved relatively easily, is presented. The analysis of the radioactive labelling of sucrose from the sieve tubes of cotyledons, in which external and intracellular sucrose had been differently labelled, revealed that at sucrose concentrations close to the natural one, 50% of sucrose is loaded directly from the external medium. The other 50% is first taken up by mesophyll and then released for uptake into the sieve tubes. No bundle tissue works as obligate, intermediate sucrose storage. The apoplast therefore definitely serves as a transit reservoir for sucrose destined to be loaded into the sieve tubes. The sieve tube sap contains glycolytic metabolites at concentrations higher than found in the hypocotyl tissue, whereas the corresponding glycolytic enzymes are missing. It is concluded that the enzymes are sequestered in the companion cell or by parietal membrane stacks. Not only the sieve tubes but nearly all cotyledonary cells are equipped with a sucrose-H(+) symporter able to achieve sucrose accumulation and sensitive to inhibition by high salt concentrations or SH reagents. A cDNA clone coding for a sucrose carrier was isolated. It is transcribed at approximately the same level in most organs of the seedling and throughout the germination period. Leaves of adult Ricinus have significantly lower levels of this transcript. Recirculation of excess, phloem-delivered solutes from the sink back to the source is shown not only to be a common feature of long-distance transport, but the only way that an imbalance between supply to and consumption of nutrients in the sink can be adjusted in the source. It is a pathway by which sink activity regulates phloem loading. Non-invasive NMR imaging revealed the flow rates and flow speeds in phloem and xylem in the intact seedling and proved directly the existence of an internal circulating solution flow. A unified model of phloem loading is proposed, based on a pump-and-leak model, where active sucrose carriers (and other carriers) accumulate solutes in the sieve tubes with a concomitant build-up of pressure resulting in mass flow. Plasmodesmata are leaks (as are the transport carriers, too), slowing down the transport rate, but they also serve as diffusion channels for substances which are produced in the neighbouring cell. Therefore, compounds, which are not made in the sieve tubes themselves are translocated together with the bulk solution of sieve tube sap.

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.

Valorization of kitchen biowaste for ethanol production via simultaneous saccharification and fermentation using co-cultures of the yeasts Saccharomyces cerevisiae and Pichia stipitis.

PubMed

Ntaikou, Ioanna; Menis, Nikolaos; Alexandropoulou, Maria; Antonopoulou, Georgia; Lyberatos, Gerasimos

2018-04-30

The biotransformation of the pre-dried and shredded organic fraction of kitchen waste to ethanol was investigated, via co-cultures of the yeasts Saccharomyces cerevisiae and Pichia stipitis (Scheffersomyces stipitis). Preliminary experiments with synthetic media were performed, in order to investigate the effect of different operational parameters on the ethanol production efficiency of the co-culture. The control of the pH and the supplementation with organic nitrogen were shown to be key factors for the optimization of the process. Subsequently, the ethanol production efficiency from the waste was assessed via simultaneous saccharification and fermentation experiments. Different loadings of cellulolytic enzymes and mixtures of cellulolytic with amylolytic enzymatic blends were tested in order to enhance the substrate conversion efficiency. It was further shown that for solids loading up to 40% waste on dry mass basis, corresponding to 170 g.L -1 initial concentration of carbohydrates, no substrate inhibition occurred, and ethanol concentration up to 45 g.L -1 was achieved. Copyright © 2018 Elsevier Ltd. All rights reserved.

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.

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.

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.

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

PubMed

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

2015-10-01

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

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.

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

PubMed

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

2016-03-01

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

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

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

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

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

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

DOE PAGES

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

2015-04-09

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

Postabsorptive hyperglucagonemia in patients with type 2 diabetes mellitus analyzed with a novel enzyme-linked immunosorbent assay.

PubMed

Matsuo, Toshihiro; Miyagawa, Jun-Ichiro; Kusunoki, Yoshiki; Miuchi, Masayuki; Ikawa, Takashi; Akagami, Takafumi; Tokuda, Masaru; Katsuno, Tomoyuki; Kushida, Akira; Inagaki, Takashi; Namba, Mitsuyoshi

2016-05-01

The aims of the present study were to investigate the performance of a novel sandwich enzyme-linked immunosorbent assay (ELISA) for measuring glucagon (1-29) with monoclonal antibodies against both the C- and N-terminal regions of glucagon (1-29), and to analyze the differences in plasma levels and responses of glucagon (1-29) to oral glucose loading in normal glucose tolerance (NGT) subjects and patients with type 2 diabetes mellitus. The cross-reactivity against proglucagon fragments using the ELISA kit and two types of conventional radioimmunoassay (RIA) kits was evaluated. A 75-g oral glucose tolerance test was carried out with NGT subjects and patients with type 2 diabetes mellitus, and the glucagon (1-29) concentration was measured using three types of kit. The ELISA kit clearly had the lowest cross-reactivity against miniglucagon (19-29) and glicentin (1-61). The oral glucose tolerance test was carried out with 30 NGT and 17 patients with type 2 diabetes mellitus. The glucagon (1-29) levels measured by the ELISA kit after glucose loading were significantly higher at all time-points in the type 2 diabetes mellitus group than in the NGT group. However, the glucagon (1-29) levels measured by one RIA kit were significantly higher in the NGT group, and those measured with the other RIA kit were approximately the same among the groups. The novel sandwich ELISA accurately determines plasma glucagon (1-29) concentrations with much less cross-reactivity against other proglucagon fragments than conventional RIA kits.

Effects of physical interventions on house dust mite allergen levels in carpet, bed, and upholstery dust in low-income, urban homes.

PubMed Central

Vojta, P J; Randels, S P; Stout, J; Muilenberg, M; Burge, H A; Lynn, H; Mitchell, H; O'Connor, G T; Zeldin, D C

2001-01-01

House dust mite allergen exposure is a postulated risk factor for allergic sensitization, asthma development, and asthma morbidity; however, practical and effective methods to mitigate these allergens from low-income, urban home environments remain elusive. The purpose of this study was to assess the feasibility and effectiveness of physical interventions to mitigate house dust mite allergens in this setting. Homes with high levels of house dust mite allergen (Der f 1 + Der p 1 > or = 10 microg/g dust by enzyme-linked immunosorbent assay) in the bed, bedroom carpet, and/or upholstered furniture were enrolled in the study. Carpets and upholstered furniture were subjected to a single treatment of either dry steam cleaning plus vacuuming (carpet only) or intensive vacuuming alone. Bed interventions consisted of complete encasement of the mattress, box spring, and pillows plus either weekly professional or in-home laundering of nonencased bedding. Dust samples were collected at baseline and again at 3 days (carpet and upholstery only) and 2, 4, and 8 weeks posttreatment. We compared pretreatment mean allergen concentrations and loads to posttreatment values and performed between-group analyses after adjusting for differences in the pretreatment means. Both dry steam cleaning plus vacuuming and vacuuming alone resulted in a significant reduction in carpet house dust mite allergen concentration and load (p < 0.05). Levels approached pretreatment values by 4 weeks posttreatment in the intensive vacuuming group, whereas steam cleaning plus vacuuming effected a decrease that persisted for up to 8 weeks. Significant decreases in bed house dust mite allergen concentration and load were obtained in response to encasement and either professional or in-home laundering (p < 0.001). Between-group analysis revealed significantly less postintervention house dust mite allergen load in professionally laundered compared to home-laundered beds (p < 0.05). Intensive vacuuming and dry steam cleaning both caused a significant reduction in allergen concentration and load in upholstered furniture samples (p < 0.005). Based on these data, we conclude that physical interventions offer practical, effective means of reducing house dust mite allergen levels in low-income, urban home environments. PMID:11564617

[Optimization on trehalose loading technique as protective conditioning for lyophilization of human platelets].

PubMed

Liu, Jing-Han; Zhou, Jun; Ouyang, Xi-Lin; Li, Xi-Jin; Lu, Fa-Qiang

2005-08-01

This study was aimed to further optimize trehalose loading technique including loading temperature, loading time, loading solution and loading concentration of trehalose, based on the established parameters. Loading efficiency in plasma was compared with that in buffer at 37 degrees C; the curves of intracellular trehalose concentration versus loading time at 37 degrees C and 16 degrees C were measured; curves of mean platelet volume (MPV) versus loading time and loading concentration were investigated and compared. According to results obtained, the loaing time, loading temperature, loading solution and trehalose concentration were ascertained for high loading efficiency of trehalose into human platelet. The results showed that the loading efficiency in plasma was markedly higher than that in buffer at 37 degrees C, the loading efficiency in plasma at 37 degrees C was significantly higher than that at 16 degrees C and reached 19.51% after loading for 4 hours, but 6.16% at 16 degrees C. MPV at 16 degrees C was increased by 43.2% than that at 37 degrees C, but had no distinct changes with loading time and loading concentration. In loading at 37 degrees C, MPV increased with loading time and loading concentration positively. Loading time and loading concentration displayed synergetic effect on MPV. MPV increased with loading time and concentration while trehalose loading concentration was above 50 mmol/L. It is concluded that the optimization parameters of trehalose loading technique are 37 degrees C (temperature), 4 hours (leading time), plasma (loading solution), 50 mmol/L (feasible trehalose concentration). The trehalose concentration can be adjusted to meet the requirement of lyophilization.

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.

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.

The Catalytic Function of Enzymes.

ERIC Educational Resources Information Center

Splittgerber, Allan G.

1985-01-01

Discusses: structure of the enzyme molecule; active site; reaction mechanism; transition state; factors affecting enzyme reaction rates, concentration of enzyme; concentration of substrate; product concentration; temperature effects and pH effects; factors causing a lowering of activation energy; proximity and orientation effects; substrate strain…

Comparison of several ethanol productions using xylanase, inorganic salts, surfactant

NASA Astrophysics Data System (ADS)

Wu, Yan; Lu, Jie; Yang, Rui-feng; Song, Wen-jing; Li, Hai-ming; Wang, Hai-song; Zhou, Jing-hui

2017-03-01

Liquid hot water (LHW) pretreatment is an effective and environmentally friendly method to produce bioethanol with lignocellulosic materials. Corn stover was pretreated with liquid hot water (LHW) and then subjected to semi-simultaneous saccharification and fermentation (S-SSF) to obtain high ethanol concentration and yield. The present study aimed to confirm the effect of several additives on the fermentation digestibility of unwashed WIS of corn stover pretreated with LHW. So we also investigated the process, such as enzyme addition, inorganic salts, surfactant and different loading Triton. Results show that high ethanol concentration is necessary to add xylanase in the stage of saccharification. The ethanol concentration increased mainly with magnesium ion on fermentation. Comparing with Tween 80, Span 80 and Polyethylene glycol, Triton is the best surfactant. In contrast to using xylanase and Triton respectively, optimization can make up the lack of stamina and improve effect of single inorganic salts.

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

PubMed

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

2015-11-18

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

A short review on SSF – an interesting process option for ethanol production from lignocellulosic feedstocks

PubMed Central

Olofsson, Kim; Bertilsson, Magnus; Lidén, Gunnar

2008-01-01

Simultaneous saccharification and fermentation (SSF) is one process option for production of ethanol from lignocellulose. The principal benefits of performing the enzymatic hydrolysis together with the fermentation, instead of in a separate step after the hydrolysis, are the reduced end-product inhibition of the enzymatic hydrolysis, and the reduced investment costs. The principal drawbacks, on the other hand, are the need to find favorable conditions (e.g. temperature and pH) for both the enzymatic hydrolysis and the fermentation and the difficulty to recycle the fermenting organism and the enzymes. To satisfy the first requirement, the temperature is normally kept below 37°C, whereas the difficulty to recycle the yeast makes it beneficial to operate with a low yeast concentration and at a high solid loading. In this review, we make a brief overview of recent experimental work and development of SSF using lignocellulosic feedstocks. Significant progress has been made with respect to increasing the substrate loading, decreasing the yeast concentration and co-fermentation of both hexoses and pentoses during SSF. Presently, an SSF process for e.g. wheat straw hydrolyzate can be expected to give final ethanol concentrations close to 40 g L-1 with a yield based on total hexoses and pentoses higher than 70%. PMID:18471273

Immobilized lysozyme for the continuous lysis of lactic bacteria in wine: Bench-scale fluidized-bed reactor study.

PubMed

Cappannella, Elena; Benucci, Ilaria; Lombardelli, Claudio; Liburdi, Katia; Bavaro, Teodora; Esti, Marco

2016-11-01

Lysozyme from hen egg white (HEWL) was covalently immobilized on spherical supports based on microbial chitosan in order to develop a system for the continuous, efficient and food-grade enzymatic lysis of lactic bacteria (Oenococcus oeni) in white and red wine. The objective is to limit the sulfur dioxide dosage required to control malolactic fermentation, via a cell concentration typical during this process. The immobilization procedure was optimized in batch mode, evaluating the enzyme loading, the specific activity, and the kinetic parameters in model wine. Subsequently, a bench-scale fluidized-bed reactor was developed, applying the optimized process conditions. HEWL appeared more effective in the immobilized form than in the free one, when the reactor was applied in real white and red wine. This preliminary study suggests that covalent immobilization renders the enzyme less sensitive to the inhibitory effect of wine flavans. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

PubMed

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

2008-01-01

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

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.

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.

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

PubMed

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

2018-04-25

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

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

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.

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.

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.

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

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.

The avidity of cross-reactive virus-specific T cells for their viral and allogeneic epitopes is variable and depends on epitope expression.

PubMed

van den Heuvel, Heleen; Heutinck, Kirstin M; van der Meer-Prins, Ellen M W; Franke-van Dijk, Marry E I; van Miert, Paula P M C; Zhang, Xiaoqian; Ten Berge, Ineke J M; Claas, Frans H J

2018-01-01

Virus-specific T cells can recognize allogeneic HLA (allo-HLA) through cross-reactivity of their T-cell receptor (TCR). In a transplantation setting, such allo-HLA cross-reactivity may contribute to harmful immune responses towards the allograft, provided that the cross-reactive T cells get sufficiently activated upon recognition of the allo-HLA. An important determinant of T-cell activation is TCR avidity, which to date, has remained largely unexplored for allo-HLA-cross-reactive virus-specific T cells. For this purpose, cold target inhibition assays were performed using allo-HLA-cross-reactive virus-specific memory CD8 + T-cell clones as responders, and syngeneic cells loaded with viral peptide and allogeneic cells as hot (radioactively-labeled) and cold (non-radioactively-labeled) targets. CD8 dependency of the T-cell responses was assessed using interferon γ (IFNγ) enzyme-linked immunosorbent assay (ELISA) in the presence and absence of CD8-blocking antibodies. At high viral-peptide loading concentrations, T-cell clones consistently demonstrated lower avidity for allogeneic versus viral epitopes, but at suboptimal concentrations the opposite was observed. In line, anti-viral reactivity was CD8 independent at high, but not at suboptimal viral-peptide-loading concentrations. The avidity of allo-HLA-cross-reactive virus-specific memory CD8 + T cells is therefore highly dependent on epitope expression, and as a consequence, can be both higher and lower for allogeneic versus viral targets under different (patho)physiological conditions. Copyright © 2017 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

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

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.

Ethanol production from sunflower meal biomass by simultaneous saccharification and fermentation (SSF) with Kluyveromyces marxianus ATCC 36907.

PubMed

Camargo, Danielle; Gomes, Simone D; Sene, Luciane

2014-11-01

The lignocellulosic materials are considered promising renewable resources for ethanol production, but improvements in the processes should be studied to reduce operating costs. Thus, the appropriate enzyme loading for cellulose saccharification is critical for process economics. This study aimed at evaluating the concentration of cellulase and β-glucosidase in the production of bioethanol by simultaneous saccharification and fermentation (SSF) of sunflower meal biomass. The sunflower biomass was pretreated with 6% H2SO4 (w/v), at 121 °C, for 20 min, for hemicellulose removal and delignificated with 1% NaOH. SSF was performed with Kluyveromyces marxianus ATCC 36907, at 38 °C, 150 rpm, for 72 h, with different enzyme concentrations (Cellulase Complex NS22086-10, 15 and 20 FPU/gsubstrate and β-Glucosidase NS22118, with a cellulase to β-glucosidase ratio of 1.5:1; 2:1 and 3:1). The best condition for ethanol production was cellulase 20 FPU/gsubstrate and β-glucosidase 13.3 CBU/gsubstrate, resulting in 27.88 g/L ethanol, yield of 0.47 g/g and productivity of 0.38 g/L h. Under this condition the highest enzymatic conversion of cellulose to glucose was attained (87.06%).

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.

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.

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

PubMed

Salvador, Armindo; Savageau, Michael A

2006-02-14

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

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.

Enhanced enzymatic hydrolysis of lignocellulose by optimizing enzyme complexes.

PubMed

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

2010-03-01

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

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

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.

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.

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

PubMed

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

2011-12-01

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

Simultaneous saccharification and co-fermentation of paper sludge to ethanol by Saccharomyces cerevisiae RWB222--Part I: kinetic modeling and parameters.

PubMed

Zhang, Jiayi; Shao, Xiongjun; Townsend, Oliver V; Lynd, Lee R

2009-12-01

A kinetic model was developed to predict batch simultaneous saccharification and co-fermentation (SSCF) of paper sludge by the xylose-utilizing yeast Saccharomyces cerevisiae RWB222 and the commercial cellulase preparation Spezyme CP. The model accounts for cellulose and xylan enzymatic hydrolysis and competitive uptake of glucose and xylose. Experimental results show that glucan and xylan enzymatic hydrolysis are highly correlated, and that the low concentrations of xylose encountered during SSCF do not have a significant inhibitory effect on enzymatic hydrolysis. Ethanol is found to not only inhibit the specific growth rate, but also to accelerate cell death. Glucose and xylose uptake rates were found to be competitively inhibitory, but this did not have a large impact during SSCF because the sugar concentrations are low. The model was used to evaluate which constants had the greatest impact on ethanol titer for a fixed substrate loading, enzyme loading, and fermentation time. The cellulose adsorption capacity and cellulose hydrolysis rate constants were found to have the greatest impact among enzymatic hydrolysis related constants, and ethanol yield and maximum ethanol tolerance had the greatest impact among fermentation related constants.

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.

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.

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).

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

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.

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.

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.

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.

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

PubMed

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

2012-08-15

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

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.

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.

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

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.

Measurements of lactate in exhaled breath condensate at rest and after maximal exercise in young and healthy subjects.

PubMed

Marek, E M; Volke, J; Hawener, I; Platen, P; Mückenhoff, K; Marek, W

2010-03-01

Arterial lactate concentrations, taken as indicators of physical fitness, in athletes as well as in patients with cardio-respiratory or metabolic diseases, are measured invasively from arterialized ear lobe blood. Currently developed micro enzyme detectors permit a non-invasive measurement of hypoxia-related metabolites such as lactate in exhaled breath condensate (EBC). The aim of our study is to prove whether this technology will replace the traditional measurement of lactate in arterialized blood. Therefore, we determined the functional relation between lactate release in EBC and lactate concentration in blood in young and healthy subjects at rest and after exhausting bicycle exercise. During resting conditions as well as after exhausting bicycle exercise, 100 L of exhaled air along with blood samples from the ear lobe was collected after stationary load conditions in 16 healthy subjects. EBC was obtained by cooling the expired air volume with an ECoScreen I (FILT GmbH, Berlin) condenser. The analysis was performed within 90 min using an ECoCheck ampere meter (FILT GmbH, Berlin). Lactate measurements were performed using a bi-enzyme sensor after lactate oxidase-induced oxidation of lactate to pyruvate and H2O2. The rates of lactate release via the exhaled air were calculated from the lactate concentration, the volume and the collection time of the EBC. The functional relation of lactate release in exhaled air and lactate concentration of arterial blood was computed. At rest, the mean lactate concentration in arterialized blood was 0.93 ± 0.30 mmol L(-1). At a resting ventilation of 11.5 ± 3.4 L min(-1), the collection time for 100 L of exhaled air, Ts, was 8.4 ± 2.9 min, and 1.68 ± 0.40 mL EBC was obtained. In EBC, the lactate concentration was 21.4 ± 7.7 µmol L(-1), and the rate of lactate release rate in collected EBC was 4.5 ± 1.7 nmol min(-1). After maximal exercise load (220 ± 20 W), the blood lactate concentration increased to 10.9 ± 1.8 mmol L(-1) and the ventilation increased to 111.6 ± 21.4 L min(-1). The EBC collection time decreased to 3.9 ± 1.9 min, and 1.20 ± 0.44 mL EBC were obtained in the recovery period after termination of exercise. The lactate concentration in EBC increased to 40.3 ± 23.0 µmol L(-1), and the lactate release in EBC increased to 13.6 ± 8.6 nmol min(-1) (p < 0.01). Assuming a volume of 4.3 mL water in 100 L of exhaled air (saturated with water at 37 °C), we calculated a lactate release at rest of 11.5 ± 4.3 nmol min(-1) and 48.6 ± 30.7 nmol min(-1) (p < 0.01) after exhausting exercise. Detectable releases of lactate in exhaled breath condensate were found already under resting conditions. During exhausting external load on a bicycle spiroergometer, an increase in the lactate concentration was found in arterialized blood along with an increased lactate release in EBC. The correlation between expiratory lactate release via EBC and lactate concentration in arterialized blood is studied in pursuing investigations.

Effects of cadmium on the performance and microbiology of laboratory-scale lagoons treating domestic sewage.

PubMed

Bonnet, J L; Bohatier, J; Pépin, D

1999-06-01

Two experiments were performed to assess the impact of cadmium on the sewage lagoon wastewater treatment process. For each one, three laboratory-scale pilot plants with one tank receiving the same raw effluent were used; one plant served as control and the other two were contaminated once only with cadmium. In the first study, the effects of a shock load of two concentrations of cadmium chloride (60 and 300 micrograms/l) on the plant performance, microbial populations (protists and bacteria) and enzyme activities were determined. Initially, most of the performance parameters were affected concentration-dependently. A reduction in the protist population density and some influence on the total bacterial population were observed, and the potential enzymatic activities were also modified. A second experiment with a lower cadmium concentration (30 micrograms/l), supplied as chloride or sulphate, still perturbed most of the parameters studied, and the effects of the two cadmium salts were identical.

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.

Association between photosynthesis and contrasting features of minor veins in leaves of summer annuals loading phloem via symplastic versus apoplastic routes.

PubMed

Muller, Onno; Cohu, Christopher M; Stewart, Jared J; Protheroe, Johanna A; Demmig-Adams, Barbara; Adams, William W

2014-09-01

Foliar vascular anatomy and photosynthesis were evaluated for a number of summer annual species that either load sugars into the phloem via a symplastic route (Cucumis sativus L. cv. Straight Eight; Cucurbita pepo L. cv. Italian Zucchini Romanesco; Citrullus lanatus L. cv. Faerie Hybrid; Cucurbita pepo L. cv. Autumn Gold) or an apoplastic route (Nicotiana tabacum L.; Solanum lycopersicum L. cv. Brandywine; Gossypium hirsutum L.; Helianthus annuus L. cv. Soraya), as well as winter annual apoplastic loaders (Spinacia oleracea L. cv. Giant Nobel; Arabidopsis thaliana (L.) Heynhold Col-0, Swedish and Italian ecotypes). For all summer annuals, minor vein cross-sectional xylem area and tracheid number as well as the ratio of phloem loading cells to phloem sieve elements, each when normalized for foliar vein density (VD), was correlated with photosynthesis. These links presumably reflect (1) the xylem's role in providing water to meet foliar transpirational demand supporting photosynthesis and (2) the importance of the driving force of phloem loading as well as the cross-sectional area for phloem sap flux to match foliar photosynthate production. While photosynthesis correlated with the product of VD and cross-sectional phloem cell area among symplastic loaders, photosynthesis correlated with the product of VD and phloem cell number per vein among summer annual apoplastic loaders. Phloem cell size has thus apparently been a target of selection among symplastic loaders (where loading depends on enzyme concentration within loading cells) versus phloem cell number among apoplastic loaders (where loading depends on membrane transporter numbers). © 2014 Scandinavian Plant Physiology Society.

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.

Enhanced cellulase hydrolysis of eucalyptus waste fibers from pulp mill by Tween80-assisted ferric chloride pretreatment.

PubMed

Chen, Liheng; Fu, Shiyu

2013-04-03

Pretreatment combining FeCl3 and Tween80 was performed for cellulose-to-ethanol conversion of eucalyptus alkaline peroxide mechanical pulping waste fibers (EAWFs). The FeCl3 pretreatment alone showed a good effect on the enzymatic hydrolysis of EAWFs, but inhibited enzyme activity to some extent. A surfactant, Tween80, added during FeCl3 pretreatment was shown to significantly enhance enzyme reaction by eluting enzymatic inhibitors such as iron(III) that are present at the surface of the pretreated biomass. Treatment temperature, liquid-solid ratio, treatment time, FeCl3 concentration, and Tween80 dosage for pretreatment were optimized as follows: 180 °C, 8:1, 30 min, 0.15 mol/L, and 1% (w/v). Pretreated EAWFs under such optimal conditions provided enzymatic glucose (based on 100 g of oven-dried feedstock) and substrate enzymatic digestibility of EAWFs of 34.8 g and 91.3% after 72 h of enzymatic hydrolysis, respectively, with an initial cellulase loading of 20 FPU/g substrate.

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.

Ethanol/water pulps from sugar cane straw and their biobleaching with xylanase from Bacillus pumilus.

PubMed

Moriya, Regina Y; Gonçalves, Adilson R; Duarte, Marta C T

2007-04-01

The influence of independent variables (temperature and time) on the cooking of sugar cane straw with ethanol/water mixtures was studied to determine operating conditions that obtain pulp with high cellulose contents and a low lignin content. An experimental 2(2) design was applied for temperatures of 185 and 215 degrees C, and time of 1 and 2.5 h with the ethanol/water mixture concentration and constant straw-to-solvent ratio. The system was scaled-up at 200 degrees C cooking temperature for 2 h with 50% ethanol-water concentration, and 1:10 (w/v) straw-to-solvent ratio to obtain a pulp with 3.14 cP viscosity, 58.09 kappa-number, and the chemical composition of the pulps were 3.2% pentosan and 31.5% lignin. Xylanase from Bacillus pumilus was then applied at a loading of 5-150 IU/g dry pulp in the sugar cane straw ethanol/water pulp at 50 degrees C for 2 and 20 h. To ethanol/water pulps, the best enzyme dosage was found to be 20 IU/g dry pulp at 20 h, and a high enzyme dosage of 150 IU/g dry pulp did not decrease the kappa-number of the pulp.

Whole cell immobilization of refractory glucose isomerase using tris(hydroxymethyl)phosphine as crosslinker for preparation of high fructose corn syrup at elevated temperature.

PubMed

Jia, Dong-Xu; Wang, Teng; Liu, Zi-Jian; Jin, Li-Qun; Li, Jia-Jia; Liao, Cheng-Jun; Chen, De-Shui; Zheng, Yu-Guo

2018-04-04

Glucose isomerase (GI) responsible for catalyzing the isomerization from d-glucose to d-fructose, was an important enzyme for producing high fructose corn syrup (HFCS). In a quest to prepare HFCS at elevated temperature and facilitate enzymatic recovery, an effective procedure for whole cell immobilization of refractory Thermus oshimai glucose isomerase (ToGI) onto Celite 545 using tris(hydroxymethyl)phosphine (THP) as crosslinker was established. The immobilized biocatalyst showed an activity of approximate 127.3 U/(g·immobilized product) via optimization in terms of cells loading, crosslinker concentration and crosslinking time. The pH optimum of the immobilized biocatalyst was displaced from pH 8.0 of native enzyme to neutral pH 7.0. Compared with conventional glutaraldehyde (GLU)-immobilized cells, it possessed the enhanced thermostability with 70.1% residual activity retaining after incubation at 90°C for 72 h. Moreover, the THP-immobilized biocatalyst exhibited superior operational stability, in which it retained 85.8% of initial activity after 15 batches of bioconversion at 85°C. This study paved a way for reducing catalysis cost for upscale preparation of HFCS with higher d-fructose concentration. Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

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.

Influence of different nanoparticles on electrochemical behavior of glucose biosensor

NASA Astrophysics Data System (ADS)

Nenkova, R. D.; Ivanov, Y. L.; Godjevargova, T. I.

2017-02-01

The influence of nanosized particles on the glucose oxidase loading and the performance of amperometric glucose bionsensors were studied. Four enzyme electrodes (Pt/PAN/GOD, Pt/PAN/NZ/GOD, Pt/PAN/NZ/MNP/GOD, Pt/PAN/NZ/MWNT/GOD) were prepared by cross-linking of glucose oxidase (GOD) on nanocomposite material. Nanocomposites were prepared by entrapping nanozeolite (NZ), multiwalled carbon nanotubes (MWNT) and magnetic nanoparticles (MNP) in polyacrylonitrile (PAN) film. Cyclic voltammetric kinetic studies have been carried out with the four biosensors and the surface concentration of the adsorbed electroactive species on the electrodes was estimated. The highest enzyme concentration on the electrode surface corresponded to the electrodes prepared by nanozeolite separate (Pt/PAN/NZ/GOD) and combined with multi-walled carbon nanotubes (Pt/PAN/NZ/MWNT/GOD). The sensitivity of these two biosensors was the highest and that is in accordance with the greater amount of the adsorbed electroactive species on the electrodes (0.373 mol.cm-2). This was indication that a good synergistic effect happened when MWNTs and NZ were combined and these greatly improve the electron transfer ability of the sensor interface. Amperometric measurement of the two glucose oxidase electrodes (Pt/PAN/NZ/GOD and Pt/PAN/NZ/MWNT/GOD) with best results was carried out. The linear concentration interval of the Pt/PAN/NZ/MWNT/GOD biosensor was up to 3 mM, the detection limit - 0.02 mM glucose and the storage stability - 81% of its initial current response after 30 days.

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

Enhanced hydrolysis of cellulose hydrogels by morphological modification.

PubMed

Alfassi, Gilad; Rein, Dmitry M; Cohen, Yachin

2017-11-01

Cellulose is one of the most abundant bio-renewable materials on earth, yet the potential of cellulosic bio-fuels is not fully exploited, primarily due to the high costs of conversion. Hydrogel particles of regenerated cellulose constitute a useful substrate for enzymatic hydrolysis, due to their porous and amorphous structure. This article describes the influence of several structural aspects of the cellulose hydrogel on its hydrolysis. The hydrogel density was shown to be directly proportional to the cellulose concentration in the initial solution, thus affecting its hydrolysis rate. Using high-resolution scanning electron microscopy, we show that the hydrogel particles in aqueous suspension exhibit a dense external surface layer and a more porous internal network. Elimination of the external surface layer accelerated the hydrolysis rate by up to sixfold and rendered the process nearly independent of cellulose concentration. These findings may be of practical relevance to saccharification processing costs, by reducing required solvent quantities and enzyme load.

Action of multi-enzyme complex on protein extraction to obtain a protein concentrate from okara.

PubMed

de Figueiredo, Vitória Ribeiro Garcia; Yamashita, Fábio; Vanzela, André Luis Laforga; Ida, Elza Iouko; Kurozawa, Louise Emy

2018-04-01

The objective of this study was to optimize the extraction of protein by applying a multi-enzymatic pretreatment to okara, a byproduct from soymilk processing. The multi-enzyme complex Viscozyme, containing a variety of carbohydrases, was used to hydrolyze the okara cell walls and facilitate extraction of proteins. Enzyme-assisted extraction was carried out under different temperatures (37-53 °C), enzyme concentrations (1.5-4%) and pH values (5.5-6.5) according to a central composite rotatable design. After extraction, the protein was concentrated by isoelectric precipitation. The optimal conditions for maximum protein content and recovery in protein concentrate were 53 °C, pH 6.2 and 4% of enzyme concentration. Under these conditions, protein content of 56% (dry weight basis) and a recovery of 28% were obtained, representing an increase of 17 and 86%, respectively, compared to the sample with no enzymatic pretreatment. The multi-enzyme complex Viscozyme hydrolyzed the structural cell wall polysaccharides, improving extraction and obtaining protein concentrate from the okara. An electrophoretic profile of the protein concentrate showed two distinct bands, corresponding to the acidic and basic subunits of the protein glycinin. There were no limiting amino acids in the protein concentrate, which had a greater content of arginine.

Preparation and characterization of PLGA-β-CD polymeric nanoparticles containing methotrexate and evaluation of their effects on T47D cell line.

PubMed

Gorjikhah, Fatemeh; Azizi Jalalian, Farid; Salehi, Roya; Panahi, Yunes; Hasanzadeh, Arash; Alizadeh, Effat; Akbarzadeh, Abolfazl; Davaran, Soodabeh

2017-05-01

Among all cancers that affect women, breast cancer has most mortality rate. It is essential to attain more safe and efficient anticancer drugs. Recent advances in medical nanotechnology and biotechnology have caused in novel improvements in breast and other cancer drug delivery. Methotrexate is an anticancer drug that prevents the dihydrofolate reductase enzyme, which inhibits in the formation of DNA, RNA and proteins which have poor water-solubility. For enhancing the solubility and stability of drugs in delivery systems, we used methotrexate-loaded PLGA- beta-cyclodextrin nanoparticles. The PLGA- beta-cyclodextrin nanoparticles were synthesized by a double emulsion method and characterized with FT-IR and SEM. T47D breast cancer cell lines were treated with equal concentrations of methotrexate-loaded PLGA- beta-cyclodextrin nanoparticles and free methotrexate. MTT assay confirmed that methotrexate-loaded PLGA- beta-cyclodextrin nanoparticles enhanced cytotoxicity and drug delivery in T47D breast cancer cells. These results indicate that encapsulated drugs could be effective in controlled drug release for a sustained period would serve the purpose for long-term treatment of many diseases such as breast cancer.

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

PubMed

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

2017-10-01

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

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.

Lactic acid production with undefined mixed culture fermentation of potato peel waste.

PubMed

Liang, Shaobo; McDonald, Armando G; Coats, Erik R

2014-11-01

Potato peel waste (PPW) as zero value byproduct generated from food processing plant contains a large quantity of starch, non-starch polysaccharide, lignin, protein, and lipid. PPW as one promising carbon source can be managed and utilized to value added bioproducts through a simple fermentation process using undefined mixed cultures inoculated from wastewater treatment plant sludge. A series of non-pH controlled batch fermentations under different conditions such as pretreatment process, enzymatic hydrolysis, temperature, and solids loading were studied. Lactic acid (LA) was the major product, followed by acetic acid (AA) and ethanol under fermentation conditions without the presence of added hydrolytic enzymes. The maximum yields of LA, AA, and ethanol were respectively, 0.22 g g(-1), 0.06 g g(-1), and 0.05 g g(-1). The highest LA concentration of 14.7 g L(-1) was obtained from a bioreactor with initial solids loading of 60 g L(-1) at 35°C. Copyright © 2014 Elsevier Ltd. All rights reserved.

Efficient elimination of nonstoichiometric enzyme inhibitors from HTS hit lists.

PubMed

Habig, Michael; Blechschmidt, Anke; Dressler, Sigmar; Hess, Barbara; Patel, Viral; Billich, Andreas; Ostermeier, Christian; Beer, David; Klumpp, Martin

2009-07-01

High-throughput screening often identifies not only specific, stoichiometrically binding inhibitors but also undesired compounds that unspecifically interfere with the targeted activity by nonstoichiometrically binding, unfolding, and/or inactivating proteins. In this study, the effect of such unwanted inhibitors on several different enzyme targets was assessed based on screening results for over a million compounds. In particular, the shift in potency on variation of enzyme concentration was used as a means to identify nonstoichiometric inhibitors among the screening hits. These potency shifts depended on both compound structure and target enzyme. The approach was confirmed by statistical analysis of thousands of dose-response curves, which showed that the potency of competitive and therefore clearly stoichiometric inhibitors was not affected by increasing enzyme concentration. Light-scattering measurements of thermal protein unfolding further verified that compounds that stabilize protein structure by stoichiometric binding show the same potency irrespective of enzyme concentration. In summary, measuring inhibitor IC(50) values at different enzyme concentrations is a simple, cost-effective, and reliable method to identify and eliminate compounds that inhibit a specific target enzyme via nonstoichiometric mechanisms.

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.

Using T-RFLP data on denitrifier community composition to inform understanding of denitrification in stream sediments (Invited)

NASA Astrophysics Data System (ADS)

Wang, S.; Somers, K.; Sudduth, E.; Hassett, B.; Bernhardt, E. S.; Urban, D. L.

2010-12-01

We used terminal restriction fragment length polymorphism (T-RFLP), a molecular fingerprinting method, to characterize denitrifier communities in sediments taken from 48 study streams in North Carolina, USA. In addition to characterizing denitrifier communities, we also used denitrification enzyme activity (DEA) assays to measure potential denitrification rates. Due to differences in watershed land-use, study streams covered a gradient of nitrogen and carbon concentrations, as well as a gradient of contaminant loading from stormwater and sanitary sewers. Nitrogen and carbon (i.e., substrate) concentrations are commonly used to make predictions about denitrification rates in streams. Such models do not take into account denitrifier community composition, which may be an important, independent control of denitrification rates, particularly under stressful conditions (e.g., high contaminant loading) that prevent communities from capitalizing on high substrate availability. Our results indicate that substrate availability by itself was a weak predictor of denitrification rates; the same was also true for denitrifier community composition. However, when both factors were incorporated in a multiple regression model, the percent variation explained increased substantially. These findings suggest that T-RFLP, a relatively cost-effective method, can be used to improve our understanding of controls on denitrification rates in streams with varying watershed land-uses.

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.

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

PubMed

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

2014-11-15

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

Influence of disposable, concentric needle electrodes on muscle enzyme and lactate serum levels.

PubMed

Finsterer, Josef; Mittendorfer, Bettina; Neuhuber, Werner; Löscher, Wolfgang N

2002-08-01

Several studies addressed the question whether needle-EMG causes elevation of muscle enzymes [aspartate-aminotransferase, alanine-aminotransferase, lactate-dehydrogenase, creatine-phosphokinase (CPK), isoenzyme-MB, aldolase] and lactate with conflicting results. However, these studies used sterilizable needle electrodes and different protocols and methods to record EMGs and determine muscle enzymes. This study examined if muscle enzymes are elevated immediately after or 24 h following EMGs with disposable, concentric needle-electrodes, and if they are dependent on age, sex, muscle, number of investigated sites and previous CPK-elevation. In 53 subjects, 24 woman, 29 men, aged 17-88 years, muscle enzymes were determined before, immediately after and 24 h following EMG with disposable, concentric needle-electrodes. Muscle enzymes were not different before, immediately after and 24 h following the EMG. Muscle enzymes were not different between patients 60 years of age. Apart from higher CPK in men than women, muscle enzymes were not different between the genders. Apart from CPK, muscle enzymes were not different between the brachial biceps and anterior tibial muscle. Muscle enzymes were not different if 20 sites were investigated and were independent on pre-EMG CPK-levels. In conclusion this study shows that muscle enzymes do not increase immediately or 24 h following EMG with disposable, concentric needle-electrodes, irrespective of age, gender, muscle, number of investigated sites and pre-EMG CK-levels.

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

A novel amperometric enzyme inhibition biosensor based on xanthine oxidase immobilised onto glassy carbon electrodes for bisphenol A determination.

PubMed

Ben Messaoud, Najib; Ghica, Mariana Emilia; Dridi, Cherif; Ben Ali, Mounir; Brett, Christopher M A

2018-07-01

A novel and simple biosensor for the determination of bisphenol A (BPA) based on xanthine oxidase (XOD) enzymatic inhibition has been developed. The biosensor was prepared from xanthine oxidase immobilised by crosslinking with glutaraldehyde, with hypoxanthine as enzyme substrate, and was successfully applied to the determination of BPA using fixed potential amperometry. Biosensor performance was optimised with respect to the applied potential, influence of pH of the electrolyte solution, XOD loading and the substrate concentration. The enzyme inhibition mechanism was evaluated from Cornish-Bowden plus Dixon plots and was found to be reversible and competitive with an apparent inhibition constant of 8.15 nM. Under optimised conditions, the determination of BPA can be achieved in the linear range up to 41 nM with a detection limit of 1.0 nM, which is equal to the lowest reported in the literature, with very good repeatability and reproducibility. The selectivity of the biosensor was evaluated by performing an interference study and found to be excellent; and stability was investigated. It was successfully applied to the detection of BPA in mineral water and in river water. Copyright © 2018 Elsevier B.V. All rights reserved.

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

PubMed

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

2015-04-21

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

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

PubMed

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

2006-01-01

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

Application of a novel type impinging streams reactor in solid-liquid enzyme reactions and modeling of residence time distribution using GDB model.

PubMed

Fatourehchi, Niloufar; Sohrabi, Morteza; Dabir, Bahram; Royaee, Sayed Javid; Haji Malayeri, Adel

2014-02-05

Solid-liquid enzyme reactions constitute important processes in biochemical industries. The isomerization of d-glucose to d-fructose, using the immobilized glucose isomerase (Sweetzyme T), as a typical example of solid-liquid catalyzed reactions has been carried out in one stage and multi-stage novel type of impinging streams reactors. Response surface methodology was applied to determine the effects of certain pertinent parameters of the process namely axial velocity (A), feed concentration (B), nozzles' flow rates (C) and enzyme loading (D) on the performance of the apparatus. The results obtained from the conversion of glucose in this reactor were much higher than those expected in conventional reactors, while residence time was decreased dramatically. Residence time distribution (RTD) in a one-stage impinging streams reactor was investigated using colored solution as the tracer. The results showed that the flow pattern in the reactor was close to that in a continuous stirred tank reactor (CSTR). Based on the analysis of flow region in the reactor, gamma distribution model with bypass (GDB) was applied to study the RTD of the reactor. The results indicated that RTD in the impinging streams reactor could be described by the latter model. Copyright © 2013 Elsevier Inc. All rights reserved.

[Coenzyme-induced slow transitions of NADP-sorbitol dehydrogenase from Gluconobacter oxydans].

PubMed

Liber, E E; Dorozhko, A I; Pomortseva, N V

1978-06-01

The kinetic properties of NADP-dependent sorbitol dehydrogenase from G. oxydans cell extract were studied at pH 8.8 and 9.3 in the direction of D-sorbitol oxydation. It was shown that the shape of the kinetic curves of NADPH accumulation in time is characterised by initial burst whose magnitude depends on the concentration of the enzyme extract used. Preincubation of the enzyme with NADP or D-sorbitol eliminated the initial burst on these curves and transformed them into straight lines coming from the start of co-ordinates. The dependence of the stationary reaction rate on the enzyme extract concentration is not a linear one. The kinetic dependences of stationary rate of the reaction catalysed by the enzyme on the concentration of D-sorbitol and NADP at pH 8.8 and 9.3 were examined under all conditions studied; the shape of these kinetic curves altered to considerable extent with the alteration of the enzyme extract concentration in the reaction mixture and pH. At pH 9.3 several intermiediate plateaux were found on the curves of the D-sorbitol concentration dependent stationary rate of the reaction. The preincubation of the enzyme extract with NADP during 1.5 h removed the intermediate plateau on these curves and made them hyperbolic. Disk-electrophoresis of the enzyme extract in PAAG concentration gradient showed that at pH 8.8 the enzyme exists in one active form, while at pH 9.3 it exists in three major and three minor active forms of the enzyme differing in their molecular weights are found. It is assumed that the enzyme from G. oxydans cell extract can exist in a great number of molecular equilibrium forms, the rate of quilibrium being comparable or significantly less than that of the enzymatic reaction. NADP significantly influences on the equilibrium of the molecular forms of the enzyme.

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

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.

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.

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.

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.

Optimization of sodium hydroxide pretreatment and enzyme loading for efficient hydrolysis of rice straw to improve succinate production by metabolically engineered Escherichia coli KJ122 under simultaneous saccharification and fermentation.

PubMed

Sawisit, Apichai; Jampatesh, Surawee; Jantama, Sirima Suvarnakuta; Jantama, Kaemwich

2018-07-01

Rice straw was pretreated with sodium hydroxide (NaOH) before subsequent use for succinate production by Escherichia coli KJ122 under simultaneous saccharification and fermentation (SSF). The NaOH pretreated rice straw was significantly enhanced lignin removal up to 95%. With the optimized enzyme loading of 4% cellulase complex + 0.5% xylanase (endo-glucanase 67 CMC-U/g, β-glucosidase 26 pNG-U/g and xylanase 18 CMC-U/g dry biomass), total sugar conversion reached 91.7 ± 0.8% (w/w). The physicochemical analysis of NaOH pretreated rice straw indicated dramatical changes in its structure, thereby favoring enzymatic saccharification. In batch SSF, succinate production of 69.8 ± 0.3 g/L with yield and productivity of 0.84 g/g pretreated rice straw and 0.76 ± 0.02 g/L/h, respectively, was obtained. Fed-batch SSF significantly improved succinate concentration and productivity to 103.1 ± 0.4 g/L and 1.37 ± 0.07 g/L/h with a comparable yield. The results demonstrated a feasibility of sequential saccharification and fermentation of rice straw as a promising process for succinate production in industrial scale. Copyright © 2018 Elsevier Ltd. All rights reserved.

Yeast Acid Phosphatase in a Student Laboratory.

ERIC Educational Resources Information Center

Barbaric, Sloeodan; Ries, Blanka

1988-01-01

Examines the influence of enzyme and substrate concentrations, pH, temperature, and inhibitors on catalytic activity. Follows the influence of different phosphate concentrations in the growth medium on enzyme activity. Studies regulation of enzyme synthesis by repression. Includes methodology for six experiments. (MVL)

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.

The inhibitory effect of convulsant agents on the enzyme in brain which inactivates nerveside.

PubMed

Toh, C C

1969-07-01

1. An enzyme which can be extracted from brain inactivates nerveside in the optimum pH range 5.8-7.0.2. The polybasic acids trypan blue and its analogue trypan red, bromphenol blue and its analogue bromthymol blue at concentrations of 0.22 mM and ethylenediaminetetra-acetic acid (EDTA) at a concentration of 1 mM are strong inhibitors of the enzyme.3. Penicillin which is a monobasic carboxylic acid also inhibits the enzyme but only if concentrations as high as 3.6 mM are used. The antibiotic streptomycin which is a basic substance does not inhibit the enzyme.4. Caffeine at a concentration of 7.2 mM only weakly inhibits the enzyme.5. Chymotrypsin and wheat germ acid phosphatase also inactivate nerveside at pH 5.9 and are inhibited by the acidic dyes and penicillin. EDTA inhibits wheat germ phosphatase but activates chymotrypsin.6. Inactivation of nerveside by the brain enzyme and by wheat germ phosphatase is different from the action of chymotrypsin. Nerveside solutions incubated with chymotrypsin completely lose all biological activity whereas if incubation is carried out with either the brain enzyme or wheat germ acid phosphatase a residual biological activity remains even when the concentration of these two enzymes is increased. This residual biological activity is due to a peptide as it is destroyed by chymotrypsin.7. The manner in which nerveside is inactivated by the brain enzyme is uncertain as the preparation of the latter contained phosphodiesterase and protease activities which were similarly inhibited by the acid dyes, penicillin and EDTA.8. Pentylenetetrazole, picrotoxin, strychnine and tetanus toxin do not inhibit the brain enzyme.9. The nerveside-inactivating enzyme is not identical with the Substance P-inactivating enzyme in brain as the former is inhibited by EDTA while the latter is not.

Dual-mode lensless imaging device for digital enzyme linked immunosorbent assay

NASA Astrophysics Data System (ADS)

Sasagawa, Kiyotaka; Kim, Soo Heyon; Miyazawa, Kazuya; Takehara, Hironari; Noda, Toshihiko; Tokuda, Takashi; Iino, Ryota; Noji, Hiroyuki; Ohta, Jun

2014-03-01

Digital enzyme linked immunosorbent assay (ELISA) is an ultra-sensitive technology for detecting biomarkers and viruses etc. As a conventional ELISA technique, a target molecule is bonded to an antibody with an enzyme by antigen-antibody reaction. In this technology, a femto-liter droplet chamber array is used as reaction chambers. Due to its small volume, the concentration of fluorescent product by single enzyme can be sufficient for detection by a fluorescent microscopy. In this work, we demonstrate a miniaturized lensless imaging device for digital ELISA by using a custom image sensor. The pixel array of the sensor is coated with a 20 μm-thick yellow filter to eliminate excitation light at 470 nm and covered by a fiber optic plate (FOP) to protect the sensor without resolution degradation. The droplet chamber array formed on a 50μm-thick glass plate is directly placed on the FOP. In the digital ELISA, microbeads coated with antibody are loaded into the droplet chamber array, and the ratio of the fluorescent to the non-fluorescent chambers with the microbeads are observed. In the fluorescence imaging, the spatial resolution is degraded by the spreading through the glass plate because the fluorescence is irradiated omnidirectionally. This degradation is compensated by image processing and the resolution of ~35 μm was achieved. In the bright field imaging, the projected images of the beads with collimated illumination are observed. By varying the incident angle and image composition, microbeads were successfully imaged.

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.

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.

[Treatment of surface burns with proteolytic enzymes: mathematic description of lysis kinetics].

PubMed

Domogatskaia, A S; Domogatskiĭ, S P; Ruuge, E K

2003-01-01

The lysis of necrotic tissue by a proteolytic enzyme applied to the surface of a burn wound was studied. A mathematical model was proposed, which describes changes in the thickness of necrotic tissue as a function of the proteolytic activity of the enzyme. The model takes into account the inward-directed diffusion of the enzyme, the counterflow of interstitial fluid (exudates) containing specific inhibitors, and the extracellular matrix proteolysis. It was shown in terms of the quasi-stationary approach that the thickness of the necrotic tissue layer decreases exponentially with time; i.e., the lysis slows down as the thickness of the necrotic tissue layer decreases. The dependence of the characteristic time of this decrease on enzyme concentration was obtained. It was shown that, at high enzyme concentrations (more than 5 mg/ml), the entire time of lysis (after the establishment of quasi-stationary equilibrium) is inversely proportional to the concentration of the enzyme.

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.

Activation and thermostabilization effects of cyclic 2, 3-diphosphoglycerate on enzymes from the hyperthermophilic Methanopyrus kandleri.

PubMed

Shima, S; Hérault, D A; Berkessel, A; Thauer, R K

1998-11-01

Enzymes involved in methane formation from carbon dioxide and dihydrogen in Methanopyrus kandleri require high concentrations (> 1 M) of lyotropic salts such as K2HPO4/KH2PO4 or (NH4)2SO4 for activity and for thermostability. The requirement correlates with high intracellular concentrations of cyclic 2,3-diphosphoglycerate (cDPG; approximately 1 M) in this hyperthermophilic organism. We report here on the effects of potassium cDPG on the activity and thermostability of the two methanogenic enzymes cyclohydrolase and formyltransferase and show that at cDPG concentrations prevailing in the cells the investigated enzymes are highly active and completely thermostable. At molar concentrations also the potassium salts of phosphate and of 2,3-bisphosphoglycerate, the biosynthetic precursor of cDPG, were found to confer activity and thermostability to the enzymes. Thermodynamic arguments are discussed as to why cDPG, rather than these salts, is present in high concentrations in the cells of Mp. kandleri.

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...

Enzymatic hydrolysis of lignocellulosic biomass by Kitasatospora sp. to produce xylo-oligosaccharides (XOS)

NASA Astrophysics Data System (ADS)

Rahmani, Nanik; Jannah, Alifah Mafatikhul; Lisdiyanti, Puspita; Prasetya, Bambang; Yopi

2017-11-01

The optimizations of enzymatic hydrolysis to produce of xylo-oligosaccharides (XOs) from three different lignocellulosic biomasses were investigated. Sugarcane bagasse, oil palm empty fruit bunch, and rice straw contain rich hemicelluloses especially hetero-xylan which can be hydrolyzes by endo-xylanase enzyme. Enzymatic hydrolysis of sugarcane bagasse by endo-xylanase from Kitasatospora sp. was optimum at temperature hydrolysis 30 °C using 16 U of enzyme concentrations and 4 % substrate concentrations, while oil palm empty fruit bunchwas optimum at temperature hydrolysis 30 °C using 16 U of enzyme concentrations and 5 % substrate concentrations, and rice straw was optimum at 40 °C temperature hydrolysis using 16 U of enzyme concentrations and 4 % substrate concentrations. The hydrolysis products were analyzed by TLC and HPLC. The main product hydrolysis for sugarcane bagasse, oil palm empty fruit bunch and rice straw are xylobiose.

Optimization of enzymatic hydrolysis for ethanol production by simultaneous saccharification and fermentation of wastepaper.

PubMed

Sangkharak, Kanokphorn

2011-11-01

The present study investigated the development of high sugar production by optimization of an enzymatic hydrolysis process using both conventional and statistical methods, as well as the production of ethanol by the selected wastepaper source. Among four sources of pretreated wastepaper including office paper, newspaper, handbills and cardboard, office paper gave the highest values of cellulose (87.12%) and holocelluloses (89.07%). The effects of the amount of wastepaper, the pretreatment method and the type of enzyme on reducing sugar production from office paper were studied using conventional methods. The highest reducing sugar production (1851.28 µg L(-1); 37.03% conversion of glucose) was obtained from the optimal condition containing 40 mg of office paper, pretreated with stream explosion and hydrolysed with the combination of cellulase from Aspergillus niger and Trichoderma viride at the fixed loading rate of 20 FPU g(-1) sample. The effects of interaction of wastepaper amount and enzyme concentration as well as incubation time were studied by a statistical method using central composite design. The optimal medium composition consisted of 43.97 µg L(-1), 28.14 FPU g(-1) sample and 53.73 h of wastepaper, enzyme concentration and incubation time, respectively, and gave the highest amount of sugar production (2184.22 µg L(-1)) and percentage conversion of glucose (43.68%). The ethanol production from pretreated office paper using Saccharomyces cerevisiae in a simultaneous saccharification and fermentation process was 21.02 g L(-1) after 36 h of cultivation, corresponding to an ethanol volumetric production rate of 0.58 g ethanol L(-1) h(-1).

Laccase-catalyzed removal of 2,4-dimethylphenol from synthetic wastewater: effect of polyethylene glycol and dissolved oxygen.

PubMed

Ghosh, J P; Taylor, K E; Bewtra, J K; Biswas, N

2008-04-01

The potential use of laccase (SP-504) in an advanced oxidation-based treatment technology to remove 2,4-dimethylphenol (DMP) from water was investigated with and without the additive, polyethylene glycol (PEG). The DMP concentration was varied between 1.0 and 5.0 mM. The optimization of pH and enzyme concentration in the presence and absence of PEG was carried out. All experiments were carried out in continuously stirred reactors for 3h at room temperature. The reaction was initiated by adding enzyme to the reaction mixture. For more than 95% removal of DMP, the presence of PEG reduced the inactivation of enzyme so that the required enzyme concentrations were reduced by about 2-fold compared to the same reactions in the absence of PEG. Finally, the PEG concentrations were optimized to obtain the minimum dose required. For higher substrate concentrations, the availability of oxygen was insufficient in achieving 95% or more removal. Therefore, the effect of increasing dissolved oxygen at higher substrate concentration was investigated. The laccase studied was capable of efficiently removing DMP at very low enzyme concentrations and hence shows great potential for cost-effective industrial applications.

Laccase-syringaldehyde-mediated degradation of trace organic contaminants in an enzymatic membrane reactor: Removal efficiency and effluent toxicity.

PubMed

Nguyen, Luong N; van de Merwe, Jason P; Hai, Faisal I; Leusch, Frederic D L; Kang, Jinguo; Price, William E; Roddick, Felicity; Magram, Saleh F; Nghiem, Long D

2016-01-01

Redox-mediators such as syringaldehyde (SA) can improve laccase-catalyzed degradation of trace organic contaminants (TrOCs) but may increase effluent toxicity. The degradation performance of 14 phenolic and 17 non-phenolic TrOCs by a continuous flow enzymatic membrane reactor (EMR) at different TrOC and SA loadings was assessed. A specific emphasis was placed on the investigation of the toxicity of the enzyme (laccase), SA, TrOCs and the treated effluent. Batch tests demonstrated significant individual and interactive toxicity of the laccase and SA preparations. Reduced removal of resistant TrOCs by the EMR was observed for dosages over 50μg/L. SA addition at a concentration of 10μM significantly improved TrOC removal, but no removal improvement was observed at the elevated SA concentrations of 50 and 100μM. The treated effluent showed significant toxicity at SA concentrations beyond 10μM, providing further evidence that higher dosage of SA must be avoided. Copyright © 2015 Elsevier Ltd. All rights reserved.

Sensitivity of bacterioplankton nitrogen metabolism to eutrophication in sub-tropical coastal waters of Key West, Florida.

PubMed

Hoch, Matthew P; Dillon, Kevin S; Coffin, Richard B; Cifuentes, Luis A

2008-05-01

Expression of intracellular ammonium assimilation enzymes were used to assess the response of nitrogen (N) metabolism in bacterioplankton to N-loading of sub-tropical coastal waters of Key West, Florida. Specific activities of glutamine synthetase (GS) and total glutamate dehydrogenase (GDHT) were measured on the bacterial size fraction (<0.8 microm) to assess N-deplete versus N-replete metabolic states, respectively. Enzyme results were compared to concentrations of dissolved organic matter and nutrients and to the biomass and production of phytoplankton and bacteria. Concentrations of dissolved inorganic N (DIN), dissolved organic N (DON), and dissolved organic carbon (DOC) positively correlated with specific activities of GDHT and negatively correlated with that of GS. Total dissolved N (TDN) concentration explained 81% of variance in bacterioplankton GDHT:GS activity ratio. The GDHT:GS ratio, TDN, DOC, and bacterial parameters decreased in magnitude along a tidally dynamic trophic gradient from north of Key West to south at the reef tract, which is consistent with the combined effects of localized coastal eutrophication and tidal exchange of seawater from the Southwest Florida Shelf and Florida Strait. The N-replete bacterioplankton north of Key West can regenerate ammonium which sustains primary production transported south to the reef. The range in GDHT:GS ratios was 5-30 times greater than that for commonly used indicators of planktonic eutrophication, which emphasizes the sensitivity of bacterioplankton N-metabolism to changes in N-bioavailability caused by nutrient pollution in sub-tropical coastal waters and utility of GDHT:GS ratio as an bioindicator of N-replete conditions.

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

Aqueous vascular endothelial growth factor and aflibercept concentrations after bimonthly intravitreal injections of aflibercept for age-related macular degeneration.

PubMed

Sawada, Tomoko; Wang, Xiying; Sawada, Osamu; Saishin, Yoshitsugu; Ohji, Masahito

2018-01-01

Clinical evidence supports the efficacy of bimonthly aflibercept injection for age-related macular degeneration. The study aimed to evaluate aqueous vascular endothelial growth factor and aflibercept concentrations and the efficacy of bimonthly aflibercept in patients with age-related macular degeneration. This study is a prospective, interventional case series. Enrolled were 35 eyes with exudative age-related macular degeneration from 35 patients. Patients received three bimonthly intravitreal aflibercept without loading doses. We collected the aqueous humor just before each injection, measured vascular endothelial growth factor and aflibercept concentrations by enzyme-linked immunosorbent assay and measured best-corrected visual acuity and central retinal subfield thickness before and after the injections. Aqueous vascular endothelial growth factor and aflibercept concentrations were measured. The vascular endothelial growth factor concentration was 135.4 ± 60.5 pg/mL (mean ± standard deviation, range 60.6-323.4) at baseline and below the lowest detectable limit in all eyes at month 2 and in 32 eyes at month 4 (P < 0.001 [month 2] and P < 0.001 [month 4]). The mean aflibercept concentration was 20.3 ng/mL at month 2 and 28.0 ng/mL at month 4. The mean logarithm of the minimum angle of resolution visual acuity improved from 0.50 ± 0.36 at baseline to 0.36 ± 0.40 at month 6 (P < 0.001). The mean central retinal subfield thickness decreased from 353 ± 100 μm at baseline to 236 ± 45 μm at month 6 (P < 0.001). Bimonthly aflibercept injections without loading doses may be considered a treatment option for age-related macular degeneration. © 2017 Royal Australian and New Zealand College of Ophthalmologists.

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.

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

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-...

Sulfhydryl oxidases: emerging catalysts of protein disulfide bond formation in eukaryotes.

PubMed

Thorpe, Colin; Hoober, Karen L; Raje, Sonali; Glynn, Nicole M; Burnside, Joan; Turi, George K; Coppock, Donald L

2002-09-01

Members of the Quiescin-sulfhydryl oxidase (QSOX) family utilize a thioredoxin domain and a small FAD-binding domain homologous to the yeast ERV1p protein to oxidize sulfhydryl groups to disulfides with the reduction of oxygen to hydrogen peroxide. QSOX enzymes are found in all multicellular organisms for which complete genomes exist and in Trypanosoma brucei, but are not found in yeast. The avian QSOX is the best understood enzymatically: its preferred substrates are peptides and proteins, not monothiols such as glutathione. Mixtures of avian QSOX and protein disulfide isomerase catalyze the rapid insertion of the correct disulfide pairings in reduced RNase. Immunohistochemical studies of human tissues show a marked and highly localized concentration of QSOX in cell types associated with heavy secretory loads. Consistent with this role in the formation of disulfide bonds, QSOX is typically found in the cell in the endoplasmic reticulum and Golgi and outside the cell. In sum, this review suggests that QSOX enzymes play a significant role in oxidative folding of a large variety of proteins in a wide range of multicellular organisms.

Lignin from bamboo shoot shells as an activator and novel immobilizing support for α-amylase.

PubMed

Gong, Weihua; Ran, Zhanxiang; Ye, Fayin; Zhao, Guohua

2017-08-01

This study examined the feasibility of α-amylase activation and immobilization, using lignin from bamboo shoot shells (BSS). Our results demonstrated that BSS lignin is an excellent α-amylase activator and it elevated α-amylase activity more than two-fold at a concentration of 5mg/ml. For immobilization of α-amylase via adsorption, BSS lignin was incubated in an α-amylase solution (5mg/ml) for 20min, and the maximum specific activity, amount of loaded protein and activity recovery were 92.4U/mg, 19.0mg/g and 111%, respectively. In contrast to its free counterpart, immobilized α-amylase improved the catalytic efficiency and storage stability, under comparable working conditions (temperature and pH). Regarding its convenient usage, immobilized enzyme can be suspended in advance, but a suspension incubated at 60°C should be used within 30min. The residual activity after 14 re-uses remained at a reasonable level (53.2%). In conclusion, this study reveals a novel support for enzyme immobilization. Copyright © 2017 Elsevier Ltd. All rights reserved.

Metronidazole loaded pectin microspheres for colon targeting.

PubMed

Vaidya, Ankur; Jain, Aviral; Khare, Piush; Agrawal, Ram K; Jain, Sanjay K

2009-11-01

A multiparticulate system having pH-sensitive property and specific enzyme biodegradability for colon-targeted delivery of metronidazole was developed. Pectin microspheres were prepared using emulsion-dehydration technique. These microspheres were coated with Eudragit(R) S-100 using oil-in-oil solvent evaporation method. The SEM was used to characterize the surface of these microspheres and a distinct coating over microspheres could be seen. The in vitro drug release studies exhibited no drug release at gastric pH, however continuous release of drug was observed from the formulation at colonic pH. Further, the release of drug from formulation was found to be higher in the presence of rat caecal contents, indicating the effect of colonic enzymes on the pectin microspheres. The in vivo studies were also performed by assessing the drug concentration in various parts of the GIT at different time intervals which exhibited the potentiality of formulation for colon targeting. Hence, it can be concluded that Eudragit coated pectin microspheres can be used for the colon specific delivery of drug. (c) 2009 Wiley-Liss, Inc. and the American Pharmacists Association

Fluorimetric urease inhibition assay on a multilayer microfluidic chip with immunoaffinity immobilized enzyme reactors.

PubMed

Zhang, Qin; Tang, Xiuwen; Hou, Fenghua; Yang, Jianping; Xie, Zhiyong; Cheng, Zhiyi

2013-10-01

We fabricated a three-layer polydimethylsiloxane (PDMS)-based microfluidic chip for realizing urease inhibition assay with sensitive fluorescence detection. Procedures such as sample prehandling, enzyme reaction, reagent mixing, fluorescence derivatization, and detection can be readily carried out. Urease reactors were prepared by adsorption of rabbit immunoglobulin G (IgG) and immunoreaction with urease-conjugated goat anti-rabbit IgG. Acetohydroxamic acid (AHA) as a competitive inhibitor of urease was tested on the chip. Microfluidically generated gradient concentrations of AHA with substrate (urea) were loaded into urease reactors. After incubation, the produced ammonia was transported out of reactors and then reacted with o-phthalaldehyde (OPA) to generate fluorescent products. Urease inhibition was indicated by a decrease in fluorescence signal detected by microplate reader. The IC50 value of AHA was determined and showed good agreement with that obtained in microplate. The presented device combines several steps of the analytical process with advantages of low reagent consumption, reduced analysis time, and ease of manipulation. This microfluidic approach can be extended to the screening of inhibitory compounds in drug discovery. Copyright © 2013 Elsevier Inc. All rights reserved.

[Research on the preparative method of Arctigenin].

PubMed

Zhang, Li-Ying; Yang, Yi-Shun; Zhang, Tong; Ding, Yue; Cai, Zhen-Zhen; Tao, Jian-Sheng

2012-03-01

To research on the preparation of Arctigenin in vitro. Took enzyme concentration, time course and substrate concentration as investigation factors, used Box-Behnken design-response surface methodology to optimize the enzyme hydrolysis path of Arctigenin. The best operational path for Arctigenin was as follows: the temperature was 50 degrees C, pH was 4.8, enzyme concentration was 0.44 U/mL, time course was 46.81 min, substrate concentration was 0.29 mg/mL, the conversion rate was 90.94%. This research can be regarded as a referencein preparing Arctigenin in vitro.

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.

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.

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.

Occurrence and transport of diazinon in the Sacramento River, California, and selected tributaries during three winter storms, January-February 2000

USGS Publications Warehouse

Dileanis, Peter D.; Bennett, Kevin P.; Domagalski, Joseph L.

2002-01-01

The organophosphate pesticide diazinon is applied as a dormant orchard spray in the Sacramento Valley, California, during the winter when the area receives a majority of its annual rainfall. Dormant spray pesticides, thus, have the potential to wash off the areas of application and migrate with storm runoff to streams in the Sacramento River Basin. Previous monitoring studies have shown that rain and associated runoff from winter storms plays an important role in the transport of diazinon from point of application to the Sacramento River and tributaries. Between January 30 and February 25, 2000, diazinon concentrations in the Sacramento River and selected tributaries were monitored on 5 consecutive days during each of three winter storms that moved through the Sacramento Valley after diazinon had been applied to orchards in the basin. Water samples were collected at 17 sites chosen to represent the effect of upstream land use at local and regional scales. Most samples were analyzed using an enzyme-linked immunosorbent assay (ELISA). Analysis by gas chromatography with electron capture detector and thermionic specific detector (GC/ECD/TSD) and gas chromatography with mass spectrometry (GC/MS) was done on split replicates from over 30 percent of the samples to confirm ELISA results and to provide lower analytical reporting limits at selected sites [30 ng/L (nanogram per liter) for ELISA, 20 ng/L for GC/ECD/TSD, and 2 ng/L for GC/MS]. Concentrations determined from ELISA analyses were consistently higher than concentrations for split samples analyzed by gas chromatography methods. Because of bias between diazinon concentrations using ELISA and gas chromatography methods, results from ELISA analyses were not compared to water-quality criteria. Load calculations using the ELISA analyses are similarly biased. Because the bias was consistent, however, the ELISA data is useful in site-to-site comparisons used to rank the relative levels and contributions of diazinon from individual subbasins in the watershed. Concentrations of diazinon in 138 samples analyzed by gas chromatography methods ranged from below detection (2 ng/L) to 2,890 ng/L with a median of 44 ng/L. Thirty percent of the samples had concentrations greater than 80 ng/L, which is considered by California as the criterion maximum concentration for the protection of aquatic habitat. Concentrations were highest in small tributaries and canals draining subbasins with predominantly agricultural land use and in a channel draining the Yuba City urban area. Load estimates using concentrations derived from GC/MS analyses indicate that about 30 percent of the diazinon in the lower Sacramento River is from the Feather River Basin. Loads estimated using ELISA analyses show a similar, but slightly higher fraction of the total load coming from that basin. The source of over half the total load measured at Sacramento River at Alamar appears to have originated in the part of the drainage basin upstream of the city of Colusa. Of the diazinon reported applied to agricultural land in Sacramento Valley (about 42,500 pounds active ingredient) just before and during the monitoring period, about 0.4 percent appeared to be transported to the lower Sacramento River during the period of monitoring. A similar percent of applied diazinon was estimated to have entered the Feather River from upstream sources. Diazinon use in the study area during the 1999-2000 dormant spray season was unusually low, about 60 percent of the average of the previous 4 years. Therefore, diazinon loadings may be higher in subsequent years, should use increase and pesticide management practices remain the same. Although diazinon was the most frequently detected pesticide and the pesticide detected at the highest concentrations, 10 other pesticides were detected in the samples collected. These included the insecticides methidathion and chlorpyrifos, and the herbicides simazine, molinate and thiobencarb.

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.

Introducing Michaelis-Menten Kinetics through Simulation

ERIC Educational Resources Information Center

Halkides, Christopher J.; Herman, Russell

2007-01-01

We describe a computer tutorial that introduces the concept of the steady state in enzyme kinetics. The tutorial allows students to produce graphs of the concentrations of free enzyme, enzyme-substrate complex, and product versus time in order to learn about the approach to steady state. By using a range of substrate concentrations and rate…

Krebs cycle metabolon formation: metabolite concentration gradient enhanced compartmentation of sequential enzymes.

PubMed

Wu, Fei; Pelster, Lindsey N; Minteer, Shelley D

2015-01-25

Dynamics of metabolon formation in mitochondria was probed by studying diffusional motion of two sequential Krebs cycle enzymes in a microfluidic channel. Enhanced directional co-diffusion of both enzymes against a substrate concentration gradient was observed in the presence of intermediate generation. This reveals a metabolite directed compartmentation of metabolic pathways.

Purification and properties of rennin-like enzyme from Aspergillus ochraceus.

PubMed

Ismail, A A; Foda, M S; Khorshid, M A

1978-01-01

An active milk-clotting enzyme was purified some 40-fold from culture supernatant of Aspergillus ochraceus. The purification steps included ammonium sulfate precipitation, G-100 Sephadex gel filtration, and ion exchange chromatography, using DEAE Cellulose column. The enzyme exhibited milk-clotting activity and proteolytic behaviour, an optimum at pH 6.0 and in the range of 7--8.5, respectively. The purified enzyme was actively proteolytic against casein, haemoglobin, and bovine serum albumin at pH 8. The milk-clotting activity was greatly enhanced by manganous ions and by increasing concentrations of calcium chloride. Copper, zinc, and ammonium ions were potent inhibitors of the milk-curdling activity of the purified enzyme. Significant inhibition was also noted with sodium chloride at concentrations of 3% or more. Under the specified reaction condition, maximum rate of proteolysis against casein was obtained at 0.4% substrate concentration, whereas the milk-clotting time was linear proportional to dry skim milk concentration in the range of 8 to 24%. The results are discussed in comparison with other microbial milk-clotting enzymes, and limitations of applicability are also presented.

Decreased Plasma Brain-Derived Neurotrophic Factor and Vascular Endothelial Growth Factor Concentrations during Military Training

PubMed Central

Nibuya, Masashi; Ishida, Toru; Yamamoto, Tetsuo; Mukai, Yasuo; Mitani, Keiji; Tsumatori, Gentaro; Scott, Daniel; Shimizu, Kunio

2014-01-01

Decreased concentrations of plasma brain-derived neurotrophic factor (BDNF) and serum BDNF have been proposed to be a state marker of depression and a biological indicator of loaded psychosocial stress. Stress evaluations of participants in military mission are critically important and appropriate objective biological parameters that evaluate stress are needed. In military circumstances, there are several problems to adopt plasma BDNF concentration as a stress biomarker. First, in addition to psychosocial stress, military missions inevitably involve physical exercise that increases plasma BDNF concentrations. Second, most participants in the mission do not have adequate quality or quantity of sleep, and sleep deprivation has also been reported to increase plasma BDNF concentration. We evaluated plasma BDNF concentrations in 52 participants on a 9-week military mission. The present study revealed that plasma BDNF concentration significantly decreased despite elevated serum enzymes that escaped from muscle and decreased quantity and quality of sleep, as detected by a wearable watch-type sensor. In addition, we observed a significant decrease in plasma vascular endothelial growth factor (VEGF) during the mission. VEGF is also neurotrophic and its expression in the brain has been reported to be up-regulated by antidepressive treatments and down-regulated by stress. This is the first report of decreased plasma VEGF concentrations by stress. We conclude that decreased plasma concentrations of neurotrophins can be candidates for mental stress indicators in actual stressful environments that include physical exercise and limited sleep. PMID:24586790

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.

Phosphate-dependent glutaminase in enterocyte mitochondria and its regulation by ammonium and other ions.

PubMed

Masola, B; Zvinavashe, E

2003-06-01

The effects of ammonium and other ions on phosphate dependent glutaminase (PDG) activity in intact rat enterocyte mitochondria were investigated. Sulphate and bicarbonate activated the enzyme in absence and presence of added phosphate. In presence of 10 mM phosphate, ammonium at concentrations <1 mM inhibited the enzyme. This inhibition was reversed by increased concentration of phosphate or sulphate. The inhibition of PDG by ammonium in presence of 10 mM phosphate was biphasic with respect to glutamine concentration, its effect being through a lowering of V(max) at glutamine concentration of

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

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.

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.

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.

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 ...

A theoretical study of concentration of profiles of primary cytochemical-enzyme reaction products in membrane-bound cell organelles and its application to lysosomal acid phosphatase.

PubMed

Cornelisse, C J; Hermens, W T; Joe, M T; Duijndam, W A; van Duijn, P

1976-11-01

A numerical method was developed for computing the steady-state concentration gradient of a diffusible enzyme reaction product in a membrane-limited compartment of a simplified theoretical cell model. In cytochemical enzyme reactions proceeding according to the metal-capture principle, the local concentration of the primary reaction product is an important factor in the onset of the precipitation process and in the distribution of the final reaction product. The following variables were incorporated into the model: enzyme activity, substrate concentration, Km, diffusion coefficient of substrate and product, particle radius and cell radius. The method was applied to lysosomal acid phosphatase. Numerical values for the variables were estimated from experimental data in the literature. The results show that the calculated phosphate concentrations inside lysosomes are several orders of magnitude lower than the critical concentrations for efficient phosphate capture found in a previous experimental model study. Reasons for this apparent discrepancy are discussed.

Statistical properties of fluctuating enzymes with dynamic cooperativity using a first passage time distribution formalism.

PubMed

Singh, Divya; Chaudhury, Srabanti

2017-04-14

We study the temporal fluctuations in catalytic rates for single enzyme reactions undergoing slow transitions between two active states. We use a first passage time distribution formalism to obtain the closed-form analytical expressions of the mean reaction time and the randomness parameter for reaction schemes where conformational fluctuations are present between two free enzyme conformers. Our studies confirm that the sole presence of free enzyme fluctuations yields a non Michaelis-Menten equation and can lead to dynamic cooperativity. The randomness parameter, which is a measure of the dynamic disorder in the system, converges to unity at a high substrate concentration. If slow fluctuations are present between the enzyme-substrate conformers (off-pathway mechanism), dynamic disorder is present at a high substrate concentration. Our results confirm that the dynamic disorder at a high substrate concentration is determined only by the slow fluctuations between the enzyme-substrate conformers and the randomness parameter is greater than unity. Slow conformational fluctuations between free enzymes are responsible for the emergence of dynamic cooperativity in single enzymes. Our theoretical findings are well supported by comparison with experimental data on the single enzyme beta-galactosidase.

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.

Improving the enzymatic hydrolysis of dilute acid pretreated wheat straw by metal ion blocking of non-productive cellulase adsorption on lignin.

PubMed

Akimkulova, Ardak; Zhou, Yan; Zhao, Xuebing; Liu, Dehua

2016-05-01

Eleven salts were selected to screen the possible metal ions for blocking the non-productive adsorption of cellulase onto the lignin of dilute acid pretreated wheat straw. Mg(2+) was screened finally as the promising candidate. The optimal concentration of MgCl2 was 1 mM, but the beneficial action was also dependent on pH, hydrolysis time and cellulase loading. Significant improvement of glucan conversion (19.3%) was observed at low cellulase loading (5 FPU/g solid). Addition of isolated lignins, tannic acid and lignin model compounds to pure cellulose hydrolysis demonstrated that phenolic hydroxyl group (Ph-OH) was the main active site blocked by Mg(2+). The interaction between Mg(2+) and Ph-OH of lignin monomeric moieties followed an order of p-hydroxyphenyl (H)>guaiacyl (G)>syringyl (S). Mg(2+) blocking made the lignin surface less negatively charged, which might weaken the hydrogen bonding and electrostatically attractive interaction between lignin and cellulase enzymes. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

NASA Astrophysics Data System (ADS)

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

2015-12-01

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

Mechanisms of Enhanced Catalysis in Enzyme-DNA Nanostructures Revealed through Molecular Simulations and Experimental Analysis.

PubMed

Gao, Yingning; Roberts, Christopher C; Toop, Aaron; Chang, Chia-En A; Wheeldon, Ian

2016-08-03

Understanding and controlling the molecular interactions between enzyme substrates and DNA nanostructures has important implications in the advancement of enzyme-DNA technologies as solutions in biocatalysis. Such hybrid nanostructures can be used to create enzyme systems with enhanced catalysis by controlling the local chemical and physical environments and the spatial organization of enzymes. Here we have used molecular simulations with corresponding experiments to describe a mechanism of enhanced catalysis due to locally increased substrate concentrations. With a series of DNA nanostructures conjugated to horseradish peroxidase, we show that binding interactions between substrates and the DNA structures can increase local substrate concentrations. Increased local substrate concentrations in HRP(DNA) nanostructures resulted in 2.9- and 2.4-fold decreases in the apparent Michaelis constants of tetramethylbenzidine and 4-aminophenol, substrates of HRP with tunable binding interactions to DNA nanostructures with dissociation constants in the micromolar range. Molecular simulations and kinetic analysis also revealed that increased local substrate concentrations enhanced the rates of substrate association. Identification of the mechanism of increased local concentration of substrates in close proximity to enzymes and their active sites adds to our understanding of nanostructured biocatalysis from which we can develop guidelines for enhancing catalysis in rationally designed systems. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Breaking The Enzymatic Latch: Do Anaerobic Conditions Constrain Decomposition In Humid Tropical Forest Soil?

NASA Astrophysics Data System (ADS)

Hall, S. J.; Silver, W. L.

2011-12-01

Anaerobic conditions have been proposed to impose a "latch" on soil organic matter decomposition by inhibiting the activity of extracellular enzymes that catalyze the transformation of organic polymers into monomers for microbial assimilation. Here, we tested the hypothesis that anaerobiosis inhibits soil hydrolytic enzyme activity in a humid tropical forest ecosystem in Puerto Rico. We sampled surface and sub-surface soil from each of 59 plots (n = 118) stratified across distinct topographical zones (ridges, slopes, and valleys) known to vary in soil oxygen (O2) concentrations, and measured the potential activity of five hydrolytic enzymes that decompose carbon (C), nitrogen (N), and phosphorus (P) substrates. We measured reduced iron (Fe (II)) concentrations in soil extractions to provide a spatially and temporally integrated index of anaerobic microbial activity, since iron oxides constitute the dominant anaerobic terminal electron acceptor in this ecosystem. Surprisingly, we observed positive relationships between Fe (II) concentrations and the activity of all enzymes that we assayed. Linear mixed effects models that included Fe (II) concentration, topographic position, and their interaction explained between 30 to 70 % of the variance of enzyme activity of β-1,4-glucosidase, β-cellobiohydrolase, β-xylosidase, N-acetylglucosaminidase, and acid phosphatase. Soils from ridges and slopes contained between 10 and 800 μg Fe (II) g-1 soil, and exhibited consistently positive relationships (p < 0.0001) between Fe (II) and enzyme activity. Valley soils did not display significant relationships between enzyme activity and Fe (II), although they displayed variation in soil Fe (II) concentrations similar to ridges and slopes. Overall, valleys exhibited lower enzyme activity and lower Fe (II) concentrations than ridges or slopes, possibly related to decreased root biomass and soil C. Our data provide no indication that anaerobiosis suppresses soil enzyme activity, but rather that high rates of decomposition induce a higher proportion of anaerobiosis soil microsites. The spatial patterns of Fe (II) concentrations that we observed also support this hypothesis. Soil Fe (II) concentrations were significantly greater in ridges than in slopes or valleys, in spite of the fact that slopes and valleys tend to experience higher soil moisture and lower bulk soil O2 concentrations. In our samples, Fe (II) concentrations correlated only weakly with ambient soil moisture, suggesting the importance of biological demand in controlling O2 availability as opposed to physical limitations on O2 diffusion imposed by soil moisture. In sum, our data suggest that anaerobic conditions do not necessarily constrain enzyme activity in humid tropical forest soils, and may not provide a proximate control on soil C storage in these ecosystems as has been recently proposed.

Nitric oxide-mediated suppression of 2,3-bisphosphoglycerate synthesis: therapeutic relevance for environmental hypoxia and sickle cell disease.

PubMed

Bertrand, R

2012-09-01

Though hemoglobin (Hb) is best known for transporting oxygen and metabolic wastes throughout the circulatory system, this erythrocyte protein also acts as a hypoxic sensor, its oxygen saturation dependent on the oxygen partial pressure (pO(2)) which varies throughout the vasculature. The production and transport of the endogenous vasodilator nitric oxide (NO) by Hb is dependent on Hb's oxygen saturation, thereby allowing the protein to auto-regulate blood flow efficiency to meet the relative demands of respiring tissues. Erythrocyte concentrations of 2,3-bisphosphoglycerate (BPG), an enhancer of oxygen off-loading from Hb, is very sensitive to changes in glycolytic rates because its synthesis by BPG synthase is dependent on the availability of the glycolytic intermediate 1,3-bisphosphoglycerate. BPG synthase, as well as some glycolytic enzymes, are also very sensitive to pH changes, and variations in BPG levels have direct consequences on the oxygen off-loading function of Hb. I hypothesize that NO may suppress BPG production by (1) inhibiting glyceraldehyde-3-phosphate dehydrogenase (G3PDH), the most critical glycolytic enzyme for the bioavailability of 1,3-bisphosphoglycerate; and to a lesser extent by (2) associated pH changes in the deoxy-Hb-catalyzed depletion of nitrite, a metabolic reservoir of NO. Both mechanisms are favored in low pO(2) environments where BPG is most needed to maximize oxygen off-loading, indicating that the auto-regulatory link between NO and Hb may have inadvertently linked Hb and BPG synthesis in an unfavorable manner. However, for reasons discussed, NO-mediated suppression of BPG may be advantageous in some circumstances; namely, for individuals living at high altitudes and those with the blood disorder sickle cell anemia. This hypothesis is thus relevant to respiratory health under both normative conditions as well as under hypoxic stress. The potential relevance of the hypothesis to comparative animal physiology and evolutionary biology is also briefly described. Copyright © 2012 Elsevier Ltd. All rights reserved.

A Model of Extracellular Enzymes in Free-Living Microbes: Which Strategy Pays Off?

PubMed Central

Thygesen, Uffe H.; Riemann, Lasse; Stedmon, Colin A.

2015-01-01

An initial modeling approach was applied to analyze how a single, nonmotile, free-living, heterotrophic bacterial cell may optimize the deployment of its extracellular enzymes. Free-living cells live in a dilute and complex substrate field, and to gain enough substrate, their extracellular enzymes must be utilized efficiently. The model revealed that surface-attached and free enzymes generate unique enzyme and substrate fields, and each deployment strategy has distinctive advantages. For a solitary cell, surface-attached enzymes are suggested to be the most cost-efficient strategy. This strategy entails potential substrates being reduced to very low concentrations. Free enzymes, on the other hand, generate a radically different substrate field, which suggests significant benefits for the strategy if free cells engage in social foraging or experience high substrate concentrations. Swimming has a slight positive effect for the attached-enzyme strategy, while the effect is negative for the free-enzyme strategy. The results of this study suggest that specific dissolved organic compounds in the ocean likely persist below a threshold concentration impervious to biological utilization. This could help explain the persistence and apparent refractory state of oceanic dissolved organic matter (DOM). Microbial extracellular enzyme strategies, therefore, have important implications for larger-scale processes, such as shaping the role of DOM in ocean carbon sequestration. PMID:26253668

A mechanism-based pharmacokinetic-enzyme model for cyclophosphamide autoinduction in breast cancer patients

PubMed Central

Hassan, M; Svensson, U S H; Ljungman, P; Björkstrand, B; Olsson, H; Bielenstein, M; Abdel-Rehim, M; Nilsson, C; Johansson, M; Karlsson, M O

1999-01-01

Aims This study investigated the pharmacokinetics of cyclophosphamide (CP) and its main metabolite 4-hydroxycyclophosphamide (4-OH-CP) in patients with breast cancer undergoing high dose chemotherapy prior to autologous stem cell transplantation. An enzyme turn-over model was also developed to study the time course of cyclophosphamide induction. Methods Fourteen patients received a combination of CP (6 g m−2), thiotepum (500 mg m−2) and carboplatin (800 mg m−2) as a 96 h infusion. Plasma concentrations of CP and 4-OH-CP were determined with h.p.l.c. and a pharmacokinetic and enzyme turn-over model applied to data using NONMEM. Results CP plasma concentrations were described by a two-compartment model with a noninducible and an inducible pathway, the latter forming 4-OH-CP. In the final enzyme model, CP affects the amount of enzymes by increasing the enzyme production rate. CP concentrations decreased during the infusion with no subsequent change in 4-OH-CP concentrations. CP inducible and noninducible clearance were estimated to 1.76 l h−1 (90% C.I. 0.92–2.58) and 1.14 l h−1 (0.31–1.85), respectively. The induction resulted in an approximately doubled CP clearance through the inducible pathway at the end of treatment. The model predicted the enzyme turn-over half-life to be 24 h. Conclusions The presented mechanism-based enzyme induction model where the pharmacokinetics of the inducer and the enzyme pool counterbalance each other successfully described CP autoinduction. It is reasonable to believe that CP affects its own elimination by increasing the enzyme production rate and thereby increasing the amount of enzyme by which CP is eliminated. PMID:10594468

Effects of resistance training using known vs unknown loads on eccentric-phase adaptations and concentric velocity.

PubMed

Hernández-Davó, J L; Sabido, R; Behm, D G; Blazevich, A J

2018-02-01

The aims of this study were to compare both eccentric- and concentric-phase adaptations in highly trained handball players to 4 weeks of twice-weekly rebound bench press throw training with varying loads (30%, 50% and 70% of one-repetition maximum [1-RM]) using either known (KL) or unknown (UL) loads and to examine the relationship between changes in eccentric- and concentric-phase performance. Twenty-eight junior team handball players were divided into two experimental groups (KL or UL) and a control group. KL subjects were told the load prior each repetition, while UL were blinded. For each repetition, the load was dropped and then a rebound bench press at maximum velocity was immediately performed. Both concentric and eccentric velocity as well as eccentric kinetic energy and musculo-articular stiffness prior to the eccentric-concentric transition were measured. Results showed similar increases in both eccentric velocity and kinetic energy under the 30% 1-RM but greater improvements under 50% and 70% 1-RM loads for UL than KL. UL increased stiffness under all loads (with greater magnitude of changes). KL improved concentric velocity only under the 30% 1-RM load while UL also improved under 50% and 70% 1-RM loads. Improvements in concentric movement velocity were moderately explained by changes in eccentric velocity (R 2 =.23-.62). Thus, UL led to greater improvements in concentric velocity, and the improvement is potentially explained by increases in the speed (as well as stiffness and kinetic energy) of the eccentric phase. Unknown load training appears to have significant practical use for the improvement of multijoint stretch-shortening cycle movements. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

On the Error of the Dixon Plot for Estimating the Inhibition Constant between Enzyme and Inhibitor

ERIC Educational Resources Information Center

Fukushima, Yoshihiro; Ushimaru, Makoto; Takahara, Satoshi

2002-01-01

In textbook treatments of enzyme inhibition kinetics, adjustment of the initial inhibitor concentration for inhibitor bound to enzyme is often neglected. For example, in graphical plots such as the Dixon plot for estimation of an inhibition constant, the initial concentration of inhibitor is usually plotted instead of the true inhibitor…

Primary enzyme quantitation

DOEpatents

Saunders, G.C.

1982-03-04

The disclosure relates to the quantitation of a primary enzyme concentration by utilizing a substrate for the primary enzyme labeled with a second enzyme which is an indicator enzyme. Enzyme catalysis of the substrate occurs and results in release of the indicator enzyme in an amount directly proportional to the amount of primary enzyme present. By quantifying the free indicator enzyme one determines the amount of primary enzyme present.

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.

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.

Hyaluronic Acid (HA)-Polyethylene glycol (PEG) as injectable hydrogel for intervertebral disc degeneration patients therapy

NASA Astrophysics Data System (ADS)

Putri Kwarta, Cityta; Widiyanti, Prihartini; Siswanto

2017-05-01

Chronic Low Back Pain (CLBP) is one health problem that is often encountered in a community. Inject-able hydrogels are the newest way to restore the disc thickness and hydration caused by disc degeneration by means of minimally invasive surgery. Thus, polymers can be combined to improve the characteristic properties of inject-able hydrogels, leading to use of Hyaluronic Acid (a natural polymer) and Polyethylene glycol (PEG) with Horse Radish Peroxide (HRP) cross linker enzymes. The swelling test results, which approaches were the ideal disc values, were sampled with variation of enzyme concentrations of 0.25 µmol/min/mL. The enzyme concentrations were 33.95%. The degradation test proved that the sample degradation increased along with the decrease of the HRP enzyme concentration. The results of the cytotoxicity assay with MTT assay method showed that all samples resulted in the 90% of living cells are not toxic. In vitro injection, models demonstrated that higher concentration of the enzymes was less state of gel which would rupture when released from the agarose gel. The functional group characterization shows the cross linking bonding in sample with enzyme adding. The conclusion of this study is PEG-HA-HRP enzyme are safe polymer composites which have a potential to be applied as an injectable hydrogel for intervertebral disc degeneration.

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.

Enhanced cellulase recovery without β-glucosidase supplementation for cellulosic ethanol production using an engineered strain and surfactant.

PubMed

Huang, Renliang; Guo, Hong; Su, Rongxin; Qi, Wei; He, Zhimin

2017-03-01

Recycling cellulases by substrate adsorption is a promising strategy for reducing the enzyme cost of cellulosic ethanol production. However, β-glucosidase has no carbohydrate-binding module (CBM). Thus, additional enzymes are required in each cycle to achieve a high ethanol yield. In this study, we report a new method of recycling cellulases without β-glucosidase supplementation using lignocellulosic substrate, an engineered strain expressing β-glucosidase and Tween 80. The cellulases and Tween 80 were added to an aqueous suspension of diluted sulfuric acid/ammonia-treated corncobs in a simultaneous saccharification and fermentation (SSF) process for ethanol production. Subsequently, the addition of fresh pretreated corncobs to the fermentation liquor and remaining solid residue provided substrates with absorbed cellulases for the next SSF cycle. This method provided excellent ethanol production in three successive SSF cycles without requiring the addition of new cellulases. For a 10% (w/v) solid loading, a cellulase dosage of 30 filter paper units (FPU)/g cellulose, 0.5% Tween 80, and 2 g/L of the engineered strain, approximately 90% of the initial ethanol concentration from the first SSF process was obtained in the next two SSF processes, with a total ethanol production of 306.27 g/kg corncobs and an enzyme productivity of 0.044 g/FPU. Tween 80 played an important role in enhancing cellulase recovery. This new enzyme recycling method is more efficient and practical than other reported methods. Biotechnol. Bioeng. 2017;114: 543-551. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

High Liver Enzyme Concentrations are Associated with Higher Glycemia, but not with Glycemic Variability, in Individuals without Diabetes Mellitus.

PubMed

Noordam, Raymond; Vermond, Debbie; Drenth, Hermijntje; Wijman, Carolien A; Akintola, Abimbola A; van der Kroef, Sabrina; Jansen, Steffy W M; Huurman, Neline C; Schutte, Bianca A M; Beekman, Marian; Slagboom, P Eline; Mooijaart, Simon P; van Heemst, Diana

2017-01-01

Elevated concentrations of liver enzymes have been associated with an increased risk of developing type 2 diabetes mellitus. However, it remains unclear to which specific aspects of diurnal glucose metabolism these associate most. We aimed to investigate the associations between liver enzyme concentrations and 24 h-glucose trajectories in individuals without diabetes mellitus from three independent cohorts. This cross-sectional study included 436 participants without diabetes mellitus from the Active and Healthy Aging Study, the Switchbox Study, and the Growing Old Together Study. Fasting blood samples were drawn to measure gamma-glutamyltransferase (GGT), alanine transaminase, and aspartate transaminase. Measures of glycemia (e.g., nocturnal and diurnal mean glucose levels) and glycemic variability (e.g., mean amplitude of glucose excursions) were derived from continuous glucose monitoring. Analyses were performed separately for the three cohorts; derived estimates were additionally meta-analyzed. After meta-analyses of the three cohorts, elevated liver enzyme concentrations, and specifically elevated GGT concentrations, were associated with higher glycemia. More specific, participants in the highest GGT tertile (GGT ≥37.9 U/L) had a 0.39 mmol/L (95% confidence interval: 0.23, 0.56) higher mean nocturnal glucose (3:00 to 6:00 a.m.) and a 0.23 mmol/L (0.10, 0.36) higher diurnal glucose (6:00 to 0:00 a.m.) than participants in the lowest GGT tertile (GGT <21.23 U/L). However, elevated liver enzyme concentrations were not associated with a higher glycemic variability. Though elevated liver enzyme concentrations did not associate with higher glycemic variability in participants without diabetes mellitus, specifically, elevated GGT concentrations associated with higher glycemia.

Charge transport model in nanodielectric composites based on quantum tunneling mechanism and dual-level traps

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

Li, Guochang; Chen, George, E-mail: gc@ecs.soton.ac.uk, E-mail: sli@mail.xjtu.edu.cn; School of Electronic and Computer Science, University of Southampton, Southampton SO17 1BJ

Charge transport properties in nanodielectrics present different tendencies for different loading concentrations. The exact mechanisms that are responsible for charge transport in nanodielectrics are not detailed, especially for high loading concentration. A charge transport model in nanodielectrics has been proposed based on quantum tunneling mechanism and dual-level traps. In the model, the thermally assisted hopping (TAH) process for the shallow traps and the tunnelling process for the deep traps are considered. For different loading concentrations, the dominant charge transport mechanisms are different. The quantum tunneling mechanism plays a major role in determining the charge conduction in nanodielectrics with high loadingmore » concentrations. While for low loading concentrations, the thermal hopping mechanism will dominate the charge conduction process. The model can explain the observed conductivity property in nanodielectrics with different loading concentrations.« less

Reactivation of triosephosphate isomerase from three trypanosomatids and human: effect of suramin.

PubMed Central

Gao, X G; Garza-Ramos, G; Saavedra-Lira, E; Cabrera, N; De Gómez-Puyou, M T; Perez-Montfort, R; Gómez-Puyou, A

1998-01-01

The reactivation of the homodimeric triosephosphate isomerases (TIMs) from Trypanosoma brucei, T. cruzi, Leishmania mexicana and humans was determined after their denaturation with guanidine hydrochloride. In the range of 2-32 microg of T. brucei TIM per ml and 0.2-5 microg of the other enzymes per ml, the rate and extent of TIM reactivation depended on protein concentration, indicating that at these protein concentrations, the rate-limiting step of reactivation is monomer association and not monomer folding. The rate of monomer association was more than one order of magnitude lower in the T. brucei enzyme than in the other three enzymes. Suramin is a drug of choice in the treatment of sleeping sickness, but its mechanism of action is not known. At micromolar concentrations, Suramin inhibited the reactivation of the four enzymes, but the extent of inhibition by Suramin decreased with increasing protein concentration as consequence of a diminution of the life time of the folded monomer. Since the life time of the monomer of T. brucei TIM is longer than that of the other enzymes, Suramin is a more effective inhibitor of the reactivation of TIM from T. brucei, particularly at monomer concentrations above 1 microg of protein per ml (monomer concentration approx. 37 nM). Compounds that are structurally related to Suramin also inhibit TIM reactivation; their effect was about five times more pronounced in the enzyme from T. brucei than in human TIM. PMID:9576855

Study of cellulase enzymes self-assembly in aqueous-acetonitrile solvent: Viscosity measurements

NASA Astrophysics Data System (ADS)

Ghaouar, N.; Aschi, A.; Belbahri, L.; Trabelsi, S.; Gharbi, A.

2009-11-01

The present study extends the viscosity measurements performed by Ghaouar et al. [Physica B, submitted for publication.] to study the conformational change of the cellulase enzymes in aqueous-acetonitrile mixture. We aim to investigate: (i) the denaturation process by measuring the specific viscosity for temperatures varying between 25 and 65 °C and acetonitrile concentrations between 0% and 50%, (ii) the enzyme-enzyme interaction by calculating the Huggins coefficient and (iii) the enzyme sizes by following the hydrodynamic radius for various temperatures. The precipitation of cellulases versus acetonitrile concentration is also considered. We show from all physical quantities measured in this work that the precipitation and the denaturation processes of cellulase enzymes exist together.

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.

User's manual for the Graphical Constituent Loading Analysis System (GCLAS)

USGS Publications Warehouse

Koltun, G.F.; Eberle, Michael; Gray, J.R.; Glysson, G.D.

2006-01-01

This manual describes the Graphical Constituent Loading Analysis System (GCLAS), an interactive cross-platform program for computing the mass (load) and average concentration of a constituent that is transported in stream water over a period of time. GCLAS computes loads as a function of an equal-interval streamflow time series and an equal- or unequal-interval time series of constituent concentrations. The constituent-concentration time series may be composed of measured concentrations or a combination of measured and estimated concentrations. GCLAS is not intended for use in situations where concentration data (or an appropriate surrogate) are collected infrequently or where an appreciable amount of the concentration values are censored. It is assumed that the constituent-concentration time series used by GCLAS adequately represents the true time-varying concentration. Commonly, measured constituent concentrations are collected at a frequency that is less than ideal (from a load-computation standpoint), so estimated concentrations must be inserted in the time series to better approximate the expected chemograph. GCLAS provides tools to facilitate estimation and entry of instantaneous concentrations for that purpose. Water-quality samples collected for load computation frequently are collected in a single vertical or at single point in a stream cross section. Several factors, some of which may vary as a function of time and (or) streamflow, can affect whether the sample concentrations are representative of the mean concentration in the cross section. GCLAS provides tools to aid the analyst in assessing whether concentrations in samples collected in a single vertical or at single point in a stream cross section exhibit systematic bias with respect to the mean concentrations. In cases where bias is evident, the analyst can construct coefficient relations in GCLAS to reduce or eliminate the observed bias. GCLAS can export load and concentration data in formats suitable for entry into the U.S. Geological Survey's National Water Information System. GCLAS can also import and export data in formats that are compatible with various commonly used spreadsheet and statistics programs.

Nutrient loadings to streams of the continental United States from municipal and industrial effluent?

USGS Publications Warehouse

Maupin, Molly A.; Ivahnenko, Tamara

2011-01-01

Data from the United States Environmental Protection Agency Permit Compliance System national database were used to calculate annual total nitrogen (TN) and total phosphorus (TP) loads to surface waters from municipal and industrial facilities in six major regions of the United States for 1992, 1997, and 2002. Concentration and effluent flow data were examined for approximately 118,250 facilities in 45 states and the District of Columbia. Inconsistent and incomplete discharge locations, effluent flows, and effluent nutrient concentrations limited the use of these data for calculating nutrient loads. More concentrations were reported for major facilities, those discharging more than 1 million gallons per day, than for minor facilities, and more concentrations were reported for TP than for TN. Analytical methods to check and improve the quality of the Permit Compliance System data were used. Annual loads were calculated using "typical pollutant concentrations" to supplement missing concentrations based on the type and size of facilities. Annual nutrient loads for over 26,600 facilities were calculated for at least one of the three years. Sewage systems represented 74% of all TN loads and 58% of all TP loads. This work represents an initial set of data to develop a comprehensive and consistent national database of point-source nutrient loads. These loads can be used to inform a wide range of water-quality management, watershed modeling, and research efforts at multiple scales.

Mineral water administration may increase kidney elimination of urea, creatinine and folic acid in a concentration-dependent fashion.

PubMed

Calomino, Francesco; Di Paolo, Nicola; Nicolai, Giulia; Miglio, Antonio

2010-05-01

In a previous experimental study we showed that the administration of a large water load in a short time increases the urinary flow and the transport capacity in the excretory tract of the rabbit ureter. In human subjects drinking a water load of 25 ml/kg(BW) in 30 minutes, diuresis, creatinine and urea clearance increase more than in those drinking the same load in 24 hours. The aim of the present study was to investigate possible correlations between percent reduction and baseline values of serum urea, creatinine, folic acid, and magnesium in humans. 20 volunteers were divided in two groups. Subjects in group 1 received a water load of 25 ml/kg(BW) in 24 hours followed by the same load in 30 minutes. Subjects in group 2 received the same water load but in inverse order. Before and after each water administration, the following variables were measured and compared: diuresis, serum urea, creatinine, folic acid and magnesium concentration, and urea and creatinine clearance. Serum urea and folic acid concentration decreased up to 40% after administration of the water load in 24 hours. Serum creatinine concentration decreased up to 20% after administration of the water load in 30 minutes. The concentration drop of these metabolites increased with increasing baseline metabolite concentrations.

Nectar loads as fuel for collecting nectar and pollen in honeybees: adjustment by sugar concentration.

PubMed

Harano, Ken-Ichi; Nakamura, Jun

2016-06-01

When honeybee foragers leave the nest, they receive nectar from nest mates for use as fuel for flight or as binding material to build pollen loads. We examined whether the concentration of nectar carried from the nest changes with the need for sugar. We found that pollen foragers had more-concentrated nectar (61.8 %) than nectar foragers (43.8 %). Further analysis revealed that the sugar concentration of the crop load increased significantly with waggle duration, an indicator of food-source distance, in both groups of foragers. Crop volume also increased with waggle duration. The results support our argument that foragers use concentrated nectar when the need for sugar is high and suggest that they precisely adjust the amount of sugar in the crop by altering both volume and nectar concentrations. We also investigated the impact of the area where foragers receive nectar on the crop load concentration at departure. Although nectar and pollen foragers tend to load nectar at different areas in the nest, area did not have a significant effect on crop load concentration. Departing foragers showed an average of 2.2 momentary (<1 s) begging trophallactic contacts before leaving the nest. They might be rejecting nectar with inappropriate concentrations during these contacts.

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.

Effects of silver nanoparticles on soil enzyme activities with and without added organic matter.

PubMed

Peyrot, Caroline; Wilkinson, Kevin J; Desrosiers, Mélanie; Sauvé, Sébastien

2014-01-01

The effects of silver nanoparticles (AgNPs) on terrestrial ecosystems need to be better understood and assessed. Cationic silver (Ag+) has well-documented toxicity against bacteria, but it is not clear what will be the effect of nanoscale Ag. In the present study, the potential effects of AgNPs were investigated in soils by measuring activity of the enzymes phosphomonoesterase, arylsulfatase, β-D-glucosidase, and leucine-aminopeptidase. The toxicity of AgNPs was compared with that of ionic Ag, and the ameliorating effects of soil organic matter were evaluated. To this end, 2 soils with different organic matter contents were artificially contaminated with either AgNPs or Ag-acetate at equivalent total Ag concentrations. In general, enzyme activities were inhibited as a function of the Ag concentration in the soil. In the AgNP exposures, only a small fraction of the AgNP was actually truly dissolved (found in the <1-nm fraction), suggesting that the particulate forms of AgNPs resulted in a significant inhibition of soil enzymes. The addition of organic matter to the soils appeared to enhance enzyme activities; however, the mechanism of organic matter action is not clear given that dissolved Ag concentrations were similar in both the organic-matter–amended and unamended soils. The present study shows that the AgNP produces significant negative effects on the soil enzyme activities tested. The Ag chemical speciation measurements suggested that the AgNP caused greater toxic effects to the soil enzymes at the low Ag concentrations. For the larger concentrations of total soil Ag, causes of the negative effects on enzyme activities are less obvious but suggest that colloidal forms of Ag play a role.

Immobilization techniques to avoid enzyme loss from oxidase-based biosensors: a one-year study.

PubMed

House, Jody L; Anderson, Ellen M; Ward, W Kenneth

2007-01-01

Continuous amperometric sensors that measure glucose or lactate require a stable sensitivity, and glutaraldehyde crosslinking has been used widely to avoid enzyme loss. Nonetheless, little data is published on the effectiveness of enzyme immobilization with glutaraldehyde. A combination of electrochemical testing and spectrophotometric assays was used to study the relationship between enzyme shedding and the fabrication procedure. In addition, we studied the relationship between the glutaraldehyde concentration and sensor performance over a period of one year. The enzyme immobilization process by glutaraldehyde crosslinking to glucose oxidase appears to require at least 24-hours at room temperature to reach completion. In addition, excess free glucose oxidase can be removed by soaking sensors in purified water for 20 minutes. Even with the addition of these steps, however, it appears that there is some free glucose oxidase entrapped within the enzyme layer which contributes to a decline in sensitivity over time. Although it reduces the ultimate sensitivity (probably via a change in the enzyme's natural conformation), glutaraldehyde concentration in the enzyme layer can be increased in order to minimize this instability. After exposure of oxidase enzymes to glutaraldehyde, effective crosslinking requires a rinse step and a 24-hour incubation step. In order to minimize the loss of sensor sensitivity over time, the glutaraldehyde concentration can be increased.

Preparation of immobilized glucose oxidase wafer enzyme on calcium-bentonite modified by surfactant

NASA Astrophysics Data System (ADS)

Widi, R. K.; Trisulo, D. C.; Budhyantoro, A.; Chrisnasari, R.

2017-07-01

Wafer glucose oxidase (GOx) enzymes was produced by addition of PAH (Poly-Allyamine Hydrochloride) polymer into immobilized GOx enzyme on modified-Tetramethylammonium Hydroxide (TMAH) 5%-calsium-bentonite. The use of surfactant molecul (TMAH) is to modify the surface properties and pore size distribution of the Ca-bentonite. These properties are very important to ensure GOx molecules can be bound on the Ca-bentonit surface to be immobilized. The addition of the polymer (PAH) is expected to lead the substrates to be adsorbed onto the enzyme. In this study, wafer enzymes were made in various concentration ratio (Ca-bentonite : PAH) which are 1:0, 1:1, 1:2 and 1:3. The effect of PAH (Poly-Allyamine Hydrochloride) polymer added with various ratios of concentrations can be shown from the capacitance value on LCR meter and enzyme activity using DNS method. The addition of the polymer (PAH) showed effect on the activity of GOx, it can be shown from the decreasing of capacitance value by increasing of PAH concentration.

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.

Energetic aspects of the light activation of two chloroplast enzymes: fructose-1,6-bisphosphatase and NADP-malate dehydrogenase.

PubMed

Miginiac-Maslow, M; Jacquot, J P; Droux, M

1985-09-01

The light energy requirements for photoactivation of two chloroplast enzymes: fructose-1,6-bisphosphatase and NADP-malate dehydrogenase were studied in a reconstituted chloroplast system. This system comprised isolated pea thylakoids, ferredoxin (Fd), ferredoxin-thioredoxin reductase (FTR) thioredoxinm and f (Tdm, Tdf) and the photoactivatable enzyme. Light-saturation curves of the photoactivation process were established with once washed thylakoids which did not require the addition of Td for light activation. They exhibited a plateau at 10 W·m(-2) under nitrogen and 50 W·m(-2) under air, while NADP photoreduction was saturated at 240 W·m(-2). Cyclic and pseudocyclic phosphorylations saturated at identical levels as enzyme photoactivations. All these observations suggested that the shift of the light saturation plateau towards higher values under air was due to competing oxygen-dependent reactions. With twice washed thylakoids, which required Td for enzyme light-activation, photophosphorylation was stimulated under N2 by the addition of the components of the photoactivation system. Its rate increased with increasing Td concentrations, just as did the enzyme photoactivation rate, while varying the target enzyme concentration had only a weak effect. Considering that Td concentrations were in a large excess over target enzyme concentrations, it may be assumed that the observed ATP synthesis was essentially dependent on the rate of Td reduction.Under air, Fd-dependent pseudo-cyclic photophosphorylation was not stimulated by the addition of the other enzyme photoactivation components, suggesting that an important site of action of O2 was located at the level of Fd.

Decolorization of Anthraquinonic Dyes from Textile Effluent Using Horseradish Peroxidase: Optimization and Kinetic Study

PubMed Central

Šekuljica, Nataša Ž.; Prlainović, Nevena Ž.; Stefanović, Andrea B.; Žuža, Milena G.; Čičkarić, Dragana Z.; Mijin, Dušan Ž.; Knežević-Jugović, Zorica D.

2015-01-01

Two anthraquinonic dyes, C.I. Acid Blue 225 and C.I. Acid Violet 109, were used as models to explore the feasibility of using the horseradish peroxidase enzyme (HRP) in the practical decolorization of anthraquinonic dyes in wastewater. The influence of process parameters such as enzyme concentration, hydrogen peroxide concentration, temperature, dye concentration, and pH was examined. The pH and temperature activity profiles were similar for decolorization of both dyes. Under the optimal conditions, 94.7% of C.I. Acid Violet 109 from aqueous solution was decolorized (treatment time 15 min, enzyme concentration 0.15 IU/mL, hydrogen peroxide concentration 0.4 mM, dye concentration 30 mg/L, pH 4, and temperature 24°C) and 89.36% of C.I. Acid Blue 225 (32 min, enzyme concentration 0.15 IU/mL, hydrogen peroxide concentration 0.04 mM, dye concentration 30 mg/L, pH 5, and temperature 24°C). The mechanism of both reactions has been proven to follow the two substrate ping-pong mechanism with substrate inhibition, revealing the formation of a nonproductive or dead-end complex between dye and HRP or between H2O2 and the oxidized form of the enzyme. Both chemical oxygen demand and total organic carbon values showed that there was a reduction in toxicity after the enzymatic treatment. This study verifies the viability of use of horseradish peroxidase for the wastewaters treatment of similar anthraquinonic dyes. PMID:25685837

Study on OELs for enzyme-containing detergent in China.

PubMed

Zhang, X D; Liang, Y X; Lee, C S; Jin, T Y

2004-01-01

This study is aimed at setting occupational exposure levels for total detergent dust and enzymes in detergent industries. The study population consisted of 795 workers from four enzyme-containing detergent manufacturing plants (A1, A2, B1 and B2), and 156 control workers from an electronic assembly factory. Work environment monitoring was conducted using high volume of air sampler fro measuring the concentration of total dust (mg/m3), and analyzing the level of enzyme (ng/m3) by ELISA method. A standard questionnaires, pulmonary function test, and skin prick test are used to assess health effects. The levels of detergent total dust varied from 0.2 mg/m3 to 12.54 mg/m3. For enzyme levels, in A1, B1 and B2, the concentration ranged from non-detectable to 9.92 ng/m3 and in A2, the concentration was analyzed by enzyme activity methods and was expressed as Gu/m3 (1 Gu/m3 = 16 ng/m3). The concentration is between 0.16-31.36 ng/m3. Non-specific irritation rates in exposed workers were significantly higher than that in controls. Based on the data collected from A1, B1 and control plants, 95% benchmark dose lower bound were calculated as 1.17 mg/m3. The difference of pulmonary function between exposed workers and controls is not significant. The results of SPT showed that neither Savinase- nor Alcalase-induced sensitization was found in controls. The prevalence rates of sensitization for Savinase and Alcalase were ranged between 3.2% and 31% in all enzyme-containing detergent manufacturers investigated. No case of occupational asthma was observed. For total dust, 1 mg/m3 is suggested as permissible concentration-time weighted average (PC-TWA), and 2 mg/m3 as permissible concentration-short term exposure limit (PC-STEL). For the enzyme Subtilisins, 15 ng/m3 is suggested as PC-TWA, and 30 ng/m3 as PC-STEL.

Extraction of Oil from Flaxseed (Linum usitatissimum L.) Using Enzyme-Assisted Three-Phase Partitioning.

PubMed

Tan, Zhi-Jian; Yang, Zi-Zhen; Yi, Yong-Jian; Wang, Hong-Ying; Zhou, Wan-Lai; Li, Fen-Fang; Wang, Chao-Yun

2016-08-01

In this study, enzyme-assisted three-phase partitioning (EATPP) was used to extract oil from flaxseed. The whole procedure is composed of two parts: the enzymolysis procedure in which the flaxseed was hydrolyzed using an enzyme solution (the influencing parameters such as the type and concentration of enzyme, temperature, and pH were optimized) and three-phase partitioning (TPP), which was conducted by adding salt and t-butanol to the crude flaxseed slurry, resulting in the extraction of flaxseed oil into alcohol-rich upper phase. The concentration of t-butanol, concentration of salt, and the temperature were optimized to maximize the extraction yield. Under optimized conditions of a 49.29 % t-butanol concentration, 30.43 % ammonium sulfate concentration, and 35 °C extraction temperature, a maximum extraction yield of 71.68 % was obtained. This simple and effective EATPP can be used to achieve high extraction yields and oil quality, and thus, it is potential for large-scale oil production.

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.

A model of extracellular enzymes in free-living microbes: which strategy pays off?

PubMed

Traving, Sachia J; Thygesen, Uffe H; Riemann, Lasse; Stedmon, Colin A

2015-11-01

An initial modeling approach was applied to analyze how a single, nonmotile, free-living, heterotrophic bacterial cell may optimize the deployment of its extracellular enzymes. Free-living cells live in a dilute and complex substrate field, and to gain enough substrate, their extracellular enzymes must be utilized efficiently. The model revealed that surface-attached and free enzymes generate unique enzyme and substrate fields, and each deployment strategy has distinctive advantages. For a solitary cell, surface-attached enzymes are suggested to be the most cost-efficient strategy. This strategy entails potential substrates being reduced to very low concentrations. Free enzymes, on the other hand, generate a radically different substrate field, which suggests significant benefits for the strategy if free cells engage in social foraging or experience high substrate concentrations. Swimming has a slight positive effect for the attached-enzyme strategy, while the effect is negative for the free-enzyme strategy. The results of this study suggest that specific dissolved organic compounds in the ocean likely persist below a threshold concentration impervious to biological utilization. This could help explain the persistence and apparent refractory state of oceanic dissolved organic matter (DOM). Microbial extracellular enzyme strategies, therefore, have important implications for larger-scale processes, such as shaping the role of DOM in ocean carbon sequestration. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Potato Peroxidase for the Study of Enzyme Properties.

ERIC Educational Resources Information Center

Shamaefsky, Brian R.

1993-01-01

Explains how the surface of a freshly sliced potato can be used for a variety of enzyme action experiments including the influence of pH on enzyme action, the enzyme denaturation potential of boiling water, the inhibition of enzymes by heavy metals, and the effects of salt concentration on enzyme effectiveness. (PR)

Pretreatment solution recycling and high-concentration output for economical production of bioethanol.

PubMed

Han, Minhee; Moon, Se-Kwon; Choi, Gi-Wook

2014-11-01

The purpose of this study was to enhance the economic efficiency of producing bioethanol. Pretreatment solution recycling is expected to increase economic efficiency by reducing the cost of pretreatment and the amount of wastewater. In addition, the production of high-concentration bioethanol could increase economic efficiency by reducing the energy cost of distillation. The pretreatment conditions were 95 °C, 0.72 M NaOH, 80 rpm twin-screw speed, and flow rate of 90 mL/min at 18 g/min of raw biomass feeding for pretreatment solution recycling. The pretreatment with NaOH solution recycling was conducted five times. All of the components and the pretreatment efficiency were similar, despite reuse. In addition, we developed a continuous biomass feeding system for production of high-concentration bioethanol. Using this reactor, the bioethanol productivity was investigated using various pretreated biomass feeding rates in a simultaneous saccharification and fermentation (SSF) process. The maximum ethanol concentration, yield, and productivity were 74.5 g/L, 89.5%, and 1.4 g/L h, respectively, at a pretreated biomass loading of approximately 25% (w/v) with an enzyme dosage of 30 FPU g/cellulose. The results presented here constitute an important contribution toward the production of bioethanol from Miscanthus.

Optimization of moistening solution concentration on xylanase activity in solid state fermentation from oil palm empty fruit bunches

NASA Astrophysics Data System (ADS)

Mardawati, Efri; Parlan; Rialita, Tita; Nurhadi, Bambang

2018-03-01

Xylanase is an enzyme used in the industrial world, including food industry. Xylanase can be utilized as a 1,4-β-xylosidic endo-hydrolysis catalyst of xylanase, a hemicellulose component for obtaining a xylose monomer. This study aims to determine the optimum concentration of the fermentation medium using Response Surface Method (RSM) in the production of xylanase enzyme from oil palm empty fruit bunches (OPEFB) through solid state fermentation process. The variables varied in this study used factor A (ammonium sulphate concentration 1.0-2.0 g/L), B (concentration of potassium dihydrogen phosphate 1.5-2.5 g/L) and C (urea concentration 0.2 – 0.5 g/L). The data was analysed by using Design Expert version 10.0.1.0 especially CCD with total 17 running including 3 times replicated of canter point. Trichoderma viride was used for the process production of xylanase enzyme. The ratio between substrate and moistening solution used was 0.63 g / mL with temperature of 32.80C, 60 h incubation time. The analysis of enzyme activity was done by DNS method with 1% xylan as substrate. Analysis of protein content in enzyme was done by Bradford method. The optimum of moistening solution concentration in this fermentation was obtained. They are, the ammonium sulphate concentration of 1.5 g/L, potassium dihydrogen phosphate 2.0 g/L and urea 0.35 g/L with activity of 684.70 U/mL, specific activity enzyme xylanase 6261.58 U/mg, protein content 0.1093 U/mg, the model was validated using experiment design with perfect reliability value 0.96.

Mediator-free interaction of glucose oxidase, as model enzyme for immobilization, with Al-doped and undoped ZnO thin films laser-deposited on polycarbonate supports.

PubMed

V T K P, Fidal; Inguva, Saikumar; Krishnamurthy, Satheesh; Marsili, Enrico; Mosnier, Jean-Paul; T S, Chandra

2017-01-01

Al doped and undoped ZnO thin films were deposited by pulsed-laser deposition on polycarbonate sheets. The films were characterized by optical transmission, Hall effect measurement, XRD and SEM. Optical transmission and surface reflectometry studies showed good transparency with thicknesses ∼100nm and surface roughness of 10nm. Hall effect measurements showed that the sheet carrier concentration was -1.44×10 15 cm -2 for AZO and -6×10 14 cm -2 for ZnO. The films were then modified by drop-casting glucose oxidase (GOx) without the use of any mediators. Higher protein concentration was observed on ZnO as compared to AZO with higher specific activity for ZnO (0.042Umg -1 ) compared to AZO (0.032Umg -1 ), and was in agreement with cyclic voltemmetry (CV). X-ray photoelectron spectroscopy (XPS) suggested that the protein was bound by dipole interactions between AZO lattice oxygen and the amino group of the enzyme. Chronoamperometry showed sensitivity of 5.5μAmM -1 cm -2 towards glucose for GOx/AZO and 2.2μAmM -1 cm -2 for GOx/ZnO. The limit of detection (LoD) was 167μM of glucose for GOx/AZO, as compared to 360μM for GOx/ZnO. The linearity was 0.28-28mM for GOx/AZO whereas it was 0.6-28mM for GOx/ZnO with a response time of 10s. Possibly due to higher enzyme loading, the decrease of impedance in presence of glucose was larger for GOx/ZnO as compared to GOx/AZO in electrochemical impedance spectroscopy (EIS). Analyses with clinical blood serum samples showed that the systems had good reproducibility and accuracy. The characteristics of novel ZnO and AZO thin films with GOx as a model enzyme, should prove useful for the future fabrication of inexpensive, highly sensitive, disposable electrochemical biosensors for high throughput diagnostics. Copyright © 2016 Elsevier Inc. All rights reserved.

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.

Comparison of multi-enzyme and thermophilic bacteria on the hydrolysis of mariculture organic waste (MOW).

PubMed

Guo, Liang; Sun, Mei; Zong, Yan; Zhao, Yangguo; Gao, Mengchun; She, Zonglian

2016-01-01

Mariculture organic waste (MOW) is rich in organic matter, which is a potential energy resource for anaerobic digestion. In order to enhance the anaerobic fermentation, the MOW was hydrolyzed by multi-enzyme and thermophilic bacteria. It was advantageous for soluble chemical oxygen demand (SCOD) release at MOW concentrations of 6 and 10 g/L with multi-enzyme and thermophilic bacteria pretreatments. For multi-enzyme, the hydrolysis was not obvious at substrate concentrations of 1 and 3 g/L, and the protein and carbohydrate increased with hydrolysis time at substrate concentrations of 6 and 10 g/L. For thermophilic bacteria, the carbohydrate was first released at 2-4 h and then consumed, and the protein increased with hydrolysis time. The optimal enzyme hydrolysis for MOW was determined by measuring the changes of SCOD, protein, carbohydrate, ammonia and total phosphorus, and comparing with acid and alkaline pretreatments.

IFCC primary reference procedures for the measurement of catalytic activity concentrations of enzymes at 37 °C. Part 9: reference procedure for the measurement of catalytic concentration of alkaline phosphatase International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) Scientific Division, Committee on Reference Systems of Enzymes (C-RSE) (1)).

PubMed

Schumann, Gerhard; Klauke, Rainer; Canalias, Francesca; Bossert-Reuther, Steffen; Franck, Paul F H; Gella, F-Javier; Jørgensen, Poul J; Kang, Dongchon; Lessinger, Jean-Marc; Panteghini, Mauro; Ceriotti, Ferruccio

2011-09-01

Abstract This paper is the ninth in a series dealing with reference procedures for the measurement of catalytic activity concentrations of enzymes at 37 °C and the certification of reference preparations. Other parts deal with: Part 1. The concept of reference procedures for the measurement of catalytic activity concentrations of enzymes; Part 2. Reference procedure for the measurement of catalytic concentration of creatine kinase; Part 3. Reference procedure for the measurement of catalytic concentration of lactate dehydrogenase; Part 4. Reference procedure for the measurement of catalytic concentration of alanine aminotransferase; Part 5. Reference procedure for the measurement of catalytic concentration of aspartate aminotransferase; Part 6. Reference procedure for the measurement of catalytic concentration of γ-glutamyltransferase; Part 7. Certification of four reference materials for the determination of enzymatic activity of γ-glutamyltransferase, lactate dehydrogenase, alanine aminotransferase and creatine kinase at 37 °C; Part 8. Reference procedure for the measurement of catalytic concentration of α-amylase. The procedure described here is derived from the previously described 30 °C IFCC reference method. Differences are tabulated and commented on in Appendix 1.

Effect of heavy metals ions on enzyme activity in the Mediterranean mussel, Donax trunculus

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

Mizrahi, L.; Achituv, Y.

Heavy metal ions strongly are bound by sulfhydryl groups of proteins. Sulfhydryl binding changes the structure and enzymatic activities of proteins and causes toxic effects evident at the whole organism level. Heavy metal ions like Cd, Cu, Hg, Zn, and Pb in sufficiently high concentrations might kill organisms or cause other adverse effects that changing aquatic community structures. Bivalves are known to be heavy metal accumulators. The aim of the present study was to examine the effects of different concentrations of each of five heavy metal ions on the activity of four enzymes in D. trunculus. As it is knownmore » that heavy metals inhibit the activity of a wide range of enzymes, the authors chose representative examples of dehydrogenases (lactate and malate dehydrogenases), respiratory enzyme (cytochrome oxidase) and digestive enzyme ({alpha}-amylase). The acute effects of different concentrations of selected metals were examined. These concentrations were higher than those found usually in the locality where the animals occur, but might be encountered during a given event of pollution.« less

Comparative study on the conventional and non thermal simultaneous saccharification and fermentation of Manihot glaziovii root starch

NASA Astrophysics Data System (ADS)

Hargono, Kumoro, Andri Cahyo; Jos, Bakti

2015-12-01

Inconventional ethanol production process, starch is converted into dextrins via liquefaction using α-amylase enzyme at high temperature (90-120°C). Then, dextrins are saccharified by glucoamylase to obtain to monomeric sugars (glucose). Recently, a granular starch hydrolyzing enzymes (GSHE), Stargen 002, was developed to convert starch into dextrins at low temperature (< 32°C) and hydrolyzes dextrins into glucose. The subject of this research was to compare ethanol production using a granular starch hydrolyzing enzymes and conventional enzymatic liquefaction and saccharification in cassava starch processing. Starch slurry concentrations were 20% w/v, and dosage of enzymes 0.50, 1.0 and 2%, respectively, were studied. After 48 hr process the final ethanol concentration for the respective enzyme concentration for conventional process were 34.90, 36.16 and 42.10 g/L, whereas for the non-thermal treatment, final ethanol concentration were 46.4, 57.62 and 59.65 g/L, respectively. By implementation of this non thermal process, the use of energy can be saved by carrying out saccharification step at lower temperature (30°C) could be realized.

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.

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.

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).

Salivary Gluten Degradation and Oral Microbial Profiles in Healthy Individuals and Celiac Disease Patients.

PubMed

Tian, Na; Faller, Lina; Leffler, Daniel A; Kelly, Ciaran P; Hansen, Joshua; Bosch, Jos A; Wei, Guoxian; Paster, Bruce J; Schuppan, Detlef; Helmerhorst, Eva J

2017-03-15

Celiac disease (CD) is a chronic immune-mediated enteropathy induced by dietary gluten in genetically predisposed individuals. Saliva harbors the second highest bacterial load of the gastrointestinal (GI) tract after the colon. We hypothesized that enzymes produced by oral bacteria may be involved in gluten processing in the intestine and susceptibility to celiac disease. The aim of this study was to investigate salivary enzymatic activities and oral microbial profiles in healthy subjects versus patients with classical and refractory CD. Stimulated whole saliva was collected from patients with CD in remission ( n = 21) and refractory CD (RCD; n = 8) and was compared to healthy controls (HC; n = 20) and subjects with functional GI complaints ( n = 12). Salivary gluten-degrading activities were monitored with the tripeptide substrate Z-Tyr-Pro-Gln-pNA and the α-gliadin-derived immunogenic 33-mer peptide. The oral microbiome was profiled by 16S rRNA-based MiSeq analysis. Salivary glutenase activities were higher in CD patients compared to controls, both before and after normalization for protein concentration or bacterial load. The oral microbiomes of CD and RCD patients showed significant differences from that of healthy subjects, e.g., higher salivary levels of lactobacilli ( P < 0.05), which may partly explain the observed higher gluten-degrading activities. While the pathophysiological link between the oral and gut microbiomes in CD needs further exploration, the presented data suggest that oral microbe-derived enzyme activities are elevated in subjects with CD, which may impact gluten processing and the presentation of immunogenic gluten epitopes to the immune system in the small intestine. IMPORTANCE Ingested gluten proteins are the triggers of intestinal inflammation in celiac disease (CD). Certain immunogenic gluten domains are resistant to intestinal proteases but can be hydrolyzed by oral microbial enzymes. Very little is known about the endogenous proteolytic processing of gluten proteins in the oral cavity. Given that this occurs prior to gluten reaching the small intestine, such enzymes are likely to contribute to the composition of the gluten digest that ultimately reaches the small intestine and causes CD. We demonstrated that endogenous salivary protease activities are incomplete, likely liberating peptides from larger gluten proteins. The potentially responsible microbes were identified. The study included refractory CD patients, who have been studied less with regard to CD pathogenesis. Copyright © 2017 American Society for Microbiology.

Incorporation of liposomes containing squid tunic ACE-inhibitory peptides into fish gelatin.

PubMed

Mosquera, Mauricio; Giménez, Begoña; Montero, Pilar; Gómez-Guillén, Maria Carmen

2016-02-01

Hydrolysates from collagen of jumbo squid (Dosidicus gigas) tunics have shown excellent angiotensin I-converting enzyme (ACE)-inhibitory activity. However, peptides directly included in food systems may suffer a decrease in activity, which could be minimized by loading them into nanoliposomes. A fraction of peptides with molecular weights <1 kDa obtained from hydrolyzed squid tunics, with reasonably high ACE-inhibitory activity (half-maximal inhibitory concentration IC50 = 0.096 g L(-1)), was encapsulated in phosphatidylcholine nanoliposomes. The peptide concentration affected the encapsulation efficiency and the stability of the resulting liposomes, which remained with a high zeta potential value (-54.3 mV) for at least 1 week at the most suitable peptide concentration. The optimal peptide concentration was established as 1.75 g L(-1). Liposomes obtained with this peptide concentration showed an encapsulation efficiency of 53%, a zeta potential of -59 mV, an average diameter of 70.3 nm and proved to be stable in the pH range 3-7 at 4 °C. Liposomes containing ACE-inhibitory peptides were incorporated in fish gelatin without detriment to the rheological properties and thermal stability of the resulting cold-induced gel. The ACE-inhibitory activity of the peptide fraction, which was not affected by the encapsulation process, conferred the bioactive potential to the nanoliposome-containing gelatin gel. © 2015 Society of Chemical Industry.

Sources and loads of nutrients in the South Platte River, Colorado and Nebraska, 1994-95

USGS Publications Warehouse

Litke, D.W.

1996-01-01

The South Platte River Basin was one of 20 river basins selected in 1991 for investigation as part of the U.S. Geological Survey's National Water- Quality Assessment (NAWQA) Program. Nationwide, nutrients have been identified as one of the primary nationwide water-quality concerns and are of particular interest in the South Platte River Basin where nutrient concentrations are large compared to concentrations in other NAWQA river basins. This report presents estimates of the magnitude of nutrient-source inputs to the South Platte River Basin, describes nutrient concen- trations and loads in the South Platte River during different seasons, and presents comparisons of nutrient inputs to instream nutrient loads. Annual nutrient inputs to the basin were estimated to be 306,000 tons of nitrogen and 41,000 tons of phosphorus. The principal nutrient sources were wastewater-treatment plants, fertilizer and manure applications, and atmospheric deposition. To characterize nutrient concentrations and loads in the South Platte River during different seasons, five nutrient synoptic samplings were conducted during 1994 and 1995. Upstream from Denver, Colorado, during April 1994 and January 1995, total nitrogen concentrations were less than 2 milligrams per liter (mg/L), and total phosphorus concentrations were less than 0.2 mg/L. The water in the river at this point was derived mostly from forested land in the mountains west of Denver. Total nutrient concentrations increased through the Denver metropolitan area, and concentration peaks occurred just downstream from each of Denver's largest wastewater-treatment plants with maximum concentrations of 13.6 mg/L total nitrogen and 2.4 mg/L total phosphorus. Nutrient concen- concentrations generally decreased downstream from Denver. Upstream from Denver during April 1994 and January 1995, total nitrogen loads were less than 1,000 pounds per day (lb/d), and total phosphorus loads were less than 125 lb/d. Total nutrient loads increased through the Denver metropolitan area, and load peaks occurred just downstream from each of Denver's largest wastewater-treatment plants, with a maximum load of 14,000 lb/d total nitrogen and 2,300 lb/d total phosphorus. In April 1994, nutrient loads generally decreased from Henderson, Colorado, to North Platte, Nebraska. In January 1995, however, nutrient loads increased from Henderson to Kersey, Colorado (maximum loads of 31,000 lb/d total nitrogen and 3,000 lb/d total phosphorus), and then decreased from Kersey to North Platte. Seasonal nutrient loads primarily were dependent on streamflow. Total nitrogen loads were largest in June 1994 and January 1995 when streamflows also were largest. During June, streamflow was large, but nitrogen concentrations were small, which indicated that snowmelt runoff diluted the available supply of nitrogen. Total phosphorus loads were largest in June, when streamflow and phosphorus concentrations were large, which indicated an additional source of phosphorus during snowmelt runoff. Streamflow along the South Platte River was smallest in April and August 1994, and nutrient loads also were smallest during these months. The downstream pattern for nutrient loads did not vary much by season. Loads were large at Henderson, decreased between Henderson and Kersey, and usually were largest at Kersey. The magnitude of the decrease in loads between Henderson and Kersey varied between synoptics and was dependent on the amount of water removed by irrigation ditches. Nutrient loads leaving the basin were very small compared to the estimated total nutrient inputs to the basin. Streamflow balances indicated that the South Platte River is a gaining river throughout much of its length; streamflow-balance residuals were as large as 15 cubic feet per second per mile. Nutrient-load balances indicated that increases in river nitrate loads were, in some places, due to nitrification and, elsewhere, were due to the influx of nitrate-enriched ground water to

Wash load and bed-material load transport in the Yellow River

USGS Publications Warehouse

Yang, C.T.; Simoes, F.J.M.

2005-01-01

It has been the conventional assumption that wash load is supply limited and is only indirectly related to the hydraulics of a river. Hydraulic engineers also assumed that bed-material load concentration is independent of wash load concentration. This paper provides a detailed analysis of the Yellow River sediment transport data to determine whether the above assumptions are true and whether wash load concentration can be computed from the original unit stream power formula and the modified unit stream power formula for sediment-laden flows. A systematic and thorough analysis of 1,160 sets of data collected from 9 gauging stations along the Middle and Lower Yellow River confirmed that the method suggested by the conjunctive use of the two formulas can be used to compute wash load, bed-material load, and total load in the Yellow River with accuracy. Journal of Hydraulic Engineering ?? ASCE.

Leptin alone and in combination with interleukin-1-beta induced cartilage degradation potentially inhibited by EPA and DHA.

PubMed

Phitak, Thanyaluck; Boonmaleerat, Kanchanit; Pothacharoen, Peraphan; Pruksakorn, Dumnoensun; Kongtawelert, Prachya

2017-09-28

Osteoarthritis (OA) is the most common form of arthritis. Obesity has been believed to be an important risk factor for OA development and the progression of not only load-bearing joints, but low-load-bearing joints as well. Increased leptin has been the focus of a link between obesity and OA. In this study, the effects of pathological (100ng/ml) or supra-pathological (10μg/ml) concentrations of leptin alone or in combination with IL1β on cartilage metabolisms were studied in porcine cartilage explant. The involved mechanisms were examined in human articular chondrocytes (HACs). Moreover, the protective effect of omega-3 polyunsaturated acids, eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) was also investigated. Leptin (10μg/ml) alone or in combination with IL1β could induce cartilage destruction, although lower concentrations had no effect. Leptin activated NFκB, ERK, JNK and p38 in HACs, which led to the induction of MMP3, MMP13 and ADAMTS4 secretions. The combined effect could further induce those enzymes through the additive effect on activation of NFκB and JNK. Interestingly, both EPA and DHA could inhibit cartilage damage induced by leptin plus IL1β by reducing the activation of NFκB and JNK, which led to the decrease of ADAMTS4 secretion. Altogether, only a supra-pathological concentration of leptin alone or in combination with IL1β could induce cartilage destruction, whereas a pathological one could not. This effect could be inhibited by EPA and DHA. To gain greater understanding of the link between leptin and OA, the effect of different levels of leptin on several states of OA cartilage requires further investigation.

Colorimetric Aptasensor Based on Enzyme for the Detection of Vibrio parahemolyticus.

PubMed

Wu, Shijia; Wang, Yinqiu; Duan, Nuo; Ma, Haile; Wang, Zhouping

2015-09-09

A simple colorimetric aptasensor system has been developed to detect Vibrio parahemolyticus. Magnetic nanoparticles (MNPs) are synthesized and conjugated with specific aptamers against target and used as capture probes. In addition, this method employs gold nanoparticles (AuNPs) as carriers of horseradish peroxidase (HRP) and aptamers, which served as signal probes. In the presence of target, a "sandwich-type" complex of AuNPs-HRP-aptamer-target-aptamer-MNPs is formed through specific recognition of aptamers and corresponding target. As a result, HRP molecules confined at the surface of the "sandwich" complexes catalyze the enzyme substrate, 3,3',5,5'-tetramethylbenzidine (TMB) and H2O2 and generate an optical signal. Under optimal conditions, the signals are linearly dependent on V. parahemolyticus concentrations from 10 to 10(6) colony-forming units (cfu)/mL in a logarithmic plot, with a limit of detection of 10 cfu/mL. Owing to AuNPs, a large amount of HRP could be loaded, resulting in an amplified signal, and the sensitivity would be improved. This strategy has the potential of being extended to the construction of simple monitor systems for a variety of biomolecules related to food safety.

Prevalence and Predictors of Abnormal Liver Enzymes in Young Women with Anorexia Nervosa

PubMed Central

Fong, Hiu-fai; DiVasta, Amy D.; DiFabio, Diane; Ringelheim, Julie; Jonas, Maureen M.; Gordon, Catherine M.

2008-01-01

Objective To determine the prevalence and predictors of abnormal liver enzyme levels in ambulatory young women with anorexia nervosa (AN). Study design In this cross-sectional study of 53 females with AN, serum concentrations of liver enzymes and hormones were measured. Anthropometric, dietary, and body composition information was collected. Correlational analyses were performed between liver enzyme concentrations and these variables. Results Elevated alanine aminotransferase (ALT) and gamma-glutamyltranspeptidase (GGT) levels were found in 14 subjects (26%) and 5 subjects (9%), respectively. ALT and GGT were inversely correlated with body mass index (r = −0.27 to −0.30, p ≤ 0.049) and percentage body fat (r = −0.36 to −0.47, p ≤ 0.007), but showed no relationship with lean body mass. Subjects with percentage body fat < 18% had higher ALT levels than those above this threshold (median 26.5 vs. 18.0 U/L, p = 0.01). Liver enzyme concentrations did not correlate with dietary variables, except for GGT and percentage of calories from protein (r = 0.28, p = 0.04). Conclusions Serum ALT and GGT concentrations are inversely related to adiposity in young women with AN. Future studies are needed to determine if these liver enzyme elevation signify unrecognized, clinically relevant liver disease. PMID:18534220

Immobilization Techniques to Avoid Enzyme Loss from Oxidase-Based Biosensors: A One-Year Study

PubMed Central

House, Jody L.; Anderson, Ellen M.; Ward, W. Kenneth

2007-01-01

Background Continuous amperometric sensors that measure glucose or lactate require a stable sensitivity, and glutaraldehyde crosslinking has been used widely to avoid enzyme loss. Nonetheless, little data is published on the effectiveness of enzyme immobilization with glutaraldehyde. Methods A combination of electrochemical testing and spectrophotometric assays was used to study the relationship between enzyme shedding and the fabrication procedure. In addition, we studied the relationship between the glutaraldehyde concentration and sensor performance over a period of one year. Results The enzyme immobilization process by glutaraldehyde crosslinking to glucose oxidase appears to require at least 24-hours at room temperature to reach completion. In addition, excess free glucose oxidase can be removed by soaking sensors in purified water for 20 minutes. Even with the addition of these steps, however, it appears that there is some free glucose oxidase entrapped within the enzyme layer which contributes to a decline in sensitivity over time. Although it reduces the ultimate sensitivity (probably via a change in the enzyme's natural conformation), glutaraldehyde concentration in the enzyme layer can be increased in order to minimize this instability. Conclusions After exposure of oxidase enzymes to glutaraldehyde, effective crosslinking requires a rinse step and a 24-hour incubation step. In order to minimize the loss of sensor sensitivity over time, the glutaraldehyde concentration can be increased. PMID:19888375

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.

Detergent effects on enzyme activity and solubilization of lipid bilayer membranes.

PubMed

Womack, M D; Kendall, D A; MacDonald, R C

1983-09-07

Over 50 detergents were tested to establish which would be most effective in releasing proteins from membrane-bounded compartments without denaturing them. Various concentrations each of detergent were tested for two activities: (1) solubilization of egg phospholipid liposomes as measured by reduction of turbidity and (2) effect of detergent concentration on the activities of soluble, hydrolytic enzymes. Those detergents most effective in solubilizing 0.2% lipid and least detrimental to enzymes were five pure, synthetic compounds recently introduced: CHAPS, CHAPSO, Zwittergents 310 and 312, and octylglucoside. Industrial detergents were generally much inferior, insofar as they solubilized membranes inefficiently and/or inactivated certain hydrolytic enzymes readily. The five detergents were characterized by (a) an unusually high critical micelle concentration and (b) a preference for forming mixed micelles with lipids instead of forming pure micelles, as indicated by an ability to solubilize lipid at concentrations of detergent significantly below the critical micelle concentration. This characteristic permits solubilization of high concentrations of membrane below the critical micelle concentration of the detergent so that protein denaturation is minimized. A generally applicable guideline that emerged from this study is that detergents should be used at approximately their critical micelle concentration which should not be exceeded by the concentration of membrane. Similar considerations should apply to the use of detergents in purifying and reconstituting intrinsic membrane proteins.

[Erythremia: the activity of erythrocyte antioxidant enzymes and the association with iron deficiency].

PubMed

Petukhov, V I; Kumerova, A O; Letse, A G; Silova, A A; Shkesters, A P; Krishchuna, M A; Mironova, N A

1997-01-01

Concentration of malonic dialdehyde (MDA) and activity of antioxidant enzymes G-6-PD, glutation peroxidase (GP), glutation reductase, catalase, superoxide dismutase were measured in red cells of patients with polycythemia vera. Plasmic ions Fe3+ were estimated by means of electron-paramagnetic resonance. MDA concentration and antioxidant enzymes (except GP) in polycythemia red cells were found increased, while the activity of selenium-dependent GP was reduced, the inhibition being greatest in severe iron deficiency. It is suggested that GP activity in red cells depends on both selenium levels in the body and concentrations of non-hematic iron.

Lactose Hydrolysis in Milk and Dairy Whey Using Microbial β-Galactosidases

PubMed Central

Dutra Rosolen, Michele; Gennari, Adriano; Volpato, Giandra; Volken de Souza, Claucia Fernanda

2015-01-01

This work aimed at evaluating the influence of enzyme concentration, temperature, and reaction time in the lactose hydrolysis process in milk, cheese whey, and whey permeate, using two commercial β-galactosidases of microbial origins. We used Aspergillus oryzae (at temperatures of 10 and 55°C) and Kluyveromyces lactis (at temperatures of 10 and 37°C) β-galactosidases, both in 3, 6, and 9 U/mL concentrations. In the temperature of 10°C, the K. lactis β-galactosidase enzyme is more efficient in the milk, cheese whey, and whey permeate lactose hydrolysis when compared to A. oryzae. However, in the enzyme reaction time and concentration conditions evaluated, 100% lactose hydrolysis was not reached using the K. lactis β-galactosidase. The total lactose hydrolysis in whey and permeate was obtained with the A. oryzae enzyme, when using its optimum temperature (55°C), at the end of a 12 h reaction, regardless of the enzyme concentration used. For the lactose present in milk, this result occurred in the concentrations of 6 and 9 U/mL, with the same time and temperature conditions. The studied parameters in the lactose enzymatic hydrolysis are critical for enabling the application of β-galactosidases in the food industry. PMID:26587283

Lactose Hydrolysis in Milk and Dairy Whey Using Microbial β-Galactosidases.

PubMed

Dutra Rosolen, Michele; Gennari, Adriano; Volpato, Giandra; Volken de Souza, Claucia Fernanda

2015-01-01

This work aimed at evaluating the influence of enzyme concentration, temperature, and reaction time in the lactose hydrolysis process in milk, cheese whey, and whey permeate, using two commercial β-galactosidases of microbial origins. We used Aspergillus oryzae (at temperatures of 10 and 55°C) and Kluyveromyces lactis (at temperatures of 10 and 37°C) β-galactosidases, both in 3, 6, and 9 U/mL concentrations. In the temperature of 10°C, the K. lactis β-galactosidase enzyme is more efficient in the milk, cheese whey, and whey permeate lactose hydrolysis when compared to A. oryzae. However, in the enzyme reaction time and concentration conditions evaluated, 100% lactose hydrolysis was not reached using the K. lactis β-galactosidase. The total lactose hydrolysis in whey and permeate was obtained with the A. oryzae enzyme, when using its optimum temperature (55°C), at the end of a 12 h reaction, regardless of the enzyme concentration used. For the lactose present in milk, this result occurred in the concentrations of 6 and 9 U/mL, with the same time and temperature conditions. The studied parameters in the lactose enzymatic hydrolysis are critical for enabling the application of β-galactosidases in the food industry.

Application of standard addition for the determination of carboxypeptidase activity in Actinomucor elegans bran koji.

PubMed

Fu, J; Li, L; Yang, X Q; Zhu, M J

2011-01-01

Leucine carboxypeptidase (EC 3.4.16) activity in Actinomucor elegans bran koji was investigated via absorbance at 507 nm after stained by Cd-nihydrin solution, with calibration curve A, which was made by a set of known concentration standard leucine, calibration B, which was made by three sets of known concentration standard leucine solutions with the addition of three concentrations inactive crude enzyme extract, and calibration C, which was made by three sets of known concentration standard leucine solutions with the addition of three concentrations crude enzyme extract. The results indicated that application of pure amino acid standard curve was not a suitable way to determine carboxypeptidase in complicate mixture, and it probably led to overestimated carboxypeptidase activity. It was found that addition of crude exact into pure amino acid standard curve had a significant difference from pure amino acid standard curve method (p < 0.05). There was no significant enzyme activity difference (p > 0.05) between addition of active crude exact and addition of inactive crude kind, when the proper dilute multiple was used. It was concluded that the addition of crude enzyme extract to the calibration was needed to eliminate the interference of free amino acids and related compounds presented in crude enzyme extract.

Inhibition of catecholamine degradation ameliorates while chemical sympathectomy aggravates the severity of acute Friend retrovirus infection in mice.

PubMed

Bloemker, Dominique; Mollerus, Sina; Gibbert, Kathrin; Dittmer, Ulf; Del Rey, Adriana; Schedlowski, Manfred; Engler, Harald

2016-05-01

Several lines of evidence indicate that the sympathetic nervous system (SNS) might be involved in the pathogenesis and progression of retroviral infections. However, experimental data are scarce and findings inconsistent. Here, we investigated the role of the SNS during acute infection with Friend virus (FV), a pathogenic murine retrovirus that causes polyclonal proliferation of erythroid precursor cells and splenomegaly in adult mice. Experimental animals were infected with FV complex, and viral load, spleen weight, and splenic noradrenaline (NA) concentration was analyzed until 25 days post infection. Results show that FV infection caused a massive but transient depletion in splenic NA during the acute phase of the disease. At the peak of the virus-induced splenomegaly, splenic NA concentration was reduced by about 90% compared to naïve uninfected mice. Concurrently, expression of the catecholamine degrading enzymes monoamine oxidase A (MAO-A) and catechol-O-methyltransferase (COMT) was significantly upregulated in immune cells of the spleen. Pharmacological inhibition of MAO-A and COMT by the selective inhibitors clorgyline and 3,5-dinitrocatechol, respectively, efficiently blocked NA degradation and significantly reduced viral load and virus-induced splenomegaly. In contrast, chemical sympathectomy prior to FV inoculation aggravated the acute infection and extended the duration of the disease. Together these findings demonstrate that catecholamine availability at the site of viral replication is an important factor affecting the course of retroviral infections. Copyright © 2016 Elsevier Inc. All rights reserved.

Chitosan-based biocatalytic nanoparticles for pollutant removal from wastewater.

PubMed

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

2017-05-01

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

PHYSICOCHEMICAL PROPERTIES OF THE PROTEOLYTIC ENZYME FROM THE LATEX OF THE MILKWEED, ASCLEPIAS SPECIOSA TORR. SOME COMPARISONS WITH OTHER PROTEASES

PubMed Central

Winnick, Theodore; Davis, Alva R.; Greenberg, David M.

1940-01-01

1. The kinetics of milk clotting by asclepain, the protease of Asclepias speciosa, were investigated. At higher concentrations of enzyme, the clotting time was inversely proportional to the enzyme concentration. 2. The digestion of casein and hemoglobin in 6.6 M urea by asclepain follows the second order reaction rate. The rate was roughly second order for casein in water. 3. Evaluation of the nature of the enzyme-substrate intermediate indicates that one molecule of asclepain combines with one molecule of casein or hemoglobin in urea solution. 4. Inhibition by the reaction products was deduced from the fact that the digestion velocity of hemoglobin in urea solution varied with the asclepain concentration in agreement with the Schütz-Borissov rule. PMID:19873155

Chemical concentrations and instantaneous loads, Green River to the Lower Duwamish Waterway near Seattle, Washington, 2013–15

USGS Publications Warehouse

Conn, Kathleen E.; Black, Robert W.; Vanderpool-Kimura, Ann M.; Foreman, James R.; Peterson, Norman T.; Senter, Craig A.; Sissel, Stephen K.

2015-12-23

Median chemical concentrations in suspended-sediment samples were greater than median chemical concentrations in fine bed sediment (less than 62.5 µm) samples, which were greater than median chemical concentrations in paired bulk bed sediment (less than 2 mm) samples. Suspended-sediment concentration, sediment particle-size distribution, and general water-quality parameters were measured concurrent with the chemistry sampling. From this discrete data, combined with the continuous streamflow record, estimates of instantaneous sediment and chemical loads from the Green River to the Lower Duwamish Waterway were calculated. For most compounds, loads were higher during storms than during baseline conditions because of high streamflow and high chemical concentrations. The highest loads occurred during dam releases (periods when stored runoff from a prior storm is released from the Howard Hanson Dam into the upper Green River) because of the high river streamflow and high suspended-sediment concentration, even when chemical concentrations were lower than concentrations measured during storm events. 

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.

Rating curve estimation of nutrient loads in Iowa rivers

USGS Publications Warehouse

Stenback, G.A.; Crumpton, W.G.; Schilling, K.E.; Helmers, M.J.

2011-01-01

Accurate estimation of nutrient loads in rivers and streams is critical for many applications including determination of sources of nutrient loads in watersheds, evaluating long-term trends in loads, and estimating loading to downstream waterbodies. Since in many cases nutrient concentrations are measured on a weekly or monthly frequency, there is a need to estimate concentration and loads during periods when no data is available. The objectives of this study were to: (i) document the performance of a multiple regression model to predict loads of nitrate and total phosphorus (TP) in Iowa rivers and streams; (ii) determine whether there is any systematic bias in the load prediction estimates for nitrate and TP; and (iii) evaluate streamflow and concentration factors that could affect the load prediction efficiency. A commonly cited rating curve regression is utilized to estimate riverine nitrate and TP loads for rivers in Iowa with watershed areas ranging from 17.4 to over 34,600km2. Forty-nine nitrate and 44 TP datasets each comprising 5-22years of approximately weekly to monthly concentrations were examined. Three nitrate data sets had sample collection frequencies averaging about three samples per week. The accuracy and precision of annual and long term riverine load prediction was assessed by direct comparison of rating curve load predictions with observed daily loads. Significant positive bias of annual and long term nitrate loads was detected. Long term rating curve nitrate load predictions exceeded observed loads by 25% or more at 33% of the 49 measurement sites. No bias was found for TP load prediction although 15% of the 44 cases either underestimated or overestimate observed long-term loads by more than 25%. The rating curve was found to poorly characterize nitrate and phosphorus variation in some rivers. ?? 2010 .

Enzyme class identification in cleaning products by hydrolysis followed by derivatization with o-phthaldialdehyde, HPLC and linear discriminant analysis.

PubMed

Beneito-Cambra, M; Bernabé-Zafón, V; Herrero-Martínez, J M; Simó-Alfonso, E F; Ramis-Ramos, G

2009-07-15

The enzymes present in raw materials of the cleaning industry (enzyme industrial concentrates) and in household cleaners were isolated by precipitation with acetone and hydrolyzed with HCl. The resulting amino acids were derivatized with o-phthaldialdehyde, and the derivatives were separated by HPLC. The peaks of 14 amino acids were observed using a C18 column and a multi-segmented gradient of acetonitrile-water in the presence of a 5 mM citric/citrate buffer of pH 6.5. Using either normalized peak areas (divided by the sum of the peak areas of the chromatogram) or ratios of pairs of peak areas as predictor variables, linear discriminant analysis models, capable of predicting the enzyme class, including proteases, lipases, amylases and cellulases, were constructed. For this purpose, both enzyme industrial concentrates and detergent bases spiked with them were included in the training set. In all cases, the enzymes of the evaluation set, including industrial concentrates, spiked detergent bases and commercial cleaners were correctly classified with assignment probabilities higher than 99%.

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

Identification and optimization of parameters for the semi-continuous production of garbage enzyme from pre-consumer organic waste by green RP-HPLC method.

PubMed

Arun, C; Sivashanmugam, P

2015-10-01

Reuse and management of organic solid waste, reduce the environmental impact on human health and increase the economic status by generating valuable products for current and novel applications. Garbage enzyme is one such product produced from fermentation of organic solid waste and it can be used as liquid fertilizer, antimicrobial agents, treatment of domestic wastewater, municipal and industrial sludge treatment, etc. The semi-continuous production of garbage enzyme in large quantity at minimal time period and at lesser cost is needed to cater for treatment of increasing quantities of industrial waste activated sludge. This necessitates a parameter for monitoring and control for the scaling up of current process on semi-continuous basis. In the present study a RP-HPLC (Reversed Phase-High Performance Liquid Chromatography) method is used for quantification of standard organic acid at optimized condition 30°C column oven temperature, pH 2.7, and 0.7 ml/min flow rate of the mobile phase (potassium dihydrogen phosphate in water) at 50mM concentration. The garbage enzyme solution collected in 15, 30, 45, 60, 75 and 90 days were used as sample to determine the concentration of organic acid. Among these, 90th day sample showed the maximum concentration of 78.14 g/l of acetic acid in garbage enzyme, whereas other organic acids concentration got decreased when compare to the 15th day sample. This result confirms that the matured garbage enzyme contains a higher concentration of acetic acid and thus it can be used as a monitoring parameter for semi-continuous production of garbage enzyme in large scale. Copyright © 2015 Elsevier Ltd. All rights reserved.

Enzyme-polyelectrolyte complexes in water-ethanol mixtures: negatively charged groups artificially introduced into alpha-chymotrypsin provide additional activation and stabilization effects.

PubMed

Kudryashova, E V; Gladilin, A K; Vakurov, A V; Heitz, F; Levashov, A V; Mozhaev, V V

1997-07-20

Formation of noncovalent complexes between alpha-chymotrypsin (CT) and a polyelectrolyte, polybrene (PB), has been shown to produce two major effects on enzymatic reactions in binary mixtures of polar organic cosolvents with water. (i) At moderate concentrations of organic cosolvents (10% to 30% v/v), enzymatic activity of CT is higher than in aqueous solutions, and this activation effect is more significant for CT in complex with PB (5- to 7-fold) than for free enzyme (1.5- to 2.5-fold). (ii) The range of cosolvent concentrations that the enzyme tolerates without complete loss of catalytic activity is much broader. For enhancement of enzyme stability in the complex with the polycation, the number of negatively charged groups in the protein has been artificially increased by using chemical modification with pyromellitic and succinic anhydrides. Additional activation effect at moderate concentrations of ethanol and enhanced resistance of the enzyme toward inactivation at high concentrations of the organic solvent have been observed for the modified preparations of CT in the complex with PB as compared with an analogous complex of the native enzyme. Structural changes behind alterations in enzyme activity in water-ethanol mixtures have been studied by the method of circular dichroism (CD). Protein conformation of all CT preparations has not changed significantly up to 30% v/v of ethanol where activation effects in enzymatic catalysis were most pronounced. At higher concentrations of ethanol, structural changes in the protein have been observed for different forms of CT that were well correlated with a decrease in enzymatic activity. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 267-277, 1997.

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.

Vicissitudes of oxidative stress biomarkers in the estuarine crab Scylla serrata with reference to dry and wet weather conditions in Ennore estuary, Tamil Nadu, India.

PubMed

Ragunathan, M G

2017-03-15

The primary objective of this study was to understand the impact of monsoon and summer seasons on the Polychlorinated Biphenyls (PCB's) and petroleum hydrocarbon compounds (PHC's) load in Ennore estuary and how the physiological response of estuarine Scylla serrata inhabiting in this estuary changed with reference to antioxidant defense. Seasonal levels of PCB's and PHC's were assessed in the water along with their bioaccumulation in gills, hemolymph, hepatopancreas and ovary of S. serrata. Concentration of PCB's and PHC's in water and their bioaccumulation was found to be higher in summer season when compared to monsoon season. Enzymic antioxidant assays [superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), glutathione reductase (GR), glutathione-S-transferase (GST)]; non-enzymic antioxidant assays [glutathione (GSH), vitamin C, vitamin E] and macromolecular alterations [membrane lipid peroxidation (LPO), and DNA Damage (strand breaks)] were assessed in the gills, hemolymph and hepatopancreas of S. serrata. There was a significant (p<0.05) upregulation in lipid peroxidation activity and DNA damage activity collected during the summer season when compared to the pre- and post-monsoon seasons. On the contrary, the enzymic and non-enzymic antioxidants exhibited significant (p<0.05) down regulation in the gills, hemolymph, hepatopancreas and ovary of S. serrata. Oxidative stress biomarkers represented a significant (p<0.05) maximum in gills when compared to hemolymph and hepatopancreas of S. serrata. Present study provided scientific evidences of how the antioxidant defense status of S. serrata responded to PCB's and PAH's stress with reference to seasonal vicissitudes, which indirectly represented the environmental health conditions of the estuary. Copyright © 2017 Elsevier Ltd. All rights reserved.

The Activity of Trypsin

ERIC Educational Resources Information Center

Russo, Salvatore F.; Holzman, Tom

1977-01-01

Describes an experiment that illustrates the following points concerning the experimental determination of trypsin activity: (1) there is a difference in basing enzyme concentration on weight, absorbance, or active sites; and (2) the method of expressing enzyme concentration determines the value of specific, molecular, and catalytic center…

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.

Nutrient Loadings to Streams of the Continental United States from Municipal and Industrial Effluent

USGS Publications Warehouse

Maupin, M.A.; Ivahnenko, T.

2011-01-01

Data from the United States Environmental Protection Agency Permit Compliance System national database were used to calculate annual total nitrogen (TN) and total phosphorus (TP) loads to surface waters from municipal and industrial facilities in six major regions of the United States for 1992, 1997, and 2002. Concentration and effluent flow data were examined for approximately 118,250 facilities in 45 states and the District of Columbia. Inconsistent and incomplete discharge locations, effluent flows, and effluent nutrient concentrations limited the use of these data for calculating nutrient loads. More concentrations were reported for major facilities, those discharging more than 1million gallons per day, than for minor facilities, and more concentrations were reported for TP than for TN. Analytical methods to check and improve the quality of the Permit Compliance System data were used. Annual loads were calculated using "typical pollutant concentrations" to supplement missing concentrations based on the type and size of facilities. Annual nutrient loads for over 26,600 facilities were calculated for at least one of the three years. Sewage systems represented 74% of all TN loads and 58% of all TP loads. This work represents an initial set of data to develop a comprehensive and consistent national database of point-source nutrient loads. These loads can be used to inform a wide range of water-quality management, watershed modeling, and research efforts at multiple scales. ?? 2011 American Water Resources Association. This article is a U.S. Government work and is in the public domain in the USA.

Analysis of Dissolved Selenium Loading for Selected Sites in the Lower Gunnison River Basin, Colorado, 1978-2005

USGS Publications Warehouse

Thomas, Judith C.; Leib, Kenneth J.; Mayo, John W.

2008-01-01

Elevated selenium concentrations in streams are a water-quality concern in western Colorado. The U.S. Geologic Survey, in cooperation with the Colorado Department of Public Health and Environment, summarized selenium loading in the Lower Gunnison River Basin to support the development of total maximum daily selenium loads at sites that represent the cumulative contribution to U.S. Environmental Protection Agency 303(d) list segments. Analysis of selenium loading included quantifying loads and determining the amount of load that would need to be reduced to bring the site into compliance, referred to as 'the load reduction,' with the State chronic aquatic-life standard for dissolved selenium [85th percentile selenium concentration not to exceed 4.6 ?g/L (micrograms per liter)], referred to as 'the water-quality standard.' Streamflow and selenium concentration data for 54 historical water-quality/water-quantity monitoring sites were compiled from U.S. Geological Survey and Colorado Department of Public Health and Environment data sources. Three methods were used for analysis of selenium concentration data to address the variable data density among sites. Mean annual selenium loads were determined for only 10 of the 54 sites due to data availability limitations. Twenty-two sites had 85th percentile selenium concentrations that exceeded the water-quality standard, 3 sites had 85th percentile selenium concentrations less than the State standard, and 29 sites could not be evaluated with respect to 85th percentile selenium concentration (sample count less than 5). To bring selenium concentrations into compliance with the water-quality standard, more than 80 percent of the mean annual selenium load would need to be reduced at Red Rock Canyon, Dry Cedar Creek, Cedar Creek, Loutzenhizer Arroyo, Sunflower Drain, and Whitewater Creek. More than 50 percent of the mean annual load would need to be reduced at Dry Creek to bring the site into compliance with the water-quality standard. The Uncompahgre River, Gunnison River at Delta, and Gunnison River near Grand Junction would require 69, 34 and 53 percent, respectively, of the mean annual load to be reduced for water years 2001 through 2005 to meet the water-quality standard. Mean annual load reductions can be further reduced by targeting the periods of time when selenium would be removed from streams by remediation. During a previous study of selenium loads in the Lower Gunnison River Basin, mean annual load reductions were estimated at the Gunnison River near Grand Junction for the 1997?2001 study period. Mean annual load reductions estimated for this study period were less than those estimated for the 2001?05 study period, emphasizing the importance of understanding that different study periods can result in different load reduction estimates.

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

A Process-Based Model of TCA Cycle Functioning to Analyze Citrate Accumulation in Pre- and Post-Harvest Fruits.

PubMed

Etienne, Audrey; Génard, Michel; Bugaud, Christophe

2015-01-01

Citrate is one of the most important organic acids in many fruits and its concentration plays a critical role in organoleptic properties. The regulation of citrate accumulation throughout fruit development, and the origins of the phenotypic variability of the citrate concentration within fruit species remain to be clarified. In the present study, we developed a process-based model of citrate accumulation based on a simplified representation of the TCA cycle to predict citrate concentration in fruit pulp during the pre- and post-harvest stages. Banana fruit was taken as a reference because it has the particularity of having post-harvest ripening, during which citrate concentration undergoes substantial changes. The model was calibrated and validated on the two stages, using data sets from three contrasting cultivars in terms of citrate accumulation, and incorporated different fruit load, potassium supply, and harvest dates. The model predicted the pre and post-harvest dynamics of citrate concentration with fairly good accuracy for the three cultivars. The model suggested major differences in TCA cycle functioning among cultivars during post-harvest ripening of banana, and pointed to a potential role for NAD-malic enzyme and mitochondrial malate carriers in the genotypic variability of citrate concentration. The sensitivity of citrate accumulation to growth parameters and temperature differed among cultivars during post-harvest ripening. Finally, the model can be used as a conceptual basis to study citrate accumulation in fleshy fruits and may be a powerful tool to improve our understanding of fruit acidity.

Effect of reactor loading on atomic oxygen concentration as measured by NO chemiluminescence

NASA Technical Reports Server (NTRS)

Lerner, N. R.

1989-01-01

It has previously been observed that the etch rate of polyethylene samples in the afterglow of an RF discharge in oxygen increases with reactor loading. This enhancement of the etch rate is attributed to reactive gas phase products of the polymer etching. In the present work, emission spectroscopy is employed to examine the species present in the gas phase during etching of polyethylene. In particular, the concentration of atomic oxygen downstream from the polyethylene samples is studied as a function of the reactor loading. It is found that the concentration of atomic oxygen increases as the reactor loading is increased. The increase of etch rate with increased reactor loading is attributed to the increase of atomic oxygen concentration in the vicinity of the sample.

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.

The kinetics of the oxidation of cytochrome c by Paracoccus cytochrome c peroxidase.

PubMed

Gilmour, R; Goodhew, C F; Pettigrew, G W; Prazeres, S; Moura, J J; Moura, I

1994-06-15

In work that is complementary to our investigation of the spectroscopic features of the cytochrome c peroxidase from Paracoccus denitrificans [Gilmour, Goodhew, Pettigrew, Prazeres, Moura and Moura (1993) Biochem. J. 294, 745-752], we have studied the kinetics of oxidation of cytochrome c by this enzyme. The enzyme, as isolated, is in the fully oxidized form and is relatively inactive. Reduction of the high-potential haem at pH 6 with ascorbate results in partial activation of the enzyme. Full activation is achieved by addition of 1 mM CaCl2. Enzyme activation is associated with formation of a high-spin state at the oxidized low-potential haem. EGTA treatment of the oxidized enzyme prevents activation after reduction with ascorbate, while treatment with EGTA of the reduced, partially activated, form abolishes the activity. We conclude that the active enzyme is a mixed-valence form with the low-potential haem in a high-spin state that is stabilized by Ca2+. Dilution of the enzyme results in a progressive loss of activity, the extent of which depends on the degree of dilution. Most of the activity lost upon dilution can be recovered after reconcentration. The M(r) of the enzyme on molecular-exclusion chromatography is concentration-dependent, with a shift to lower values at lower concentrations. Values of M(r) obtained are intermediate between those of a monomer (39,565) and a dimer. We propose that the active form of the enzyme is a dimer which dissociates at high dilution to give inactive monomers. From the activity of the enzyme at different dilutions, a KD of 0.8 microM can be calculated for the monomerdimer equilibrium. The cytochrome c peroxidase oxidizes horse ferrocytochrome c with first-order kinetics, even at high ferrocytochrome c concentrations. The maximal catalytic-centre activity ('turnover number') under the assay conditions used is 62,000 min-1, with a half-saturating ferrocytochrome c concentration of 3.3 microM. The corresponding values for the Paracoccus cytochrome c-550 (presumed to be the physiological substrate) are 85,000 min-1 and 13 microM. However, in this case, the kinetics deviate from first-order progress curves at all ferrocytochrome c concentrations. Consideration of the periplasmic environment in Paracoccus denitrificans leads us to propose that the enzyme will be present as the fully active dimer supplied with saturating ferrocytochrome c-550.

Reliability of the Load-Velocity Relationship Obtained Through Linear and Polynomial Regression Models to Predict the One-Repetition Maximum Load.

PubMed

Pestaña-Melero, Francisco Luis; Haff, G Gregory; Rojas, Francisco Javier; Pérez-Castilla, Alejandro; García-Ramos, Amador

2017-12-18

This study aimed to compare the between-session reliability of the load-velocity relationship between (1) linear vs. polynomial regression models, (2) concentric-only vs. eccentric-concentric bench press variants, as well as (3) the within-participants vs. the between-participants variability of the velocity attained at each percentage of the one-repetition maximum (%1RM). The load-velocity relationship of 30 men (age: 21.2±3.8 y; height: 1.78±0.07 m, body mass: 72.3±7.3 kg; bench press 1RM: 78.8±13.2 kg) were evaluated by means of linear and polynomial regression models in the concentric-only and eccentric-concentric bench press variants in a Smith Machine. Two sessions were performed with each bench press variant. The main findings were: (1) first-order-polynomials (CV: 4.39%-4.70%) provided the load-velocity relationship with higher reliability than second-order-polynomials (CV: 4.68%-5.04%); (2) the reliability of the load-velocity relationship did not differ between the concentric-only and eccentric-concentric bench press variants; (3) the within-participants variability of the velocity attained at each %1RM was markedly lower than the between-participants variability. Taken together, these results highlight that, regardless of the bench press variant considered, the individual determination of the load-velocity relationship by a linear regression model could be recommended to monitor and prescribe the relative load in the Smith machine bench press exercise.

Enzyme-mediated bacterial biodegradation of an azo dye (C.I. Acid blue 113): reuse of treated dye wastewater in post-tanning operations.

PubMed

Senthilvelan, T; Kanagaraj, J; Panda, R C

2014-11-01

"Dyeing" is a common practice used to color the hides during the post-tanning operations in leather processing generating plenty of wastewater. The waste stream containing dye as pollutant is severely harmful to living beings. An azo dye (C.I. Acid Blue 113) has been biodegraded effectively by bacterial culture mediated with azoreductase enzyme to reduce the pollution load in the present investigation. The maximum rate of dye degradation was found to be 96 ± 4 and 92 ± 4 % for the initial concentrations of 100 and 200 mg/l, respectively. The enzyme activity was measured using NADH as a substrate. Fourier transform infrared spectroscopy (FT-IR) analysis was confirmed that the transformation of azo linkage could be transformed into N2 or NH3 or incorporated into complete biomass. Breaking down of dye molecules to various metabolites (such as aniline, naphthalene-1,4-diamine, 3-aminobenzenesulfonic acid, naphthalene-1-sulfonic acid, 8-aminonaphthalene-1-sulfonic acid, 5,8-diaminonaphthalene-1-sulfonic acid) was confirmed by gas chromatography and mass spectra (GC-MS) and mass (electrospray ionization (ESI)) spectra analysis. The treated wastewater could be reused for dyeing operation in the leather processing, and the properties of produced leather were evaluated by conventional methods that revealed to have improved dye penetration into the grain layer of experimental leather sample and resulted in high levelness of dyeing, which helps to obtain the desired smoothness and soft leather properties.

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.

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.

Selected Contribution: Skeletal muscle focal adhesion kinase, paxillin, and serum response factor are loading dependent

NASA Technical Reports Server (NTRS)

Gordon, S. E.; Fluck, M.; Booth, F. W.

2001-01-01

This investigation examined the effect of mechanical loading state on focal adhesion kinase (FAK), paxillin, and serum response factor (SRF) in rat skeletal muscle. We found that FAK concentration and tyrosine phosphorylation, paxillin concentration, and SRF concentration are all lower in the lesser load-bearing fast-twitch plantaris and gastrocnemius muscles compared with the greater load-bearing slow-twitch soleus muscle. Of these three muscles, 7 days of mechanical unloading via tail suspension elicited a decrease in FAK tyrosine phosphorylation only in the soleus muscle and decreases in FAK and paxillin concentrations only in the plantaris and gastrocnemius muscles. Unloading decreased SRF concentration in all three muscles. Mechanical overloading (via bilateral gastrocnemius ablation) for 1 or 8 days increased FAK and paxillin concentrations in the soleus and plantaris muscles. Additionally, whereas FAK tyrosine phosphorylation and SRF concentration were increased by < or =1 day of overloading in the soleus muscle, these increases did not occur until somewhere between 1 and 8 days of overloading in the plantaris muscle. These data indicate that, in the skeletal muscles of rats, the focal adhesion complex proteins FAK and paxillin and the transcription factor SRF are generally modulated in association with the mechanical loading state of the muscle. However, the somewhat different patterns of adaptation of these proteins to altered loading in slow- vs. fast-twitch skeletal muscles indicate that the mechanisms and time course of adaptation may partly depend on the prior loading state of the muscle.

Metabolite concentrations, fluxes and free energies imply efficient enzyme usage

DOE PAGES

Park, Junyoung O.; Rubin, Sara A.; Xu, Yi -Fan; ...

2016-05-02

In metabolism, available free energy is limited and must be divided across pathway steps to maintain a negative Δ G throughout. For each reaction, Δ G is log proportional both to a concentration ratio (reaction quotient to equilibrium constant) and to a flux ratio (backward to forward flux). In this paper, we use isotope labeling to measure absolute metabolite concentrations and fluxes in Escherichia coli, yeast and a mammalian cell line. We then integrate this information to obtain a unified set of concentrations and Δ G for each organism. In glycolysis, we find that free energy is partitioned so asmore » to mitigate unproductive backward fluxes associated with Δ G near zero. Across metabolism, we observe that absolute metabolite concentrations and Δ G are substantially conserved and that most substrate (but not inhibitor) concentrations exceed the associated enzyme binding site dissociation constant ( K m or K i). Finally, the observed conservation of metabolite concentrations is consistent with an evolutionary drive to utilize enzymes efficiently given thermodynamic and osmotic constraints.« less

Impact of limited solvent capacity on metabolic rate, enzyme activities, and metabolite concentrations of S. cerevisiae glycolysis.

PubMed

Vazquez, Alexei; de Menezes, Marcio A; Barabási, Albert-László; Oltvai, Zoltan N

2008-10-01

The cell's cytoplasm is crowded by its various molecular components, resulting in a limited solvent capacity for the allocation of new proteins, thus constraining various cellular processes such as metabolism. Here we study the impact of the limited solvent capacity constraint on the metabolic rate, enzyme activities, and metabolite concentrations using a computational model of Saccharomyces cerevisiae glycolysis as a case study. We show that given the limited solvent capacity constraint, the optimal enzyme activities and the metabolite concentrations necessary to achieve a maximum rate of glycolysis are in agreement with their experimentally measured values. Furthermore, the predicted maximum glycolytic rate determined by the solvent capacity constraint is close to that measured in vivo. These results indicate that the limited solvent capacity is a relevant constraint acting on S. cerevisiae at physiological growth conditions, and that a full kinetic model together with the limited solvent capacity constraint can be used to predict both metabolite concentrations and enzyme activities in vivo.

Impact of Limited Solvent Capacity on Metabolic Rate, Enzyme Activities, and Metabolite Concentrations of S. cerevisiae Glycolysis

PubMed Central

Vazquez, Alexei; de Menezes, Marcio A.; Barabási, Albert-László; Oltvai, Zoltan N.

2008-01-01

The cell's cytoplasm is crowded by its various molecular components, resulting in a limited solvent capacity for the allocation of new proteins, thus constraining various cellular processes such as metabolism. Here we study the impact of the limited solvent capacity constraint on the metabolic rate, enzyme activities, and metabolite concentrations using a computational model of Saccharomyces cerevisiae glycolysis as a case study. We show that given the limited solvent capacity constraint, the optimal enzyme activities and the metabolite concentrations necessary to achieve a maximum rate of glycolysis are in agreement with their experimentally measured values. Furthermore, the predicted maximum glycolytic rate determined by the solvent capacity constraint is close to that measured in vivo. These results indicate that the limited solvent capacity is a relevant constraint acting on S. cerevisiae at physiological growth conditions, and that a full kinetic model together with the limited solvent capacity constraint can be used to predict both metabolite concentrations and enzyme activities in vivo. PMID:18846199

Treating Wastewater With Immobilized Enzymes

NASA Technical Reports Server (NTRS)

Jolly, Clifford D.

1991-01-01

Experiments show enzymes are immobilized on supporting materials to make biocatalyst beds for treatment of wastewater. With suitable combination of enzymes, concentrations of various inorganic and organic contaminants, including ammonia and urea, reduced significantly.

Enzyme leaps fuel antichemotaxis

PubMed Central

Jee, Ah-Young; Dutta, Sandipan; Cho, Yoon-Kyoung

2018-01-01

There is mounting evidence that enzyme diffusivity is enhanced when the enzyme is catalytically active. Here, using superresolution microscopy [stimulated emission-depletion fluorescence correlation spectroscopy (STED-FCS)], we show that active enzymes migrate spontaneously in the direction of lower substrate concentration (“antichemotaxis”) by a process analogous to the run-and-tumble foraging strategy of swimming microorganisms and our theory quantifies the mechanism. The two enzymes studied, urease and acetylcholinesterase, display two families of transit times through subdiffraction-sized focus spots, a diffusive mode and a ballistic mode, and the latter transit time is close to the inverse rate of catalytic turnover. This biochemical information-processing algorithm may be useful to design synthetic self-propelled swimmers and nanoparticles relevant to active materials. Executed by molecules lacking the decision-making circuitry of microorganisms, antichemotaxis by this run-and-tumble process offers the biological function to homogenize product concentration, which could be significant in situations when the reactant concentration varies from spot to spot. PMID:29255047

Polysaccharide nano-vesicular multidrug carriers for synergistic killing of cancer cells

NASA Astrophysics Data System (ADS)

Pramod, P. S.; Shah, Ruchira; Chaphekar, Sonali; Balasubramanian, Nagaraj; Jayakannan, Manickam

2014-09-01

Multi-drug delivery based on polymer nano-scaffolds is an essential protocol to be developed for better administration of anticancer drugs to enhance their therapeutic efficacies against cancer cells. Here, we report dual delivery polysaccharide nano-vesicles that are capable of loading and delivering both water soluble and water insoluble drugs together in a single polymer scaffold. The selective rupture of the nano-vesicular assembly under intracellular enzyme conditions allowed the simultaneous delivery of a hydrophobic drug camptothecin (CPT) and hydrophilic drug doxorubicin (DOX) supporting their synergistic killing of breast and colon cancer cells. The polysaccharide nano-vesicles have allowed us to address a few important questions regarding the need for multiple drug administration in cancer cells including (a) the role of simultaneous drug release, (b) antagonistic versus synergistic effects of drug combinations and (c) how these are affected by the ratio of drugs. Further, evaluation of the role of caveolae in endocytosis of these polymer scaffolds was also made. The vesicular scaffolds were found to preserve and deliver DOX resulting in 50-60% better killing of cancer cells than the free drug. Additionally, dual loaded nano-vesicles when compared to drug cocktails with individual drugs in separate nano-vesicles (at comparable molar ratios) suggest the relative drug concentration following release and mode of delivery to be both important in cancer cell killing. Results from these experiments have revealed newly developed polysaccharide nano-vesicles loaded with DOX and CPT drugs as potential candidates for improved breast cancer cell killing. Thus, these custom-designed polysaccharide nano-vesicles provide a new perspective on multi-anticancer drug delivery systems and their efficacy.Multi-drug delivery based on polymer nano-scaffolds is an essential protocol to be developed for better administration of anticancer drugs to enhance their therapeutic efficacies against cancer cells. Here, we report dual delivery polysaccharide nano-vesicles that are capable of loading and delivering both water soluble and water insoluble drugs together in a single polymer scaffold. The selective rupture of the nano-vesicular assembly under intracellular enzyme conditions allowed the simultaneous delivery of a hydrophobic drug camptothecin (CPT) and hydrophilic drug doxorubicin (DOX) supporting their synergistic killing of breast and colon cancer cells. The polysaccharide nano-vesicles have allowed us to address a few important questions regarding the need for multiple drug administration in cancer cells including (a) the role of simultaneous drug release, (b) antagonistic versus synergistic effects of drug combinations and (c) how these are affected by the ratio of drugs. Further, evaluation of the role of caveolae in endocytosis of these polymer scaffolds was also made. The vesicular scaffolds were found to preserve and deliver DOX resulting in 50-60% better killing of cancer cells than the free drug. Additionally, dual loaded nano-vesicles when compared to drug cocktails with individual drugs in separate nano-vesicles (at comparable molar ratios) suggest the relative drug concentration following release and mode of delivery to be both important in cancer cell killing. Results from these experiments have revealed newly developed polysaccharide nano-vesicles loaded with DOX and CPT drugs as potential candidates for improved breast cancer cell killing. Thus, these custom-designed polysaccharide nano-vesicles provide a new perspective on multi-anticancer drug delivery systems and their efficacy. Electronic supplementary information (ESI) available: Synthesis scheme, DLS histogram, FE-SEM image, AFM image, TEM image of DEX-PDP-5, AFM image of VDOX+CPT, AFM image of VDOX, characterization of VCPT, characterization of VRHO, DOX nuclear localization, characterization of dual drug loaded vesicles, fluorescent microscopic image of VDOX-CPT, cumulative drug release profile from dual drug loaded vesicles, rate constant determination, and cumulative release profile of DOX and CPT from VDOX+CPT (1 : 4). See DOI: 10.1039/c4nr03514c

Targeted quantification of functional enzyme dynamics in environmental samples for microbially mediated biogeochemical processes: Targeted quantification of functional enzyme dynamics

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

Li, Minjing; Gao, Yuqian; Qian, Wei-Jun

Microbially mediated biogeochemical processes are catalyzed by enzymes that control the transformation of carbon, nitrogen, and other elements in environment. The dynamic linkage between enzymes and biogeochemical species transformation has, however, rarely been investigated because of the lack of analytical approaches to efficiently and reliably quantify enzymes and their dynamics in soils and sediments. Herein, we developed a signature peptide-based technique for sensitively quantifying dissimilatory and assimilatory enzymes using nitrate-reducing enzymes in a hyporheic zone sediment as an example. Moreover, the measured changes in enzyme concentration were found to correlate with the nitrate reduction rate in a way different frommore » that inferred from biogeochemical models based on biomass or functional genes as surrogates for functional enzymes. This phenomenon has important implications for understanding and modeling the dynamics of microbial community functions and biogeochemical processes in environments. Our results also demonstrate the importance of enzyme quantification for the identification and interrogation of those biogeochemical processes with low metabolite concentrations as a result of faster enzyme-catalyzed consumption of metabolites than their production. The dynamic enzyme behaviors provide a basis for the development of enzyme-based models to describe the relationship between the microbial community and biogeochemical processes.« less

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.

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 cervical mucus plug inhibits, but does not block, the passage of ascending bacteria from the vagina during pregnancy.

PubMed

Hansen, Lea K; Becher, Naja; Bastholm, Sara; Glavind, Julie; Ramsing, Mette; Kim, Chong J; Romero, Roberto; Jensen, Jørgen S; Uldbjerg, Niels

2014-01-01

To evaluate the microbial load and the inflammatory response in the distal and proximal parts of the cervical mucus plug. Experimental research. Twenty women with a normal, singleton pregnancy. Vaginal swabs and specimens from the distal and proximal parts of the cervical mucus plug. Immunohistochemistry, enzyme-linked immunosorbent assay, quantitative polymerase chain reaction and histology. The total bacterial load (16S rDNA) was significantly lower in the cervical mucus plug compared with the vagina (p = 0.001). Among women harboring Ureaplasma parvum, the median genome equivalents/g were 1574 (interquartile range 2526) in the proximal part, 657 (interquartile range 1620) in the distal part and 60,240 (interquartile range 96,386) in the vagina. Histological examinations and quantitative polymerase chain reaction revealed considerable amounts of lactobacilli and inflammatory cells in both parts of the cervical mucus plug. The matrix metalloproteinase-8 concentration was decreased in the proximal part of the plug compared with the distal part (p = 0.08). The cervical mucus plug inhibits, but does not block, the passage of Ureaplasma parvum during its ascending route from the vagina through the cervical canal. © 2013 Nordic Federation of Societies of Obstetrics and Gynecology.

Robust enzymatic hydrolysis of Formiline-pretreated oil palm empty fruit bunches (EFB) for efficient conversion of polysaccharide to sugars and ethanol.

PubMed

Cui, Xingkai; Zhao, Xuebing; Zeng, Jing; Loh, Soh Kheang; Choo, Yuen May; Liu, Dehua

2014-08-01

Oil palm empty fruit bunch (EFB) was pretreated by Formiline process to overcome biomass recalcitrance and obtain hemicellulosic syrup and lignin. Higher formic acid concentration led to more lignin removal but also higher degree of cellulose formylation. Cellulose digestibility could be well recovered after deformylation with a small amount of lime. After digested by enzyme loading of 15 FPU+10 CBU/g solid for 48 h, the polysaccharide conversion could be over 90%. Simultaneous saccharification and fermentation (SSF) results demonstrated that ethanol concentration reached 83.6 g/L with approximate 85% of theoretic yield when performed at an initial dry solid consistency of 20%. A mass balance showed that via Formiline pretreatment 0.166 kg of ethanol could be produced from 1 kg of dry EFB with co-production of 0.14 kg of high-purity lignin and 5.26 kg hemicellulosic syrup containing 2.8% xylose. Formiline pretreatment thus can be employed as an entry for biorefining of EFB. Copyright © 2014 Elsevier Ltd. All rights reserved.

Cholinesterase assay for monitoring the kinetics of the JD6. 5 organophosphorus acid anhydrase in detoxification of diisopropylfluorophosphate. Final report, January-June 1988

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

Yeh, H.R.; Cheng, T.C.; DeFrank, J.J.

1992-06-01

In the present studies, cholinesterase was used for monitoring the enzymatic activities of the JD6.5 organophosphorus acid anhydrase. The kinetic data indicated that: (1) the first order of kinetic constants (k) and Vmax values of the enzymatic reactions increased as the concentrations of the enzyme increased; (2) while the half-life (tl/2) of diisopropylfluorophosphate (DFP) hydrolysis decreased as the enzyme concentrations increased; (3) the minimum time required for hydrolysis of 9mM of DFP was 3 min at the concentrations of the enzyme present; Km values of DFP were found to be in range of 5mM; and (4) both MnCl2 and NaClmore » were found to be required for the optimal activity of the enzyme.« less

Lead and PCB's in canvasback ducks: Relationship between enzyme levels and residues in blood

USGS Publications Warehouse

Dieter, M.P.; Perry, M.C.; Mulhern, B.M.

1976-01-01

Blood samples were taken for two successive years from canvasback ducks trapped in the Chesapeake Bay. The first winter (1972?1973) five plasma enzymes known to respond to organochlorine poisoning were examined. Abnormal enzyme elevations suggested that 20% of the population sampled (23/115 ducks) might contain organochlorine contaminants, but no residue analyses were performed. The second winter (1974) two of the same enzymes, aspartate aminotransferase and lactate dehydrogenase, and a third enzyme known to be specifically inhibited by lead, delta-aminolevulinic acid dehydratase, were assayed in 95 blood samples. Blood residues of organochlorine compounds and of lead were determined in representative samples, and the correlations between residue levels and enzyme changes were examined. The enzyme bioassays in 1974 indicated that lead was a more prevalent environmental contaminant than organochlorine compounds in canvasback ducks; 17% of the blood samples had less than one-half of the normal delta-aminolevulinic acid dehydratase activity, but only 11% exhibited abnormal aspartate aminotransferase or lactate dehydrogenase activities. These findings were confirmed by residue analyses that demonstrated lead concentrations four times higher than background levels, but only relatively low organochlorine concentrations. There was a highly significant inverse correlation between delta-aminolevulinic acid dehydratase activity and blood lead concentrations (P<0.01), and a weaker but significant correlation between plasma aspartate aminotransferase activity and blood PCB concentrations (P<0.05). It was apparent that delta-aminolevulinic acid dehydratase activity in the blood provided a sensitive and precise estimate of lead contamination in waterfowl. In canvasback ducks 200 ppb of lead in the blood caused a 75% decrease in delta-aminolevulinic acid dehydratase activity, a magnitude of enzyme inhibition that disturbs heme synthesis and is regarded as detrimental in humans.

Atypical profiles and modulations of heme-enzymes catalyzed outcomes by low amounts of diverse additives suggest diffusible radicals' obligatory involvement in such redox reactions.

PubMed

Manoj, Kelath Murali; Parashar, Abhinav; Venkatachalam, Avanthika; Goyal, Sahil; Satyalipsu; Singh, Preeti Gunjan; Gade, Sudeep K; Periyasami, Kalaiselvi; Jacob, Reeba Susan; Sardar, Debosmita; Singh, Shanikant; Kumar, Rajan; Gideon, Daniel A

2016-06-01

Peroxidations mediated by heme-enzymes have been traditionally studied under a single-site (heme distal pocket), non-sequential (ping-pong), two-substrates binding scheme of Michaelis-Menten paradigm. We had reported unusual modulations of peroxidase and P450 reaction outcomes and explained it invoking diffusible reactive species [Manoj, 2006; Manoj et al., 2010; Andrew et al., 2011, Parashar et al., 2014 & Venkatachalam et al., 2016]. A systematic investigation of specific product formation rates was undertaken to probe the hypothesis that involvement of diffusible reactive species could explain undefined substrate specificities and maverick modulations (sponsored by additives) of heme-enzymes. When the rate of specific product formation was studied as a function of reactants' concentration or environmental conditions, we noted marked deviations from normal profiles. We report that heme-enzyme mediated peroxidations of various substrates are inhibited (or activated) by sub-equivalent concentrations of diverse redox-active additives and this is owing to multiple redox equilibriums in the milieu. At low enzyme and peroxide concentrations, the enzyme is seen to recycle via a one-electron (oxidase) cycle, which does not require the substrate to access the heme centre. Schemes are provided that explain the complex mechanistic cycle, kinetics & stoichiometry. It is not obligatory for an inhibitor or substrate to interact with the heme centre for influencing overall catalysis. Roles of diffusible reactive species explain catalytic outcomes at low enzyme and reactant concentrations. The current work highlights the scope/importance of redox enzyme reactions that could occur "out of the active site" in biological or in situ systems. Copyright © 2016 Elsevier B.V. and Société française de biochimie et biologie Moléculaire (SFBBM). All rights reserved.

Quality of wet deposition in the Grand Calumet River watershed, northwestern Indiana, June 30, 1992-August 31, 1993

USGS Publications Warehouse

Willoughby, T.C.

1995-01-01

Northwestern Indiana is one of the most heavily industrialized and largest steel-producing areas in the United States. High temperature processes, such as fossil-fuel combustion and steel production, release contaminants to the atmosphere that may result in wet deposition being a major contributor to major ion and trace-metal loadings in north- western Indiana and Lake Michigan. A wet-deposition collection site was established at the Gary (Indiana) Regional Airport in June 1992 to monitor the chemical quality of wet deposition. Weekly samples were collected at this site from June 30, 1992, through August 31, 1993, and were analyzed for pH, specific conductance, and selected major ions and trace metals. Forty-eight samples collected during the study were of sufficient volumes for some of the determinations to be performed. Median constituent concentrations were determined for samples collected during warm weather and cold weather (November 1 through March 31). Median concentrations were substituted for missing values from samples with insufficient volumes for analysis of all the constituents of interest. Constituent concentrations were converted to weekly loadings. Two values were calculated to provide a range for the weekly loading for samples with measured concentrations of constituents less than the method reporting limit. The minimum weekly loading was computed by substituting zero for the constituent concentration; the maximum weekly loading was computed by substituting the method reporting limit for the concentration. If all of the sample concentrations measured were greater than the method reporting limit, an annual loading value was computed. The annual loadings could be used to assist in estimating the contribution of wet deposition to the total annual constituent loadings in the Grand Calumet River in northwestern Indiana.

Association between maternal micronutrient status, oxidative stress, and common genetic variants in antioxidant enzymes at 15 weeks׳ gestation in nulliparous women who subsequently develop preeclampsia.

PubMed

Mistry, Hiten D; Gill, Carolyn A; Kurlak, Lesia O; Seed, Paul T; Hesketh, John E; Méplan, Catherine; Schomburg, Lutz; Chappell, Lucy C; Morgan, Linda; Poston, Lucilla

2015-01-01

Preeclampsia is a pregnancy-specific condition affecting 2-7% of women and a leading cause of perinatal and maternal morbidity and mortality. Deficiencies of specific micronutrient antioxidant activities associated with copper, selenium, zinc, and manganese have previously been linked to preeclampsia at the time of disease. Our aims were to investigate whether maternal plasma micronutrient concentrations and related antioxidant enzyme activities are altered before preeclampsia onset and to examine the dependence on genetic variations in these antioxidant enzymes. Predisease plasma samples (15±1 weeks׳ gestation) were obtained from women enrolled in the international Screening for Pregnancy Endpoints (SCOPE) study who subsequently developed preeclampsia (n=244) and from age- and BMI-matched normotensive controls (n=472). Micronutrient concentrations were measured by inductively coupled plasma mass spectrometry; associated antioxidant enzyme activities, selenoprotein-P, ceruloplasmin concentration and activity, antioxidant capacity, and markers of oxidative stress were measured by colorimetric assays. Sixty-four tag-single-nucleotide polymorphisms (SNPs) within genes encoding the antioxidant enzymes and selenoprotein-P were genotyped using allele-specific competitive PCR. Plasma copper and ceruloplasmin concentrations were modestly but significantly elevated in women who subsequently developed preeclampsia (both P<0.001) compared to controls (median (IQR), copper, 1957.4 (1787, 2177.5) vs 1850.0 (1663.5, 2051.5) µg/L; ceruloplasmin, 2.5 (1.4, 3.2) vs 2.2 (1.2, 3.0) µg/ml). There were no differences in other micronutrients or enzymes between groups. No relationship was observed between genotype for SNPs and antioxidant enzyme activity. This analysis of a prospective cohort study reports maternal micronutrient concentrations in combination with associated antioxidant enzymes and SNPs in their encoding genes in women at 15 weeks׳ gestation that subsequently developed preeclampsia. The modest elevation in copper may contribute to oxidative stress, later in pregnancy, in those women that go on to develop preeclampsia. The lack of evidence to support the hypothesis that functional SNPs influence antioxidant enzyme activity in pregnant women argues against a role for these genes in the etiology of preeclampsia. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

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.

Improving of enzyme immunoassay for detection and quantification of the target molecules using silver nanoparticles

NASA Astrophysics Data System (ADS)

Syrvatka, Vasyl J.; Slyvchuk, Yurij I.; Rozgoni, Ivan I.; Gevkan, Ivan I.; Overchuk, Marta O.

2014-02-01

Modern routine enzyme immunoassays for detection and quantification of biomolecules have several disadvantages such as high cost, insufficient sensitivity, complexity and long-term execution. The surface plasmon resonance of silver nanoparticles gives reasons of creating new in the basis of simple, highly sensitive and low cost colorimetric assays that can be applied to the detection of small molecules, DNA, proteins and pollutants. The main aim of the study was the improving of enzyme immunoassay for detection and quantification of the target molecules using silver nanoparticles. For this purpose we developed method for synthesis of silver nanoparticles with hyaluronic acid and studied possibility of use these nanoparticles in direct determination of target molecules concentration (in particular proteins) and for improving of enzyme immunoassay. As model we used conventional enzyme immunoassays for determination of progesterone and estradiol concentration. We obtained the possibility to produce silver nanoparticles with hyaluronan homogeneous in size between 10 and 12 nm, soluble and stable in water during long term of storage using modified procedure of silver nanoparticles synthesis. New method allows to obtain silver nanoparticles with strong optical properties at the higher concentrations - 60-90 μg/ml with the peak of absorbance at the wavelength 400 nm. Therefore surface plasmon resonance of silver nanoparticles with hyaluronan and ultraviolet-visible spectroscopy provide an opportunity for rapid determination of target molecules concentration (especial protein). We used silver nanoparticles as enzyme carriers and signal enhancers. Our preliminary data show that silver nanoparticles increased absorbance of samples that allows improving upper limit of determination of estradiol and progesterone concentration.

Delivery of Formulated Industrial Enzymes with Acoustic Technology.

PubMed

Hwang, Jennifer Dorcas; Ortiz-Maldonado, Mariliz; Paramonov, Sergey

2016-02-01

Industrial enzymes are instrumental in many applications, including carbohydrate processing, fabric and household care, biofuels, food, and animal nutrition, among others. Enzymes have to be active and stable not only in harsh application conditions, but also during shipment and storage. In protein stability studies, formulated concentrated enzyme solutions are frequently diluted gravimetrically prior to enzyme activity measurements, making it challenging to move toward more high-throughput techniques using conventional robotic equipment. Current assay methods pose difficulties when measuring highly concentrated proteins. For example, plastic pipette tips can introduce error because proteins adsorb to the tip surface, despite the presence of detergents, decreasing precision and overall efficiency of protein activity assays. Acoustic liquid handling technology, frequently used for various dilute small-molecule assays, may overcome such problems. Originally shown to effectively deliver dilute solutions of small molecules, this technology is used here as an effective alternative to the aforementioned challenge with viscous concentrated protein solutions. Because the acoustic liquid handler transfers nanoliter quantities of liquids without using pipette tips and without sample loss, it rapidly and uniformly prepares assay plates for enzyme activity measurements within minutes. This increased efficiency transforms the nature of enzyme stability studies toward high precision and throughput. © 2015 Society for Laboratory Automation and Screening.

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.

Liver function in cats with hyperthyroidism before and after 131I therapy.

PubMed

Berent, Allyson C; Drobatz, Kenneth J; Ziemer, Lisa; Johnson, Victoria S; Ward, Cynthia R

2007-01-01

The clinical significance of high serum concentration or activity of markers of liver damage in cats with hyperthyroidism is unknown. To evaluate serum markers of liver function and damage, and ultrasonographic changes in cats with hyperthyroidism and with high liver enzymes, and to determine if abnormalities resolve after treatment with 131I. Nineteen cats with hyperthyroidism (15 with high serum activities of liver enzymes) and 4 age-matched healthy control cats. Serum bile acids, albumin, ammonia, cholesterol, and blood urea nitrogen concentrations, and activities of liver-derived enzymes, and blood glucose concentrations were measured before and after 131I therapy. These values were compared with those of cats that were euthyroid. In addition, gross liver parenchymal changes detected by abdominal ultrasonographic examination, before and after 131I therapy were evaluated. High serum liver enzyme activities were not associated with abnormalities in hepatic parenchyma and liver functional variables, regardless of the degree of increase. Serum liver enzyme activities return to normal after control of hyperthyroidism with 131I therapy. Cats with hyperthyroidism have a significantly higher serum fasting ammonia concentration than cats who were euthyroid (P = .019). Cats with hyperthyroidism also have significantly lower serum cholesterol (P = .005) and glucose (P = .002) concentrations before compared with after 131I therapy. Nine of 19 cats with hyperthyroidism had trace ketonuria. These results demonstrate that extensive examination for hepatobiliary disease in most cats with hyperthyroidism is unnecessary.

Closed-form analysis of fiber-matrix interface stresses under thermo-mechanical loadings

NASA Technical Reports Server (NTRS)

Naik, Rajiv A.; Crews, John H., Jr.

1992-01-01

Closed form techniques for calculating fiber matrix (FM) interface stresses, using repeating square and diamond regular arrays, were presented for a unidirectional composite under thermo-mechanical loadings. An Airy's stress function micromechanics approach from the literature, developed for calculating overall composite moduli, was extended in the present study to compute FM interface stresses for a unidirectional graphite/epoxy (AS4/3501-6) composite under thermal, longitudinal, transverse, transverse shear, and longitudinal shear loadings. Comparison with finite element results indicate excellent agreement of the FM interface stresses for the square array. Under thermal and longitudinal loading, the square array has the same FM peak stresses as the diamond array. The square array predicted higher stress concentrations under transverse normal and longitudinal shear loadings than the diamond array. Under transverse shear loading, the square array had a higher stress concentration while the diamond array had a higher radial stress concentration. Stress concentration factors under transverse shear and longitudinal shear loadings were very sensitive to fiber volume fraction. The present analysis provides a simple way to calculate accurate FM interface stresses for both the square and diamond array configurations.

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

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.

Differences in the Load-Velocity Profile Between 4 Bench-Press Variants.

PubMed

García-Ramos, Amador; Pestaña-Melero, Francisco Luis; Pérez-Castilla, Alejandro; Rojas, Francisco Javier; Haff, Guy Gregory

2018-03-01

To compare the load-velocity relationship between 4 variants of the bench-press (BP) exercise. The full load-velocity relationship of 30 men was evaluated by means of an incremental loading test starting at 17 kg and progressing to the individual 1-repetition maximum (1RM) in 4 BP variants: concentric-only BP, concentric-only BP throw (BPT), eccentric-concentric BP, and eccentric-concentric BPT. A strong and fairly linear relationship between mean velocity (MV) and %1RM was observed for the 4 BP variants (r 2  > .96 for pooled data and r 2  > .98 for individual data). The MV associated with each %1RM was significantly higher in the eccentric-concentric technique than in the concentric-only technique. The only significant difference between the BP and BPT variants was the higher MV with the light to moderate loads (20-70%1RM) in the BPT using the concentric-only technique. MV was significantly and positively correlated between the 4 BP variants (r = .44-.76), which suggests that the subjects with higher velocities for each %1RM in 1 BP variant also tend to have higher velocities for each %1RM in the 3 other BP variants. These results highlight the need for obtaining specific equations for each BP variant and the existence of individual load-velocity profiles.

A Simulation Game for the Study of Enzyme Kinetics and Inhibition.

ERIC Educational Resources Information Center

Chayoth, Reuben; Cohen, Annette

1996-01-01

Presents a simulation game that facilitates understanding of the concepts of enzyme kinetics and inhibition. The first part of the game deals with the relationship between enzyme activity and substrate concentration while the second part deals with characterization of competitive and noncompetitive inhibition of enzyme activity. (JRH)

The limit of photoreceptor sensitivity: molecular mechanisms of dark noise in retinal cones.

PubMed

Holcman, David; Korenbrot, Juan I

2005-06-01

Detection threshold in cone photoreceptors requires the simultaneous absorption of several photons because single photon photocurrent is small in amplitude and does not exceed intrinsic fluctuations in the outer segment dark current (dark noise). To understand the mechanisms that limit light sensitivity, we characterized the molecular origin of dark noise in intact, isolated bass single cones. Dark noise is caused by continuous fluctuations in the cytoplasmic concentrations of both cGMP and Ca(2+) that arise from the activity in darkness of both guanylate cyclase (GC), the enzyme that synthesizes cGMP, and phosphodiesterase (PDE), the enzyme that hydrolyzes it. In cones loaded with high concentration Ca(2+) buffering agents, we demonstrate that variation in cGMP levels arise from fluctuations in the mean PDE enzymatic activity. The rates of PDE activation and inactivation determine the quantitative characteristics of the dark noise power density spectrum. We developed a mathematical model based on the dynamics of PDE activity that accurately predicts this power spectrum. Analysis of the experimental data with the theoretical model allows us to determine the rates of PDE activation and deactivation in the intact photoreceptor. In fish cones, the mean lifetime of active PDE at room temperature is approximately 55 ms. In nonmammalian rods, in contrast, active PDE lifetime is approximately 555 ms. This remarkable difference helps explain why cones are noisier than rods and why cone photocurrents are smaller in peak amplitude and faster in time course than those in rods. Both these features make cones less light sensitive than rods.

Denitrification and mixing in a stream-aquifer system: Effects on nitrate loading to surface water

USGS Publications Warehouse

McMahon, P.B.; Böhlke, J.K.

1996-01-01

Ground water in terrace deposits of the South Platte River alluvial aquifer near Greeley, Colorado, USA, had a median nitrate concentration of 1857 ??mol l-1. Median nitrate concentrations in ground water from adjacent floodplain deposits (468 ??mol l-1) and riverbed sediments (461 ??mol l-1), both of which are downgradient from the terrace deposits, were lower than the median concentration in the terrace deposits. The concentrations and ??15N values of nitrate and N2 in ground water indicated that denitrifying activity in the floodplain deposits and riverbed sediments accounted for 15- 30% of the difference in nitrate concentrations. Concentrations of Cl- and SiO2 indicated that mixing between river water and ground water in the floodplain deposits and riverbed sediments accounted for the remainder of the difference in nitrate concentrations. River flux measurements indicated that ground-water discharge in a 7.5 km segment of river had a nitrate load of 1718 kg N day-1 and accounted for about 18% of the total nitrate load in the river at the downstream end of that segment. This nitrate load was 70% less than the load predicted on the basis of the median nitrate concentration in the terrace deposits and assuming no denitrification or mixing in the aquifer. Water exchange between the river and aquifer caused ground water that originally discharged to the river to reenter denitrifying sediments in the riverbed and floodplain, thereby further decreasing the nitrate load in this stream-aquifer system. Results from this study indicated that denitrification and mixing within alluvial aquifer sediments may substantially decrease the nitrate load added to rivers by discharging ground water.

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.

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

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.

Threonine deaminase from extremely halophilic bacteria - Cooperative substrate kinetics and salt dependence.

NASA Technical Reports Server (NTRS)

Lieberman, M. M.; Lanyi, J. K.

1972-01-01

The effect of salt on the activity, stability, and allosteric properties of catabolic threonine deaminase from Halobacterium cutirubrum was studied. The enzyme exhibits sigmoidal kinetics with the substrate, threonine. The Hill slope is 1.55 at pH 10. The enzyme is activated by ADP at low substrate concentrations. In the presence of this effector, sigmoidal kinetics are no longer observed. At pH 10, in the absence of ADP, enzyme activity increases with increasing NaCl concentration from 0 to 4 M.

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.

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

Concentrations and loads of suspended sediment and trace element pollutants in a small semi-arid urban tributary, San Francisco Bay, California.

PubMed

McKee, Lester J; Gilbreath, Alicia N

2015-08-01

Water-quality policy documents throughout the world often identify urban stormwater as a large and controllable impact to sensitive ecosystems, yet there is often limited data to characterize concentrations and loads especially for rare and more difficult to quantify pollutants. In response, concentrations of suspended sediments and silver, mercury and selenium including speciation, and other trace elements were measured in dry and wet weather stormwater flow from a 100% urban watershed near San Francisco. Suspended sediment concentrations ranged between 1.4 and 2700 mg/L and varied with storm intensity. Turbidity was shown to correlate strongly with suspended sediments and most trace elements and was used as a surrogate with regression to estimate concentrations during unsampled periods and to compute loads. Mean suspended sediment yield was 31.5 t/km(2)/year. Total mercury ranged between 1.4 and 150 ng/L and was, on average, 92% particulate, 0.9% methylated, and 1.2% acid labile. Total mercury yield averaged 5.7 μg/m(2)/year. Total selenium ranged between non-detect and 2.9 μg/L and, on average, the total load (0.027 μg/m(2)/year) was 61% transported in dissolved phase. Selenate (Se(VI)) was the dominant species. Silver concentrations ranged between non-detect and 0.11 μg/L. Concentrations and loads of other trace elements were also highly variable and were generally similar to other urban systems with the exceptions of Ag and As (seldom reported) and Cr and Zn which exhibited concentrations and loads in the upper range of those reported elsewhere. Consistent with the semi-arid climatic setting, >95% of suspended sediment, 94% of total Hg, and 85-95 % of all other trace element loads were transported during storm flows with the exception of selenium which showed an inverse relationship between concentration and flow. Treatment of loads is made more challenging in arid climate settings due to low proportions of annual loads and greater dissolved phase during low flow conditions. This dataset fills an important local data gap for highly urban watersheds of San Francisco Bay. The field and interpretative methods, the uniqueness of the analyte list, and resulting information have general applicability for managing pollutant concentrations and loads in urban watersheds in other parts of the world and may have particularly useful application in more arid climates.

The effect of sampling frequency on the accuracy of nitrogen load estimates from a drained loblolly pine plantation in eastern North Carolina

Treesearch

George M. Chescheir; François Birgand; Shiying Tian; Mohamed A. Youssef; Devendra M. Amatya

2010-01-01

Nutrient loading in drainage outflow is estimated from measured flows and nutrient concentrations in the drainage water. The loading function is ideally continuous, representing the product of continuously measured outflows and nutrient concentrations in drainage water. However, loading is often estimated as the product of continuously measured outflow and nutrient...

Biocatalytic induction of supramolecular order

NASA Astrophysics Data System (ADS)

Hirst, Andrew R.; Roy, Sangita; Arora, Meenakshi; Das, Apurba K.; Hodson, Nigel; Murray, Paul; Marshall, Stephen; Javid, Nadeem; Sefcik, Jan; Boekhoven, Job; van Esch, Jan H.; Santabarbara, Stefano; Hunt, Neil T.; Ulijn, Rein V.

2010-12-01

Supramolecular gels, which demonstrate tunable functionalities, have attracted much interest in a range of areas, including healthcare, environmental protection and energy-related technologies. Preparing these materials in a reliable manner is challenging, with an increased level of kinetic defects observed at higher self-assembly rates. Here, by combining biocatalysis and molecular self-assembly, we have shown the ability to more quickly access higher-ordered structures. By simply increasing enzyme concentration, supramolecular order expressed at molecular, nano- and micro-levels is dramatically enhanced, and, importantly, the gelator concentrations remain identical. Amphiphile molecules were prepared by attaching an aromatic moiety to a dipeptide backbone capped with a methyl ester. Their self-assembly was induced by an enzyme that hydrolysed the ester. Different enzyme concentrations altered the catalytic activity and size of the enzyme clusters, affecting their mobility. This allowed structurally diverse materials that represent local minima in the free energy landscape to be accessed based on a single gelator structure.

Stress concentrations for straight-shank and countersunk holes in plates subjected to tension, bending, and pin loading

NASA Technical Reports Server (NTRS)

Shivakumar, K. N.; Newman, J. C., Jr.

1992-01-01

A three dimensional stress concentration analysis was conducted on straight shank and countersunk (rivet) holes in a large plate subjected to various loading conditions. Three dimensional finite element analysis were performed with 20 node isoparametric elements. The plate material was assumed to be linear elastic and isotropic, with a Poisson ratio of 0.3. Stress concentration along the bore of the hole were computed for several ratios of hole radius to plate thickness (0.1 to 2.5) and ratios of countersink depth to plate thickness (0.25 to 1). The countersink angles were varied from 80 to 100 degrees in some typical cases, but the angle was held constant at 100 degrees for most cases. For straight shank holes, three types of loading were considered: remote tension, remote bending, and wedge loading in the hole. Results for remote tension and wedge loading were used to estimate stress concentration for simulated rivet in pin loading. For countersunk holes only remote tension and bending were considered. Based on the finite element results, stress concentration equations were developed. Whenever possible, the present results were compared with other numerical solutions and experimental results from the literature.

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

DOE PAGES

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

2015-02-09

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

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

PubMed

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

2011-01-01

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

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

PubMed

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

2018-02-01

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

An Additional Method for Analyzing the Reversible Inhibition of an 
Enzyme Using Acid Phosphatase as a Model.

PubMed

Baumhardt, Jordan M; Dorsey, Benjamin M; McLauchlan, Craig C; Jones, Marjorie A

2015-08-01

Using wheat germ acid phosphatase and sodium orthovanadate as a competitive inhibitor, a novel method for analyzing reversible inhibition was carried out. Our alternative approach involves plotting the initial velocity at which product is formed as a function of the ratio of substrate concentration to inhibitor concentration at a constant enzyme concentration and constant assay conditions. The concept of initial concentrations driving equilibrium leads to the chosen axes. Three apparent constants can be derived from this plot: K max , K min , and K inflect . K max and K min represent the substrate to inhibitor concentration ratio for complete inhibition and minimal inhibition, respectively. K inflect represents the substrate to inhibitor concentration ratio at which the enzyme-substrate complex is equal to the inhibitory complex. These constants can be interpolated from the graph or calculated using the first and second derivative of the plot. We conclude that a steeper slope and a shift of the line to the right (increased x-axis values) would indicate a better inhibitor. Since initial velocity is not a linear function of the substrate/inhibitor ratio, this means that inhibition changes more quickly with the change in the [S]/ [I] ratio. When preincubating the enzyme with substrate before the addition of inhibitor, preincubating the enzyme with inhibitor before the addition of substrate or with concurrent addition of both substrate and inhibitor, modest changes in the slopes and y-intercepts were obtained. This plot appears useful for known competitive and non-competitive inhibitors and may have general applicability.

Concentrations and Loads of Nutrients and Suspended Sediments in Englesby Brook and Little Otter Creek, Lake Champlain Basin, Vermont, 2000-2005

USGS Publications Warehouse

Medalie, Laura

2007-01-01

The effectiveness of best-management practices (BMPs) in improving water quality in Lake Champlain tributaries was evaluated from 2000 through 2005 on the basis of analysis of data collected on concentrations of total phosphorus and suspended sediment in Englesby Brook, an urban stream in Burlington, and Little Otter Creek, an agricultural stream in Ferrisburg. Data also were collected on concentrations of total nitrogen in the Englesby Brook watershed. In the winter of 2001-2002, one of three planned structural BMPs was installed in the urban watershed. At approximately the same time, a set of barnyard BMPs was installed in the agricultural watershed; however, the other planned BMPs, which included streambank fencing and nutrient management, were not implemented within the study period. At Englesby Brook, concentrations of phosphorus ranged from 0.024 to 0.3 milligrams per liter (mg/L) during base-flow and from 0.032 to 11.8 mg/L during high-flow conditions. Concentrations of suspended sediment ranged from 3 to 189 mg/L during base-flow and from 5 to 6,880 mg/L during high-flow conditions. An assessment of the effectiveness of an urban BMP was made by comparing concentrations and loads of phosphorus and suspended sediment before and after a golf-course irrigation pond in the Englesby Brook watershed was retrofitted with the objective of reducing sediment transport. Results from a modified paired watershed study design showed that the BMP reduced concentrations of phosphorus and suspended sediment during high-flow events - when average streamflow was greater than 3 cubic feet per second. While construction of the BMP did not reduce storm loads of phosphorus or suspended sediment, an evaluation of changes in slope of double-mass curves showing cumulative monthly streamflow plotted against cumulative monthly loads indicated a possible reduction in cumulative loads of phosphorus and suspended sediment after BMP construction. Results from the Little Otter Creek assessment of agricultural BMPs showed that concentrations of phosphorus ranged from 0.016 to 0.141 mg/L during base-flow and from 0.019 to 0.565 mg/L during high-flow conditions at the upstream monitoring station. Concentrations of suspended sediment ranged from 2 to 13 mg/L during base-flow and from 1 to 473 mg/L during high-flow conditions at the upstream monitoring station. Concentrations of phosphorus ranged from 0.018 to 0.233 mg/L during base-flow and from 0.019 to 1.95 mg/L during high-flow conditions at the downstream monitoring station. Concentrations of suspended sediment ranged from 10 to 132 mg/L during base-flow and from 8 to 1,190 mg/L during high-flow conditions at the downstream monitoring station. Annual loads of phosphorus at the downstream monitoring station were significantly larger than loads at the upstream monitoring station, and annual loads of suspended sediment at the downstream monitoring station were larger than loads at the upstream monitoring station for 4 out of 6 years. On a monthly basis, loads of phosphorus and suspended sediment at the downstream monitoring station were significantly larger than loads at the upstream monitoring station. Pairs of concentrations of phosphorus and monthly loads of phosphorus and suspended sediment from the upstream and downstream monitoring stations were evaluated using the paired watershed study design. The only significant reduction between the calibration and treatment periods was for monthly loads of phosphorus; all other evaluations showed no change between periods.

Space shuttle solid rocket booster recovery system definition. Volume 3: SRB water impact loads computer program, user's manual

NASA Technical Reports Server (NTRS)

1973-01-01

This user's manual describes the FORTRAN IV computer program developed to compute the total vertical load, normal concentrated pressure loads, and the center of pressure of typical SRB water impact slapdown pressure distributions specified in the baseline configuration. The program prepares the concentrated pressure load information in punched card format suitable for input to the STAGS computer program. In addition, the program prepares for STAGS input the inertia reacting loads to the slapdown pressure distributions.

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.

Use of isoelectric focusing and a chromophoric organomercurial to monitor urea-induced conformational changes of yeast phosphoglycerate kinase.

PubMed Central

Stinson, R A

1977-01-01

The effects of urea in concentrations from 0 to 6M on the following properties of yeast phosphoglycerate kinase were studied: the kinetics of inactivation of the enzyme, the spectrum of 2-chloromercuri-4-nitrophenol bound to the single thiol group of the enzyme, the rate of reaction between the mercurial and enzyme, and the isoelectric point. The enzyme was inactivated by as much as 30% in 1M-urea, and the other data were interpreted as a possible 'tightening' of enzyme structure. The catalytic behaviour of the enzyme in 2M-urea was time-dependent, the initial effects being similar to those in 1M-urea. Polyacrylamide-gel isoelectric focusing of the enzyme in the presence of 2M-urea showed a single species of enzyme with an isoelectric point intermediate between those in 1M- and 3M-urea; a species with an identical isoelectric point was obtained after an 11-day exposure at 4 degrees C to the denaturant at 2M. The enzyme was rapidly inactivated in 3M-urea, with the thiol group fully exposed and the isoelectric point 0.9pH unit higher than in the absence of urea. No further conformational changes could be demonstrated with urea concentrations of 4M or greater. It is suggested that the equilibrium species that exists in 2M-urea has one of two buried lysine residues exposed. The second lysine residue is exposed in 3M or greater concentrations of the denaturant. Images Fig. 2. PMID:337969

Enzyme stabilization by glass-derived silicates in glass-exposed aqueous solutions

USGS Publications Warehouse

Ives, J.A.; Moffett, J.R.; Arun, P.; Lam, D.; Todorov, T.I.; Brothers, A.B.; Anick, D.J.; Centeno, J.; Namboodiri, M.A.A.; Jonas, W.B.

2010-01-01

Objectives: To analyze the solutes leaching from glass containers into aqueous solutions, and to show that these solutes have enzyme activity stabilizing effects in very dilute solutions. Methods: Enzyme assays with acetylcholine esterase were used to analyze serially succussed and diluted (SSD) solutions prepared in glass and plastic containers. Aqueous SSD preparations starting with various solutes, or water alone, were prepared under several conditions, and tested for their solute content and their ability to affect enzyme stability in dilute solution. Results: We confirm that water acts to dissolve constituents from glass vials, and show that the solutes derived from the glass have effects on enzymes in the resultant solutions. Enzyme assays demonstrated that enzyme stability in purified and deionized water was enhanced in SSD solutions that were prepared in glass containers, but not those prepared in plastic. The increased enzyme stability could be mimicked in a dose-dependent manner by the addition of silicates to the purified, deionized water that enzymes were dissolved in. Elemental analyses of SSD water preparations made in glass vials showed that boron, silicon, and sodium were present at micromolar concentrations. Conclusions: These results show that silicates and other solutes are present at micromolar levels in all glass-exposed solutions, whether pharmaceutical or homeopathic in nature. Even though silicates are known to have biological activity at higher concentrations, the silicate concentrations we measured in homeopathic preparations were too low to account for any purported in vivo efficacy, but could potentially influence in vitro biological assays reporting homeopathic effects. ?? 2009 The Faculty of Homeopathy.

Formate Dehydrogenase from Clostridium acidiurici

PubMed Central

Kearny, James J.; Sagers, Richard D.

1972-01-01

Partial purification of formate dehydrogenase from Clostridium acidiurici has been accomplished, and some properties of the enzyme have been determined. The molecular weight of the protein is at least 200,000 daltons. The enzyme showed marked instability to freezing and thawing and was inhibited strongly by oxygen and by light. Such inhibition was not reversed by incubation in the presence of thiol compounds. Cyanide inhibited the enzyme 90% at 0.1 mm concentrations, but ethylenediaminetetraacetate produced only slight inhibition at concentrations as high as 50 mm. The purified enzyme showed no ferredoxin activity in the Clostridium pasteurianum clastic system during pyruvate oxidation. Crude preparations of the enzyme could be coupled through ferredoxin to the reduction of nicotinamide adenine dinucleotide during formate oxidation, but the purified enzyme could not catalyze the reduction of pyridine nucleotides by formate in the presence of ferredoxin. Formate oxidation with the purified enzyme was readily coupled to benzyl viologen reduction, in which case ferredoxin was not required. An exchange between formate and bicarbonate was catalyzed by both crude and purified preparations of the enzyme, but the net synthesis of formate from CO2 was not accomplished. PMID:4333376

Immobilized Hydrolytic Enzymes Exhibit Antibiofilm Activity Against Escherichia coli at Sub-Lethal Concentrations.

PubMed

Villa, Federica; Secundo, Francesco; Polo, Andrea; Cappitelli, Francesca

2015-07-01

The effects of two commercially available immobilized enzymes (namely the glycosidase pectinase and the protease subtilisin A) at sub-lethal concentrations were investigated in terms of their influence on biofilm genesis, on the composition of the biofilm matrix, and their antibiotic synergy against Escherichia coli biofilm, used as a model system of bacterial biofilms. The best antibiofilm performance of solid-supported hydrolases was obtained at the surface concentration of 0.022 and 0.095 U/cm(2) with a reduction of 1.2 and 2.3 log CFU/biofilm for pectinase and subtilisin, respectively. At these enzyme surface concentrations, the biocatalysts affected the structural composition of the biofilm matrix, impacting biofilm thickness. Finally, the immobilized hydrolases enhanced biofilm sensitivity to a clinically relevant concentration of the antibiotic ampicillin. At the final antibiotic concentration of 0.1 mg/ml, a reduction of 2 and 3.5 log10 units in presence of 0.022 Upectinase/cm(2) and 0.095 Usubtilisin/cm(2) was obtained, respectively, in comparison the antibiotic alone. Immobilized pectinase and subtilisin at sub-lethal concentrations demonstrated a great potential for antibiofilm applications.

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.

The alteration of interelemental ratios in myocardium under the congenital heart disease (SRXRF)

NASA Astrophysics Data System (ADS)

Trunova, V. A.; Zvereva, V. V.; Okuneva, G. N.; Levicheva, E. N.

2007-05-01

It is the myocardium that bears the basic functional loading during heart working, including muscle contractility and enzyme activity. The elemental concentrations in myocardium tissue of heart were determined by SRXRF technique. Our investigation is systematical: the elemental content in each compartment (left and right ventricles, left and right auricles) of hearts of healthy and diseased children (congenital heart diseases, transposition of main vessels (TMV)) was analyzed. The elemental distribution in myocardium of four heart chambers of human fetuses was also analyzed. Following elements were determined: S, Cl, K, Ca, Cr, Mn, Fe, Ni, Cu, Zn, As, Se, Br, Rb, Sr. It was revealed that the elemental concentrations in myocardium of both ventricles are almost constant in heart of fetuses and healthy children. The transition from pre-natal study (fetus) to post-natal study is accompanied by the redistribution of chemical elements in myocardium. The higher concentrations of S, Fe, Ca, Sr and Cu in myocardium of children are observed, the content of K, Br, Rb and especially Se is lower than in heart of fetuses. The elemental distribution in myocardium of children TMV is considerably different in comparison with the healthy children: the higher levels of Cu are observed. The content of Se is lower.

Variation in oxidative stress indices of two green seaweeds growing under different heavy metal stresses.

PubMed

Ismail, Gehan A; Ismail, Mona M

2017-02-01

Concentrations of nine heavy metals (Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb, and Zn) were determined in the green seaweed species Cladophora glomerata and Ulva compressa collected from El-Mex and Sidi Kirayr locations. The heavy metal concentrations in algal tissues were in direct correlation with their soluble concentrations in seawater with the descending order: Fe

Concentrations and transport of atrazine in the Delaware River-Perry Lake system, northeast Kansas, July 1993 through September 1995

USGS Publications Warehouse

Pope, L.M.; Brewer, L.D.; Foley, G.A.; Morgan, S.C.

1996-01-01

A study of the distribution and transport of atrazine in surface water in the 1,117 square-mile Delaware River Basin in northeast Kansas was conducted from July 1992 through September 1995. The purpose of this report is to present information to assess the present (1992-95) conditions and possible future changes in the distribution and magnitude of atrazine concentrations, loads, and yields spatially, temporally, and in relation to hydrologic conditions and land-use characteristics. A network of 11 stream-monitoring and sample-collection sites was established within the basin. Stream- water samples were collected during a wide range of hydrologic conditions throughout the study. Nearly 5,000 samples were analyzed by enzyme- linked immunosorbent assay (ELISA) for triazine herbicide concentrations. Daily mean triazine herbicide concentrations were calculated for all sampling sites and subsequently used to estimate daily mean atrazine concentrations with a linear- regression relation between ELISA-derived triazine concentrations and atrazine concentrations determined by gas chromatography/mass spectrometry for 141 dual-analyzed surface-water samples. During May, June, and July, time-weighted, daily mean atrazine concentrations in streams in the Delaware River Basin commonly exceeded the value of 3.0-ug/L (micrograms per liter) annual mean Maximum Contaminant Level (MCL) established by the U.S. Environmental Protection Agency for drinking-water supplies. Time-weighted, daily mean concentrations equal to or greater than 20 ug/L were not uncommon. However, most time- weighted, daily mean concentrations were less than 1.0 ug/L from August through April. The largest time-weighted, monthly mean atrazine concentrations occurred during May, June, and July. Most monthly mean concentrations between August and April were less than 0.50 ug/L. Large differences were documented in monthly mean concentrations within the basin. Sites receiving runoff from the northern and northeastern parts of the Delaware River Basin had the largest monthly and annual mean atrazine concentrations. Time- weighted, annual mean atrazine concentrations did not exceed the MCL in water from any sampling site for either the 1993 or 1994 crop years (April-March); however, concentrations were during 1994 than during 1993. Time-weighted, annual mean concentrations in water from among the 11 sampling sites during the 1993 crop year ranged from 0.27 to 1.5 ug/L and from 0.36 to 2.8 ug/L during the 1994 crop year. Furthermore, concentrations in samples from the outflow of Perry Lake were larger during the first 6 months of the 1995 crop year than during the previous year. Flow-weighted, annual mean atrazine concentrations were larger than time-weighted, annual mean concentrations in water from all sampling sites upstream of Perry Lake, and samples from several sites had concentrations were substantially larger than the MCL. This difference explained why time-weighted, annual mean concentrations in the outflow of Perry Lake were larger than corresponding time-weighted concentrations in water from sampling sites upstream of Perry Lake. Flow- weighted, annual mean concentrations in water from among the 11 sampling sites during the 1993 crop year ranged from 1.0 to 4.4 ug/L and from 1.0 to 8.9 ug/L during the 1994 crop year. Statistically significant linear-regression equations were identified relating the percentage of subbasin in cropland to time- and flow-weighted, average annual mean atrazine concentrations. The relations indicate that time-weighted, average annual mean atrazine concentrations may not exceed the MCL in water from subbasins with at least about 70-percent cropland. However, flow-weighted, average annual mean atrazine concentrations may exceed the MCL when the percentage of cropland is greater than about 40 percent. Approximately 90 percent of the annual atrazine load is transport from May through July. Atrazine loads and yields were larger during the 1993 cro

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

Dependence of yield of nuclear track-biosensors on track radius and analyte concentration

NASA Astrophysics Data System (ADS)

García-Arellano, H.; Muñoz H., G.; Fink, D.; Vacik, J.; Hnatowicz, V.; Alfonta, L.; Kiv, A.

2018-04-01

In swift heavy ion track-based polymeric biosensor foils with incorporated enzymes one exploits the correlation between the analyte concentration and the sensor current, via the enrichment of charged enzymatic reaction products in the track's confinement. Here we study the influence of the etched track radius on the biosensor's efficiency. These sensors are analyte-specific only if both the track radii and the analyte concentration exceed certain threshold values of ∼15 nm and ∼10-6 M (for glucose sensing), respectively. Below these limits the sensor signal stems un-specifically from any charge carrier. In its proper working regime, the inner track walls are smoothly covered by enzymes and the efficiency is practically radius independent. Theory shows that the measured current should be slightly sub-proportional to the analyte concentration; the measurements roughly reconfirm this. Narrower tracks (∼5-15 nm radius) with reduced enzyme coverage lead to decreasing efficiency. Tiny signals visible when the tracks are etched to effective radii between 0 and ∼5 nm are tentatively ascribed to enzymes bonded to surface-near nano-cracks in the polymer foil, resulting from its degradation due to aging, rather than to the tracks. Precondition for this study was the accurate determination of the etched track radii, which is possible only by a nanofluidic approach. This holds to some extent even for enzyme-covered tracks, though in this case most of the wall charges are compensated by enzyme bonding.

Effects of dietary Pb on accumulation, histopathology, and digestive enzyme activities in the digestive system of tilapia (Oreochromis niloticus).

PubMed

Dai, Wei; Du, Huahua; Fu, Linglin; Jin, Chengguan; Xu, Zirong; Liu, Huitao

2009-02-01

With the increasing occurrence of dietary lead (Pb) contamination in aquatic environment, threat of the dietary Pb toxicity to aquatic organisms attracted more attention. In this study, after being exposed to dietary Pb at concentrations of 0, 100, 400, and 800-microg/g dry weight for 60 days, the groups of tilapia (Oreochromis niloticus) were sacrificed and sampled to analyze the effects of dietary Pb on accumulation, histopathology, and digestive enzyme activities in tissues of the digestive system. The results showed that the Pb accumulation in tissues increased with the dietary Pb concentrations. Moreover, Pb accumulated in sampled tissues in the following order: intestine > stomach > liver. By observation of liver histological sections in optical microscope, lesions could be detected in the Pb-contaminated groups. It was also demonstrated that the inhibitory effect of dietary Pb on digestive enzyme activities was dietary Pb concentration dependent. Different degrees of inhibition of enzyme activities were exhibited in sampled tissues. It was indicated that digestive enzyme activities in the digestive system might be considered as the potential biomarkers of dietary Pb contamination in tilapia.

[Spectroscopic analysis of the interaction of ethanol and acid phosphatase from wheat germ].

PubMed

Xu, Dong-mei; Liu, Guang-shen; Wang, Li-ming; Liu, Wei-ping

2004-11-01

Conformational and activity changes of acid phosphatase from wheat germ in ethanol solutions of different concentrations were measured by fluorescence spectra and differential UV-absorption spectra. The effect of ethanol on kinetics of acid phosphatase was determined by using the double reciprocal plot. The results indicate the ethanol has a significant effect on the activity and conformation of acid phosphatase. The activity of acid phosphatase decreased linearly with increasing the concentration of ethanol. Differential UV-absorption spectra of the enzyme denatured in ethanol solutions showed two positive peaks at 213 and 234 nm, respectively. The peaks on the differential UV-absorption spectra suggested that the conformation of enzyme molecule changed from orderly structure to out-of-order crispation. The fluorescence emission peak intensity of the enzyme gradually strengthened with increasing ethanol concentration, which is in concordance with the conformational change of the microenvironments of tyrosine and tryptophan residues. The results indicate that the expression of the enzyme activity correlates with the stability and integrity of the enzyme conformation to a great degree. Ethanol is uncompetitive inhibitor of acid phosphatase.

Beneficial therapeutic effects of Nigella sativa and/or Zingiber officinale in HCV patients in Egypt

PubMed Central

Abdel-Moneim, Adel; Morsy, Basant M.; Mahmoud, Ayman M.; Abo-Seif, Mohamed A.; Zanaty, Mohamed I.

2013-01-01

Hepatitis C is a major global health burden and Egypt has the highest prevalence of hepatitis C virus (HCV) worldwide. The current study was designed to evaluate the beneficial therapeutic effects of ethanolic extracts of Nigella sativa, Zingiber officinale and their mixture in Egyptian HCV patients. Sixty volunteer patients with proven HCV and fifteen age matched healthy subjects were included in this study. Exclusion criteria included patients on interferon alpha (IFN-α) therapy, infection with hepatitis B virus, drug-induced liver diseases, advanced cirrhosis, hepatocellular carcinoma (HCC) or other malignancies, blood picture abnormalities and major severe illness. Liver function enzymes, albumin, total bilirubin, prothrombin time and concentration, international normalized ratio, alpha fetoprotein and viral load were all assessed at baseline and at the end of the study. Ethanolic extracts of Nigella sativa and Zingiber officinale were prepared and formulated into gelatinous capsules, each containing 500 mg of Nigella sativa and/or Zingiber officinale. Clinical response and incidence of adverse drug reactions were assessed initially, periodically, and at the end of the study. Both extracts as well as their mixture significantly ameliorated the altered viral load, alpha fetoprotein, liver function parameters; with more potent effect for the combined therapy. In conclusion, administration of Nigella sativa and/or Zingiber officinale ethanolic extracts to HCV patients exhibited potential therapeutic benefits via decreasing viral load and alleviating the altered liver function, with more potent effect offered by the mixture. PMID:27298610

Beneficial therapeutic effects of Nigella sativa and/or Zingiber officinale in HCV patients in Egypt.

PubMed

Abdel-Moneim, Adel; Morsy, Basant M; Mahmoud, Ayman M; Abo-Seif, Mohamed A; Zanaty, Mohamed I

2013-01-01

Hepatitis C is a major global health burden and Egypt has the highest prevalence of hepatitis C virus (HCV) worldwide. The current study was designed to evaluate the beneficial therapeutic effects of ethanolic extracts of Nigella sativa, Zingiber officinale and their mixture in Egyptian HCV patients. Sixty volunteer patients with proven HCV and fifteen age matched healthy subjects were included in this study. Exclusion criteria included patients on interferon alpha (IFN-α) therapy, infection with hepatitis B virus, drug-induced liver diseases, advanced cirrhosis, hepatocellular carcinoma (HCC) or other malignancies, blood picture abnormalities and major severe illness. Liver function enzymes, albumin, total bilirubin, prothrombin time and concentration, international normalized ratio, alpha fetoprotein and viral load were all assessed at baseline and at the end of the study. Ethanolic extracts of Nigella sativa and Zingiber officinale were prepared and formulated into gelatinous capsules, each containing 500 mg of Nigella sativa and/or Zingiber officinale. Clinical response and incidence of adverse drug reactions were assessed initially, periodically, and at the end of the study. Both extracts as well as their mixture significantly ameliorated the altered viral load, alpha fetoprotein, liver function parameters; with more potent effect for the combined therapy. In conclusion, administration of Nigella sativa and/or Zingiber officinale ethanolic extracts to HCV patients exhibited potential therapeutic benefits via decreasing viral load and alleviating the altered liver function, with more potent effect offered by the mixture.

Biosensor reveals multiple sources for mitochondrial NAD⁺.

PubMed

Cambronne, Xiaolu A; Stewart, Melissa L; Kim, DongHo; Jones-Brunette, Amber M; Morgan, Rory K; Farrens, David L; Cohen, Michael S; Goodman, Richard H

2016-06-17

Nicotinamide adenine dinucleotide (NAD(+)) is an essential substrate for sirtuins and poly(adenosine diphosphate-ribose) polymerases (PARPs), which are NAD(+)-consuming enzymes localized in the nucleus, cytosol, and mitochondria. Fluctuations in NAD(+) concentrations within these subcellular compartments are thought to regulate the activity of NAD(+)-consuming enzymes; however, the challenge in measuring compartmentalized NAD(+) in cells has precluded direct evidence for this type of regulation. We describe the development of a genetically encoded fluorescent biosensor for directly monitoring free NAD(+) concentrations in subcellular compartments. We found that the concentrations of free NAD(+) in the nucleus, cytoplasm, and mitochondria approximate the Michaelis constants for sirtuins and PARPs in their respective compartments. Systematic depletion of enzymes that catalyze the final step of NAD(+) biosynthesis revealed cell-specific mechanisms for maintaining mitochondrial NAD(+) concentrations. Copyright © 2016, American Association for the Advancement of Science.

Effects of oregano essential oil with or without feed enzymes on growth performance, digestive enzyme, nutrient digestibility, lipid metabolism and immune response of broilers fed on wheat-soybean meal diets.

PubMed

Basmacioğlu Malayoğlu, H; Baysal, S; Misirlioğlu, Z; Polat, M; Yilmaz, H; Turan, N

2010-02-01

1. The study was conducted to determine the effects of dietary supplementation of enzyme and oregano essential oil at two levels, alone or together, on performance, digestive enzyme, nutrient digestibility, lipid metabolism and immune response of broilers fed on wheat-soybean meal based diets. 2. The following dietary treatments were used from d 0 to 21. Diet 1 (control, CONT): a commercial diet containing no enzyme or oregano essential oil, diet 2 (ENZY): supplemented with enzyme, diet 3 (EO250): supplemented with essential oil at 250 mg/kg feed, diet 4 (EO500): supplemented with essential oil at 500 mg/kg feed, diet 5 (ENZY + EO250): supplemented with enzyme and essential oil at 250 mg/kg, and diet 6 (ENZY + EO500): supplemented with enzyme and essential oil at 500 mg/kg. 3. Birds fed on diets containing ENZY, EO250 and ENZY + EO250 had significantly higher weight gain than those given CONT diet from d 0 to 7. No significant effects on feed intake, feed conversion ratio, mortality, organ weights except for jejunum weight and intestinal lengths was found with either enzyme or essential oil, alone or in combination, over the 21-d growth period. The supplementation of essential oil together with enzyme decreased jejunum weight compared with essential oil alone. 4. Supplementation with enzyme significantly decreased viscosity and increased dry matter of digesta, but did not alter pH of digesta. There was no effect of essential oil alone at either concentration on viscosity, dry matter or pH of digesta. A significant decrease in viscosity of digesta appeared when essential oil was used with together enzyme. 5. The supplementation of essential oil at both levels with or without enzyme significantly increased chymotrypsin activity in the digestive system, and improved crude protein digestibility. 6. The higher concentration of essential oil with and without enzyme significantly increased serum total cholesterol concentrations. No significant effect on immune response was found with either enzyme or essential oil, alone or together. 7. Enzymes and essential oil had different modes of actions. The supplementation of enzyme with essential oil in diets is likely more effective in view of performance, nutrient digestibility, enzyme activities and immune system.

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.

Methods for Estimating Annual Wastewater Nutrient Loads in the Southeastern United States

USGS Publications Warehouse

McMahon, Gerard; Tervelt, Larinda; Donehoo, William

2007-01-01

This report describes an approach for estimating annual total nitrogen and total phosphorus loads from point-source dischargers in the southeastern United States. Nutrient load estimates for 2002 were used in the calibration and application of a regional nutrient model, referred to as the SPARROW (SPAtially Referenced Regression On Watershed attributes) watershed model. Loads from dischargers permitted under the National Pollutant Discharge Elimination System were calculated using data from the U.S. Environmental Protection Agency Permit Compliance System database and individual state databases. Site information from both state and U.S. Environmental Protection Agency databases, including latitude and longitude and monitored effluent data, was compiled into a project database. For sites with a complete effluent-monitoring record, effluent-flow and nutrient-concentration data were used to develop estimates of annual point-source nitrogen and phosphorus loads. When flow data were available but nutrient-concentration data were missing or incomplete, typical pollutant-concentration values of total nitrogen and total phosphorus were used to estimate load. In developing typical pollutant-concentration values, the major factors assumed to influence wastewater nutrient-concentration variability were the size of the discharger (the amount of flow), the season during which discharge occurred, and the Standard Industrial Classification code of the discharger. One insight gained from this study is that in order to gain access to flow, concentration, and location data, close communication and collaboration are required with the agencies that collect and manage the data. In addition, the accuracy and usefulness of the load estimates depend on the willingness of the states and the U.S. Environmental Protection Agency to provide guidance and review for at least a subset of the load estimates that may be problematic.

[Optimization of trehalose loading in red blood cells before freeze-drying].

PubMed

Zhuang, Yuan; Liu, Jing-Han; Ouyang, Xi-Lin; Chen, Lin-Feng; Che, Ji

2007-04-01

The key points for better protection of trehalose in freeze-drying red blood cells (RBCs) are to resolve non-osmosis of trehalose to red blood cells and to make cytoplasmic trehalose to reach effective concentration. This study was aimed to investigate the regularity of loading RBCs with trehalose, screen out optimal loading condition and evaluate the effect of trehalose on physico-chemical parameters of RBCs during the period of loading. The cytoplasmic trehalose concentration in red blood cells, free hemoglobin and ATP level were determined at different incubation temperatures (4, 22 and 37 degrees C), different trehaolse concentrations (0, 200, 400, 600, 800 and 1000 mmol/L) and different incubation times (2, 4, 6, 8 and 10 hours), the cytoplasmic trehalose, free hemoglobin (FHb), hemoglobin (Hb) and mean corpuscular volume (MCV) in fresh RBCs and RBCs stored for 72 hours at 4 degrees C were compared, when loading condition was ensured. The results showed that with increase of incubation temperature, time and extracellular trehalose concentration, the loading of trehalose in RBCs also increased. Under the optimal loading condition, cytoplasmic trehalose concentration and free hemoglobin level of fresh RBCs and RBCs stored for 72 hours at 4 degrees C were 65.505 +/- 6.314 mmol/L, 66.2 +/- 5.002 mmol/L and 6.567 +/- 2.568 g/L, 16.168 +/- 3.922 g/L respectively. It is concluded that the most optimal condition of loading trehalose is that fresh RBCs incubate in 800 mmol/L trehalose solution for 8 hours at 37 degrees C. This condition can result in a efficient cytoplasmic trehalose concentration. The study provides an important basis for long-term preservation of RBCs.

Ecotoxicological effects of copper and selenium combined pollution on soil enzyme activities in planted and unplanted soils.

PubMed

Hu, Bin; Liang, Dongli; Liu, Juanjuan; Xie, Junyu

2013-04-01

The present study explored the joint effects of Cu and Se pollution mechanisms on soil enzymes to provide references for the phytoremediation of contaminated areas and agricultural environmental protection. Pot experiments and laboratory analyses were carried out to study the individual and combined influences of Cu and Se on soil enzyme activities. The activities of four soil enzymes (urease, catalase, alkaline phosphatase, and nitrate reductase) were chosen. All soil enzyme activities tested were inhibited by Cu and Se pollution, either individually or combined, in varying degrees, following the order nitrate reductase>urease>catalase>alkaline phosphatase. Growing plants stimulated soil enzyme activity in a similar trend compared with treatments without plants. The joint effects of Cu and Se on catalase activity showed synergism at low concentrations and antagonism at high concentrations, whereas the opposite was observed for urease activity. However, nitrate reductase activity showed synergism both with and without plant treatments. The half maximal effective concentration (EC50) of exchangeable fractions had a similar trend with the EC50 of total content and was lower than that of total content. The EC50 values of nitrate reductase and urease activities were significantly lower for both Se and Cu (p<0.05), which indicated that they were more sensitive than the other two enzymes. Copyright © 2013 SETAC.

Frog brain uridine diphosphate galactose–N-acetylgalactosaminyl-N-acetylneuraminylgalactosylglucosylceramide galactosyltransferase

PubMed Central

Yip, Morris C. M.; Dain, Joel A.

1970-01-01

1. The enzyme that catalyses the transfer of galactose from UDP-galactose to N-acetylgalactosaminyl-(1→4)-N-acetylneuraminyl-(2→3)-galactosyl-(1→4)-glucosylceramide (GM2) was found mainly in the heavy- and light-microsomal fractions of the adult frog brain. 2. The subcellular distribution of the enzyme, UDP-galactose–GM2 galactosyltransferase, parallels that of gangliosides in adult frog brain. 3. The enzymic activity was first detected at late gastrulation (Shumway stage 11½) and increased until the completion of the operculum (Shumway stage 25) and then decreased in the tadpoles. 4. In adult frog brain, the enzyme exhibited a pH optimum of 7.2–7.3 in both cacodylate and tris buffers. The enzyme required 10mm-Mn2+ for maximal activity and the Km for Mn2+ was determined as 2.2mm. The half-maximal velocity was obtained at a GM2 concentration of 0.18mm. Inhibition of the enzymic reaction was found when the GM2 concentration was greater than 1.38mm. 5. The enzymic activity was also inhibited by the products in the pathway of ganglioside synthesis, i.e. either by a mixture of gangliosides or by individual ganglioside components. The most active inhibitor was disialoganglioside. The degree of inhibition is a function of the individual ganglioside concentration. 6. A product-inhibition mechanism for the regulation of ganglioside biosynthesis is discussed. PMID:5484669

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.

Relationships between environmental organochlorine contaminant residues, plasma corticosterone concentrations, and intermediary metabolic enzyme activities in Great Lakes herring gull embryos.

PubMed Central

Lorenzen, A; Moon, T W; Kennedy, S W; Glen, G A

1999-01-01

Experiments were conducted to survey and detect differences in plasma corticosterone concentrations and intermediary metabolic enzyme activities in herring gull (Larus argentatus) embryos environmentally exposed to organochlorine contaminants in ovo. Unincubated fertile herring gull eggs were collected from an Atlantic coast control site and various Great Lakes sites in 1997 and artificially incubated in the laboratory. Liver and/or kidney tissues from approximately half of the late-stage embryos were analyzed for the activities of various intermediary metabolic enzymes known to be regulated, at least in part, by corticosteroids. Basal plasma corticosterone concentrations were determined for the remaining embryos. Yolk sacs were collected from each embryo and a subset was analyzed for organochlorine contaminants. Regression analysis of individual yolk sac organochlorine residue concentrations, or 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalents (TEQs), with individual basal plasma corticosterone concentrations indicated statistically significant inverse relationships for polychlorinated dibenzo-p-dioxins/polychlorinated dibenzofurans (PCDDs/PCDFs), total polychlorinated biphenyls (PCBs), non-ortho PCBs, and TEQs. Similarly, inverse relationships were observed for the activities of two intermediary metabolic enzymes (phosphoenolpyruvate carboxykinase and malic enzyme) when regressed against PCDDs/PCDFs. Overall, these data suggest that current levels of organochlorine contamination may be affecting the hypothalamo-pituitary-adrenal axis and associated intermediary metabolic pathways in environmentally exposed herring gull embryos in the Great Lakes. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:10064546

Assessing the suitability of the OECD 29 guidance document to investigate the transformation and dissolution of silver nanoparticles in aqueous media.

PubMed

Wasmuth, Claus; Rüdel, Heinz; Düring, Rolf-Alexander; Klawonn, Thorsten

2016-02-01

The OECD guidance document No. 29 was designed to determine the rate and extend to which metals can produce soluble available ionic metal species. This transformation/dissolution protocol was applied to silver nanomaterials. The results prove that concentrations of released Ag(+) at pH 8 were nearly similar at all three different loadings. At pH 6, the concentration of Ag(+) was almost the same at loadings of 10 and 100 mg L(-1) AgNPs. However, the study showed changes in concentrations of nanoparticles and aggregates (operationally defined as the fraction passing a 0.2 µm filter). At the higher pH both the concentrations in the test medium of Ag(+) and of AgNPs (fraction < 0.2 µm) decreased. After 7 days of test duration, 71 µg L(-1) of Ag(+) was found in pH 6 medium (initial loading of 100 mg L(-1)). In pH 8 medium a maximum concentration of 29 µg L(-1) Ag(+) was measured (initial loading of 10 mg L(-1)). The maximum transformation from AgNPs to Ag(+) was 2.7% (27 µg L(-1)) in pH 8 medium (loading of 1 mg L(-1)) after 7 days. At an initial loading of 100 mg L(-1) AgNPs in medium at pH 8, only 0.03% (30 µg L(-1)) were transformed to Ag(+) after 7 days. At the loading of 1 mg L(-1) AgNPs all silver concentrations remain relatively constant for the duration of the test after 7 until 28 days. The results reveal that only low concentrations of Ag(+) are released from AgNPs under the applied conditions. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Estimating discharge and non-point source nitrate loading to streams from three end-member pathways using high-frequency water quality and streamflow data

NASA Astrophysics Data System (ADS)

Miller, M. P.; Tesoriero, A. J.; Hood, K.; Terziotti, S.; Wolock, D.

2017-12-01

The myriad hydrologic and biogeochemical processes taking place in watersheds occurring across space and time are integrated and reflected in the quantity and quality of water in streams and rivers. Collection of high-frequency water quality data with sensors in surface waters provides new opportunities to disentangle these processes and quantify sources and transport of water and solutes in the coupled groundwater-surface water system. A new approach for separating the streamflow hydrograph into three components was developed and coupled with high-frequency specific conductance and nitrate data to estimate time-variable watershed-scale nitrate loading from three end-member pathways - dilute quickflow, concentrated quickflow, and slowflow groundwater - to two streams in central Wisconsin. Time-variable nitrate loads from the three pathways were estimated for periods of up to two years in a groundwater-dominated and a quickflow-dominated stream, using only streamflow and in-stream water quality data. The dilute and concentrated quickflow end-members were distinguished using high-frequency specific conductance data. Results indicate that dilute quickflow contributed less than 5% of the nitrate load at both sites, whereas 89±5% of the nitrate load at the groundwater-dominated stream was from slowflow groundwater, and 84±13% of the nitrate load at the quickflow-dominated stream was from concentrated quickflow. Concentrated quickflow nitrate concentrations varied seasonally at both sites, with peak concentrations in the winter that were 2-3 times greater than minimum concentrations during the growing season. Application of this approach provides an opportunity to assess stream vulnerability to non-point source nitrate loading and expected stream responses to current or changing conditions and practices in watersheds.

Testing Dose-Dependent Effects of the Nectar Alkaloid Anabasine on Trypanosome Parasite Loads in Adult Bumble Bees.

PubMed

Anthony, Winston E; Palmer-Young, Evan C; Leonard, Anne S; Irwin, Rebecca E; Adler, Lynn S

2015-01-01

The impact of consuming biologically active compounds is often dose-dependent, where small quantities can be medicinal while larger doses are toxic. The consumption of plant secondary compounds can be toxic to herbivores in large doses, but can also improve survival in parasitized herbivores. In addition, recent studies have found that consuming nectar secondary compounds may decrease parasite loads in pollinators. However, the effect of compound dose on bee survival and parasite loads has not been assessed. To determine how secondary compound consumption affects survival and pathogen load in Bombus impatiens, we manipulated the presence of a common gut parasite, Crithidia bombi, and dietary concentration of anabasine, a nectar alkaloid produced by Nicotiana spp. using four concentrations naturally observed in floral nectar. We hypothesized that increased consumption of secondary compounds at concentrations found in nature would decrease survival of uninfected bees, but improve survival and ameliorate parasite loads in infected bees. We found medicinal effects of anabasine in infected bees; the high-anabasine diet decreased parasite loads and increased the probability of clearing the infection entirely. However, survival time was not affected by any level of anabasine concentration, or by interactive effects of anabasine concentration and infection. Crithidia infection reduced survival time by more than two days, but this effect was not significant. Our results support a medicinal role for anabasine at the highest concentration; moreover, we found no evidence for a survival-related cost of anabasine consumption across the concentration range found in nectar. Our results suggest that consuming anabasine at the higher levels of the natural range could reduce or clear pathogen loads without incurring costs for healthy bees.

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

PubMed

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

1989-07-01

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

Optimization of 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.

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

Cytotoxic immune responses in the lungs correlate to disease severity in patients with hantavirus infection.

PubMed

Rasmuson, J; Pourazar, J; Mohamed, N; Lejon, K; Evander, M; Blomberg, A; Ahlm, C

2016-04-01

Hantavirus infections may cause severe and sometime life-threatening lung failure. The pathogenesis is not fully known and there is an urgent need for effective treatment. We aimed to investigate the association between pulmonary viral load and immune responses, and their relation to disease severity. Bronchoscopy with sampling of bronchoalveolar lavage (BAL) fluid was performed in 17 patients with acute Puumala hantavirus infection and 16 healthy volunteers acting as controls. Lymphocyte subsets, granzyme concentrations, and viral load were determined by flow cytometry, enzyme-linked immunosorbent assay (ELISA), and quantitative reverse transcription polymerase chain reaction (RT-PCR), respectively. Analyses of BAL fluid revealed significantly higher numbers of activated CD8(+) T cells and natural killer (NK) cells, as well as higher concentrations of the cytotoxins granzymes A and B in hantavirus-infected patients, compared to controls. In patients, Puumala hantavirus RNA was detected in 88 % of BAL cell samples and correlated inversely to the T cell response. The magnitude of the pulmonary cytotoxic lymphocyte response correlated to the severity of disease and systemic organ dysfunction, in terms of need for supplemental oxygen treatment, hypotension, and laboratory data indicating renal failure, cardiac dysfunction, vascular leakage, and cell damage. Regulatory T cell numbers were significantly lower in patients compared to controls, and may reflect inadequate immune regulation during hantavirus infection. Hantavirus infection elicits a pronounced cytotoxic lymphocyte response in the lungs. The magnitude of the immune response was associated with disease severity. These results give insights into the pathogenesis and possibilities for new treatments.

Vocal exercise may attenuate acute vocal fold inflammation

PubMed Central

Abbott, Katherine Verdolini; Li, Nicole Y.K.; Branski, Ryan C.; Rosen, Clark A.; Grillo, Elizabeth; Steinhauer, Kimberly; Hebda, Patricia A.

2012-01-01

Objectives/Hypotheses The objective was to assess the utility of selected “resonant voice” exercises for the reduction of acute vocal fold inflammation. The hypothesis was that relatively large-amplitude, low-impact exercises associated with resonant voice would reduce inflammation more than spontaneous speech and possibly more than voice rest. Study Design The study design was prospective, randomized, double-blind. Methods Nine vocally healthy adults underwent a 1-hr vocal loading procedure, followed by randomization to (a) a spontaneous speech condition, (b) a vocal rest condition, or (c) a resonant voice exercise condition. Treatments were monitored in clinic for 4 hr, and continued extra-clinically until the next morning. At baseline, immediately following loading, after the 4-hr in-clinic treatment, and 24 hr post baseline, secretions were suctioned from the vocal folds bilaterally and submitted to enzyme-linked immunosorbent assay (ELISA) to estimate concentrations of key markers of tissue injury and inflammation: IL-1β, IL-6, IL-8, TNF-α, MMP-8, and IL-10. Results Complete data sets were obtained for 3 markers -- IL-1β, IL-6, and MMP-8 -- for one subject in each treatment condition. For those markers, results were poorest at 24-hr follow-up in the spontaneous speech condition, sharply improved in the voice rest condition, and best in the resonant voice condition. Average results for all markers, for all responsive subjects with normal baseline mediator concentrations, revealed an almost identical pattern. Conclusions Some forms of tissue mobilization may be useful to attenuate acute vocal fold inflammation. PMID:23177745

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

Micromotors Powered by Enzyme Catalysis.

PubMed

Dey, Krishna K; Zhao, Xi; Tansi, Benjamin M; Méndez-Ortiz, Wilfredo J; Córdova-Figueroa, Ubaldo M; Golestanian, Ramin; Sen, Ayusman

2015-12-09

Active biocompatible systems are of great current interest for their possible applications in drug or antidote delivery at specific locations. Herein, we report the synthesis and study of self-propelled microparticles powered by enzymatic reactions and their directed movement in substrate concentration gradient. Polystyrene microparticles were functionalized with the enzymes urease and catalase using a biotin-streptavidin linkage procedure. The motion of the enzyme-coated particles was studied in the presence of the respective substrates, using optical microscopy and dynamic light scattering analysis. The diffusion of the particles was found to increase in a substrate concentration dependent manner. The directed chemotactic movement of these enzyme-powered motors up the substrate gradient was studied using three-inlet microfluidic channel architecture.

Effects of Mg2+ and adenine nucleotides on thymidylate synthetase from different mouse tumors.

PubMed

Rode, W; Jastreboff, M M

1984-01-01

Magnesium ions variably influenced activity of highly purified thymidylate synthetase preparations from different mouse tumors, activating the enzyme from Ehrlich ascites carcinoma (EAC) cells and inhibiting the enzyme from L1210 and L5178Y cells and from 5-fluorodeoxyuridine (FdUrd)-resistant EAC cells. In the presence of Mg2+ in a concentration resulting in either maximum activation or inhibition (25-30 mM) the enzymes from both the sensitive and FdUrd-resistant EAC lines and L5178Y cells were activated by ATP. Under the same conditions of Mg2+ concentration ADP and AMP inhibited the enzyme from the parental but not from the FdUrd-resistant EAC cells.

Hydrogen-Induced Plastic Deformation in ZnO

NASA Astrophysics Data System (ADS)

Lukáč, F.; Čížek, J.; Vlček, M.; Procházka, I.; Anwand, W.; Brauer, G.; Traeger, F.; Rogalla, D.; Becker, H.-W.

In the present work hydrothermally grown ZnO single crystals covered with Pd over-layer were electrochemically loaded with hydrogen and the influence of hydrogen on ZnO micro structure was investigated by positron annihilation spectroscopy (PAS). Nuclear reaction analysis (NRA) was employed for determination of depth profile of hydrogen concentration in the sample. NRA measurements confirmed that a substantial amount of hydrogen was introduced into ZnO by electrochemical charging. The bulk hydrogen concentration in ZnO determined by NRA agrees well with the concentration estimated from the transported charge using the Faraday's law. Moreover, a subsurface region with enhanced hydrogen concentration was found in the loaded crystals. Slow positron implantation spectroscopy (SPIS) investigations of hydrogen-loaded crystal revealed enhanced concentration of defects in the subsurface region. This testifies hydrogen-induced plastic deformation of the loaded crystal. Absorbed hydrogen causes a significant lattice expansion. At low hydrogen concentrations this expansion is accommodated by elastic straining, but at higher concentrations hydrogen-induced stress exceeds the yield stress in ZnO and plastic deformation of the loaded crystal takes place. Enhanced hydrogen concentration detected in the subsurface region by NRA is, therefore, due to excess hydrogen trapped at open volume defects introduced by plastic deformation. Moreover, it was found that hydrogen-induced plastic deformation in the subsurface layer leads to typical surface modification: formation of hexagonal shape pyramids on the surface due to hydrogen-induced slip in the [0001] direction.

Interactions between the concentration of non-starch polysaccharides in wheat and the addition of an enzyme mixture in a broiler digestibility and performance trial.

PubMed

Smeets, N; Nuyens, F; Van Campenhout, L; Delezie, E; Niewold, T A

2018-06-01

Two broiler trials were designed to investigate the relationship between the concentration of non-starch polysaccharides (NSP) in wheat and 1) its nutritional value for broilers and 2) the efficacy of exogenous enzymes. In a balance trial, diets were formulated with 3 wheat cultivars (Rustic and Viscount-medium NSP, Centenaire-high NSP) and were tested with or without the addition of an exogenous enzyme mixture. The diets were fed to 144 male Ross 308 broiler chickens housed in digestibility cages. Total tract nutrient digestibilities and AMEn were measured from 18 to 22 d of age. In a performance trial, diets were formulated with wheat (medium NSP diet) or with wheat mixed with rye and barley (high NSP diet) and were tested with or without the addition of an exogenous enzyme mixture. The diets were fed to 960 male Ross 308 broilers housed in pens and broiler performance during starter, grower and finisher periods was measured.In the balance trial, wheat cultivar did not affect nutrient digestibility or AMEn. Enzyme addition caused a significant increase in nutrient digestibilities and AMEn for the diet formulated with the high NSP wheat Centenaire only. In the performance trial, feeding the high NSP diet resulted in a higher feed conversion ratio and lower final body weight compared to the medium NSP diet. The largest improvements by enzyme addition were observed in the high NSP diet.In conclusion, the study was not able to show a consistent relationship between the NSP concentration of wheat and its nutritional value, but did demonstrate that the effect of an enzyme mixture on nutrient digestibility or broiler performance depends upon the NSP concentration in the diet.

Interactions between the concentration of non-starch polysaccharides in wheat and the addition of an enzyme mixture in a broiler digestibility and performance trial

PubMed Central

Smeets, N; Nuyens, F; Van Campenhout, L; Delezie, E; Niewold, T A

2018-01-01

ABSTRACT Two broiler trials were designed to investigate the relationship between the concentration of non-starch polysaccharides (NSP) in wheat and 1) its nutritional value for broilers and 2) the efficacy of exogenous enzymes. In a balance trial, diets were formulated with 3 wheat cultivars (Rustic and Viscount—medium NSP, Centenaire—high NSP) and were tested with or without the addition of an exogenous enzyme mixture. The diets were fed to 144 male Ross 308 broiler chickens housed in digestibility cages. Total tract nutrient digestibilities and AMEn were measured from 18 to 22 d of age. In a performance trial, diets were formulated with wheat (medium NSP diet) or with wheat mixed with rye and barley (high NSP diet) and were tested with or without the addition of an exogenous enzyme mixture. The diets were fed to 960 male Ross 308 broilers housed in pens and broiler performance during starter, grower and finisher periods was measured. In the balance trial, wheat cultivar did not affect nutrient digestibility or AMEn. Enzyme addition caused a significant increase in nutrient digestibilities and AMEn for the diet formulated with the high NSP wheat Centenaire only. In the performance trial, feeding the high NSP diet resulted in a higher feed conversion ratio and lower final body weight compared to the medium NSP diet. The largest improvements by enzyme addition were observed in the high NSP diet. In conclusion, the study was not able to show a consistent relationship between the NSP concentration of wheat and its nutritional value, but did demonstrate that the effect of an enzyme mixture on nutrient digestibility or broiler performance depends upon the NSP concentration in the diet. PMID:29471412

Sensitivity of small myosin II ensembles from different isoforms to mechanical load and ATP concentration.

PubMed

Erdmann, Thorsten; Bartelheimer, Kathrin; Schwarz, Ulrich S

2016-11-01

Based on a detailed crossbridge model for individual myosin II motors, we systematically study the influence of mechanical load and adenosine triphosphate (ATP) concentration on small myosin II ensembles made from different isoforms. For skeletal and smooth muscle myosin II, which are often used in actomyosin gels that reconstitute cell contractility, fast forward movement is restricted to a small region of phase space with low mechanical load and high ATP concentration, which is also characterized by frequent ensemble detachment. At high load, these ensembles are stalled or move backwards, but forward motion can be restored by decreasing ATP concentration. In contrast, small ensembles of nonmuscle myosin II isoforms, which are found in the cytoskeleton of nonmuscle cells, are hardly affected by ATP concentration due to the slow kinetics of the bound states. For all isoforms, the thermodynamic efficiency of ensemble movement increases with decreasing ATP concentration, but this effect is weaker for the nonmuscle myosin II isoforms.

Sediment concentrations and loads in the Loxahatchee River estuary, Florida, 1980-82

USGS Publications Warehouse

Sonntag, Wayne H.; McPherson, Benjamin F.

1984-01-01

This study was conducted to estimate the magnitude of sediment loads and the general spatial and temporal patterns of sediment transport in the Loxahatchee River estuary, Florida. Mean concentrations of suspended sediment generally were higher in the Jupiter Inlet area than in the remainder of the embayment area. Concentrations of suspended sediment varied with season and weather conditions. Concentrations in selected tributaries following Tropical Storm Dennis in August 1981 immediately increased as much as 16 times over concentrations before the storm. Suspended-sediment loads from the tributaries were also highly seasonal and storm related. During a 61-day period of above-average rainfall that included Tropical Storm Dennis, 5 major tributaries discharged 926 tons (short) of suspended sediment to the estuary, accounting for 74 percent of the input for the 1981 water year and 49 percent of the input for the 20-month study period. Suspended-sediment loads at Jupiter Inlet and at the mouth of the estuary embayment on both incoming and outgoing tides far exceeded tributary loads, but the direction of long-term, net tidal transport was not determined. (USGS)

Gas hold-up and oxygen mass transfer in three pneumatic bioreactors operating with sugarcane bagasse suspensions.

PubMed

Esperança, M N; Cunha, F M; Cerri, M O; Zangirolami, T C; Farinas, C S; Badino, A C

2014-05-01

Sugarcane bagasse is a low-cost and abundant by-product generated by the bioethanol industry, and is a potential substrate for cellulolytic enzyme production. The aim of this work was to evaluate the effects of air flow rate (QAIR), solids loading (%S), sugarcane bagasse type, and particle size on the gas hold-up (εG) and volumetric oxygen transfer coefficient (kLa) in three different pneumatic bioreactors, using response surface methodology. Concentric tube airlift (CTA), split-cylinder airlift (SCA), and bubble column (BC) bioreactor types were tested. QAIR and %S affected oxygen mass transfer positively and negatively, respectively, while sugarcane bagasse type and particle size (within the range studied) did not influence kLa. Using large particles of untreated sugarcane bagasse, the loop-type bioreactors (CTA and SCA) exhibited higher mass transfer, compared to the BC reactor. At higher %S, SCA presented a higher kLa value (0.0448 s−1) than CTA, and the best operational conditions in terms of oxygen mass transfer were achieved for %S < 10.0 g L−1 and QAIR > 27.0 L min−1. These results demonstrated that pneumatic bioreactors can provide elevated oxygen transfer in the presence of vegetal biomass, making them an excellent option for use in three-phase systems for cellulolytic enzyme production by filamentous fungi.

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.

Block copolymer micelles for controlled delivery of glycolytic enzyme inhibitors.

PubMed

Akter, Shanjida; Clem, Brian F; Lee, Hyun Jin; Chesney, Jason; Bae, Younsoo

2012-03-01

To develop block copolymer micelles as an aqueous dosage form for a potent glycolytic enzyme inhibitor, 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO). The micelles were prepared from poly(ethylene glycol)-poly(aspartate hydrazide) [PEG-p(HYD)] block copolymers to which 3PO was conjugated through an acid-labile hydrazone bond. The optimal micelle formulation was determined following the screening of block copolymer library modified with various aromatic and aliphatic pendant groups. Both physical drug entrapment and chemical drug conjugation methods were tested to maximize 3PO loading in the micelles during the screening. Particulate characterization showed that the PEG-p(HYD) block copolymers conjugated with 3PO (2.08∼2.21 wt.%) appeared the optimal polymer micelles. Block copolymer compositions greatly affected the micelle size, which was 38 nm and 259 nm when 5 kDa and 12 kDa PEG chains were used, respectively. 3PO release from the micelles was accelerated at pH 5.0, potentiating effective drug release in acidic tumor environments. The micelles retained biological activity of 3PO, inhibiting various cancer cells (Jurkat, He-La and LLC) in concentration ranges similar to free 3PO. A novel micelle formulation for controlled delivery of 3PO was successfully prepared.

Metabolomics reveals metabolic changes in male reproductive cells exposed to thirdhand smoke

NASA Astrophysics Data System (ADS)

Xu, Bo; Chen, Minjian; Yao, Mengmeng; Ji, Xiaoli; Mao, Zhilei; Tang, Wei; Qiao, Shanlei; Schick, Suzaynn F.; Mao, Jian-Hua; Hang, Bo; Xia, Yankai

2015-10-01

Thirdhand smoke (THS) is a new term for the toxins in cigarette smoke that linger in the environment long after the cigarettes are extinguished. The effects of THS exposure on male reproduction have not yet been studied. In this study, metabolic changes in male germ cell lines (GC-2 and TM-4) were analyzed after THS treatment for 24 h. THS-loaded chromatography paper samples were generated in a laboratory chamber system and extracted in DMEM. At a paper: DMEM ratio of 50 μg/ml, cell viability in both cell lines was normal, as measured by the MTT assay and markers of cytotoxicity, cell cycle, apoptosis and ROS production were normal as measured by quantitative immunofluorescence. Metabolomic analysis was performed on methanol extracts of GC-2 and TM-4 cells. Glutathione metabolism in GC-2 cells, and nucleic acid and ammonia metabolism in TM-4 cells, was changed significantly by THS treatment. RT-PCR analyses of mRNA for enzyme genes Gss and Ggt in GC-2 cells, and TK, SMS and Glna in TM-4 cells reinforced these findings, showing changes in the levels of enzymes involved in the relevant pathways. In conclusion, exposure to THS at very low concentrations caused distinct metabolic changes in two different types of male reproductive cell lines.

A novel amperometric biosensor based on gold nanoparticles anchored on reduced graphene oxide for sensitive detection of l-lactate tumor biomarker.

PubMed

Azzouzi, Sawsen; Rotariu, Lucian; Benito, Ana M; Maser, Wolfgang K; Ben Ali, Mounir; Bala, Camelia

2015-07-15

In this work, a novel amperometric biosensor based on gold nanoparticles anchored on reduced graphene oxide (RGO-AuNPs) and l-lactate dehydrogenase (LDH) was developed for the sensing of l-lactate. Firstly, the RGO-AuNPs modified screen printed electrodes were tested for NADH detection showing a wide dynamic range and a low detection limit. Next, the biosensor was constructed by incorporating both enzyme and RGO-AuNPs in a sol gel matrix derived from tetrametoxysilane and methyltrimetoxysilane. The enzyme loading, working pH, and coenzyme concentration were optimized. The biosensor linearly responded to l-lactate in the range of 10µM-5mM and showed a good specific sensitivity of 154µA/mMcm(2) with a detection limit of 0.13µM. This was accompanied by good reproducibility and operational stability. Tests on artificial serum proved that l-lactate can be determined practically without interferences from commonly interfering compounds such as urate, paracetamol and l-ascorbate. Our LDH/RGO-AuNPs/SPCE based biosensor thus performs as electrochemical device for the detection of l-lactate as a viable early cancer bio-marker. Copyright © 2015 Elsevier B.V. All rights reserved.

Solution Behavior and Activity of a Halophilic Esterase under High Salt Concentration

PubMed Central

Rao, Lang; Zhao, Xiubo; Pan, Fang; Li, Yin; Xue, Yanfen; Ma, Yanhe; Lu, Jian R.

2009-01-01

Background Halophiles are extremophiles that thrive in environments with very high concentrations of salt. Although the salt reliance and physiology of these extremophiles have been widely investigated, the molecular working mechanisms of their enzymes under salty conditions have been little explored. Methodology/Principal Findings A halophilic esterolytic enzyme LipC derived from archeaon Haloarcula marismortui was overexpressed from Escherichia coli BL21. The purified enzyme showed a range of hydrolytic activity towards the substrates of p-nitrophenyl esters with different alkyl chains (n = 2−16), with the highest activity being observed for p-nitrophenyl acetate, consistent with the basic character of an esterase. The optimal esterase activities were found to be at pH 9.5 and [NaCl] = 3.4 M or [KCl] = 3.0 M and at around 45°C. Interestingly, the hydrolysis activity showed a clear reversibility against changes in salt concentration. At the ambient temperature of 22°C, enzyme systems working under the optimal salt concentrations were very stable against time. Increase in temperature increased the activity but reduced its stability. Circular dichroism (CD), dynamic light scattering (DLS) and small angle neutron scattering (SANS) were deployed to determine the physical states of LipC in solution. As the salt concentration increased, DLS revealed substantial increase in aggregate sizes, but CD measurements revealed the maximal retention of the α-helical structure at the salt concentration matching the optimal activity. These observations were supported by SANS analysis that revealed the highest proportion of unimers and dimers around the optimal salt concentration, although the coexistent larger aggregates showed a trend of increasing size with salt concentration, consistent with the DLS data. Conclusions/Significance The solution α-helical structure and activity relation also matched the highest proportion of enzyme unimers and dimers. Given that all the solutions studied were structurally inhomogeneous, it is important for future work to understand how the LipC's solution aggregation affected its activity. PMID:19759821

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.

The mechanism of phloem loading in rice (Oryza sativa).

PubMed

Eom, Joon-Seob; Choi, Sang-Bong; Ward, John M; Jeon, Jong-Seong

2012-05-01

Carbohydrates, mainly sucrose, that are synthesized in source organs are transported to sink organs to support growth and development. Phloem loading of sucrose is a crucial step that drives long-distance transport by elevating hydrostatic pressure in the phloem. Three phloem loading strategies have been identified, two active mechanisms, apoplastic loading via sucrose transporters and symplastic polymer trapping, and one passive mechanism. The first two active loading mechanisms require metabolic energy, carbohydrate is loaded into the phloem against a concentration gradient. The passive process, diffusion, involves equilibration of sucrose and other metabolites between cells through plasmodesmata. Many higher plant species including Arabidopsis utilize the active loading mechanisms to increase carbohydrate in the phloem to higher concentrations than that in mesophyll cells. In contrast, recent data revealed that a large number of plants, especially woody species, load sucrose passively by maintaining a high concentration in mesophyll cells. However, it still remains to be determined how the worldwide important cereal crop, rice, loads sucrose into the phloem in source organs. Based on the literature and our results, we propose a potential strategy of phloem loading in rice. Elucidation of the phloem loading mechanism should improve our understanding of rice development and facilitate its manipulation towards the increase of crop productivity.

Activation of a chloroplast type of fructose bisphosphatase from Chlamydomonas reinhardtii by light-mediated agents

NASA Technical Reports Server (NTRS)

Huppe, H. C.; Buchanan, B. B.

1989-01-01

A chloroplast type of fructose-1,6-bisphosphatase, a central regulatory enzyme of photosynthetic carbon metabolism, has been partially purified from Chlamydomonas reinhardtii. Unlike its counterpart from spinach chloroplasts, the algal FBPase showed a strict requirement for a dithiol reductant irrespective of Mg2+ concentration. The enzymes from the two sources resembled each other immunologically, in subunit molecular mass and response to pH. In the presence of dithiothreitol, the pH optimum for both the algal and spinach enzymes shifted from 8.5 to a more physiologic value of 8.0 as the Mg2+ concentration was increased from 1 to 16 mM. At 1 mM Mg2+, a concentration estimated to be close to physiological, the Chlamydomonas FBPase was active only in the presence of reduced thioredoxin and was most active with Chlamydomonas thioredoxin f. Under these conditions, the enzyme showed a pH optimum of 8.0. The data suggest that the Chlamydomonas enzyme resembles its spinach counterpart in most respects, but it has a stricter requirement for reduction and less strict reductant specificity. A comparison of the properties of the FBPases from Chlamydomonas and spinach will be helpful for elucidating the mechanism of the reductive activation of this enzyme.

Enhancement of Biogas Production from Rice Husk by NaOH and Enzyme Pretreatment

NASA Astrophysics Data System (ADS)

Syafrudin; Nugraha, Winardi Dwi; Agnesia, Shandy Sarima; Matin, Hashfi Hawali Abdul; Budiyono

2018-02-01

Biogas is a renewable energy source that can be used as an alternative fuel to replace fossil fuel such as oil and natural gas. This research aims to analyze the impact of NaOH (Sodium hydroxide) and enzyme usage on the production of rice husk biogas using Solid State Anaerobic Digestion (SS-AD). Generally, SS-AD occurs at solid concentrations higher than 15%. The waste of rice husk are used as substrate with a C/N ratio of 25% and the total of solid that are used is 21%. Rice husk contains high lignin, therefore it is handled with chemical and biological treatment. The chemical preliminary treatment was using NaOH with various concentrations from 3%, 6% and 9% while the biological preliminary treatment was using enzyme with various concentration from 5%, 8%, and 11%. The biogas that is produced then measured every two days during 60 days of research with the biogas volume as a parameter observed. The result of the research shows that preliminary treatment with NaOH and enzyme can increase the production of biogas. The highest biogas production was obtained by the NaOH pretreatment using 6% NaOH which was 497 ml and by enzyme pretreatment using 11% enzyme which was 667,5 ml.

Purification and characterization of chromatin-bound DNA-dependent RNA polymerase I from parsley (Petroselinum crispum). Influence of nucleoside triphosphates.

PubMed Central

Grossmann, K; Friedrich, H; Seitz, U

1980-01-01

The isolation and purification of DNA-dependent RNA polymerase I (EC 2.7.7.6) from parsley (Petroselinum crispum) callus cells grown in suspension culture is described. The enzyme was solubilized from isolated chromatin. Purification was achieved by using DEAE- and phospho-cellulose in batches, followed by column chromatography on DEAE- and phospho-cellulose (two columns) and density-gradient centrifugation. The highly purified enzyme was stable over several months. The properties of purified parsley RNA polymerase I were investigated. Optimum concentration for Mn2+ was 1 mM, and for Mg2+ 4-6 mM, Mn2+ was slightly more stimulatory than Mg2+. The enzyme was most active at low ionic strengths [10-20 mM-(NH4)SO4]. The influence of various phosphates was tested: pyrophosphate inhibited RNA polymerase at low concentrations, whereas orthophosphate had no effect on the enzyme activity. ADP was slightly inhibitory, and AMP had no effect on the enzyme reaction. Nucleoside triphosphates and bivalent cations in equimolar concentrations in the range 4-11 mM did not influence the RNA synthesis in vitro. Free nucleoside triphosphates in excess of this 1:1 ratio inhibited the enzyme activity, unlike free bivalent cations, which stimulated RNA polymerase I. PMID:7470092

Enzyme-responsive nanocomposites for wound infection prophylaxis in burn management: in vitro evaluation of their compatibility with healing processes

PubMed Central

Grützner, Verena; Unger, Ronald E; Baier, Grit; Choritz, Lars; Freese, Christian; Böse, Thomas; Landfester, Katharina; Kirkpatrick, C James

2015-01-01

Responsive, theranostic nanosystems, capable of both signaling and treating wound infections, is a sophisticated approach to reduce the most common and potentially traumatizing side effects of burn wound treatment: slowed wound healing due to prophylactic anti-infective drug exposure as well as frequent painful dressing changes. Antimicrobials as well as dye molecules have been incorporated into biodegradable nanosystems that release their content only in the presence of pathogens. Following nanocarrier degradation by bacterial enzymes, any infection will thus emit a visible signal and be effectively treated at its source. In this study, we investigated the effect of fluorescent-labeled hyaluronan nanocapsules containing polyhexanide biguanide and poly-L-lactic acid nanoparticles loaded with octenidine on primary human dermal microvascular endothelial cells, which play a major role in cutaneous wound healing. Microscopic and flow cytometric analysis indicated a time-dependent uptake of both the nanocapsules and the nanoparticles. However, enzyme immunoassays showed no significant influence on the expression of pro-inflammatory cell adhesion molecules and cytokines by the endothelial cells. Under angiogenic-stimulating conditions, the potential to form capillary-like structures in co-culture with dermal fibroblasts was not inhibited. Furthermore, cytotoxicity studies (the MTS and crystal violet assay) after short- and long-term exposure to the materials demonstrated that both systems exhibited less toxicity than solutions of the antiseptic agents alone in comparable concentrations. The results indicate that responsive antimicrobial nanocomposites could be used as an advanced drug delivery system and a promising addition to current best practice wound infection prophylaxis with few side effects. PMID:26150717

Polyluminol/hydrogel composites as new electrochemiluminescent-active sensing layers.

PubMed

Leca-Bouvier, Béatrice D; Sassolas, Audrey; Blum, Loïc J

2014-09-01

This paper reports on electrochemiluminescent sensors and biosensors based on polyluminol/hydrogel composite sensing layers using chemical or biological membranes as hydrogel matrices. In this work, luminol is electropolymerized under near-neutral conditions onto screen-printed electrode (SPE)-supported hydrogel films. The working electrode coated with a hydrogel film is soaked in a solution containing monomeric luminol units, allowing the monomeric luminol units to diffuse inside the porous matrix to the electrode surface where they are electropolymerized by cyclic voltammetry (CV). Sensors and enzymatic biosensors for H2O2 and choline detection, respectively, have been developed, using choline oxidase (ChOD) as a model enzyme. In this case, hydrogel is used both as the enzymatic immobilization matrix and as a template for the electrosynthesis of polyluminol. The enzyme was immobilized by entrapment in the gel matrix during its formation before electropolymerization of the monomer. Several parameters have been optimized in terms of polymerization conditions, enzyme loading, and average pore size. Using calcium alginate or tetramethoxysilane (TMOS)-based silica as porous matrix, H2O2 and choline detection are reported down to micromolar concentrations with three orders of magnitude wide dynamic ranges starting from 4 × 10(-7) M. Polyluminol/hydrogel composites appear as suitable electrochemiluminescence (ECL)-active sensing layers for the design of new reagentless and disposable easy-to-use optical sensors and biosensors, using conventional TMOS-based silica gel or the more original and easier to handle calcium alginate, reported here for the first time in such a configuration, as the biocompatible hydrogel matrix.

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.

Effect of solubilizing agents on mupirocin loading into and release from PEGylated nanoliposomes.

PubMed

Cern, Ahuva; Nativ-Roth, Einat; Goldblum, Amiram; Barenholz, Yechezkel

2014-07-01

Mupirocin was identified by quantitative structure property relationship models as a good candidate for remote liposomal loading. Mupirocin is an antibiotic that is currently restricted to topical administration because of rapid hydrolysis in vivo to its inactive metabolite. Formulating mupirocin in PEGylated nanoliposomes may potentially expand its use to parenteral administration by protecting it from degradation in the circulation and target it (by the enhanced permeability effect) to the infected tissue. Mupirocin is slightly soluble in aqueous medium and its solubility can be increased using solubilizing agents. The effect of the solubilizing agents on mupirocin remote loading was studied when the solubilizing agents were added to the drug loading solution. Propylene glycol was found to increase mupirocin loading, whereas polyethylene glycol 400 showed no effect. Hydroxypropyl-β-cyclodextrin (HPCD) showed a concentration-dependent effect on mupirocin loading; using the optimal HPCD concentration increased loading, but higher concentrations inhibited it. The inclusion of HPCD in the liposome aqueous phase while forming the liposomes resulted in increased drug loading and substantially inhibited drug release in serum. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

Release Profile of Andrographis paniculata Leaf Extract Nanocapsule as α-Glucosidase Inhibitors

NASA Astrophysics Data System (ADS)

Zahrani, K.; Imansari, F.; Utami, T. S.; Arbianti, R.

2017-07-01

Andrographis paniculata is one of 13 leading commodities Indonesian medicinal plants through the Ditjen POM. Andrographolide as main active compound has been shown to have many pharmacological activities, one of which is as α-glucosidase enzyme inhibitors which has clinical potential as an antitumor, antiviral, antidiabetic, and immunoregulator agents. This study aims to do nanoencapsulation of Andrographis paniculatar leaf extract to increase its active compound bioavailability and get a release profile through synthetic fluids media simulation. Nanoencapsulation with ionic gelation method result the encapsulation efficiency and loading capacity values of 73.47% and 46.29% at 2%: 1% of chitosan: STPP ratio. The maximum α-glucosidase inhibition of 37.17% was obtained at 16% concentration. Burst release at gastric pH conditions indicate that most of the drug (in this study is an Andrographis paniculata leaf extract) adsorbed on the surface of the nanoparticles an indicates that the kind of nanoparticle formed is nanosphere.

Mesoporous silicas synthesis and application for lignin peroxidase immobilization by covalent binding method.

PubMed

Hu, Zunfang; Xu, Longqian; Wen, Xianghua

2013-01-01

Immobilization of enzymes on mesoporous silicas (MS) allows for good reusability. MS with two-dimensional hexagonal pores in diameter up to 14.13 nm were synthesized using Pluronic P123 as template and 1,3,5-triisopropylbenzene as a swelling agent in acetate buffer. The surface of MS was modified by the silanization reagents 3-aminopropyltriethoxysilane. Lignin peroxidase (LiP) was successfully immobilized on the modified MS through covalent binding method by four agents: glutaraldehyde, 1,4-phenylene diisothiocyanate, cyanotic chloride and water-soluble carbodiimide. Results showed that cyanotic chloride provided the best performance for LIP immobilization. The loaded protein concentration was 12.15 mg/g and the immobilized LiP activity was 812.9 U/L. Immobilized LiP had better pH stability. Acid Orange II was used to examine the reusability of immobilized LiP, showing more than 50% of the dye was decolorized at the fifth cycle.

Encapsulation of basic fibroblast growth factor by polyelectrolyte multilayer microcapsules and its controlled release for enhancing cell proliferation.

PubMed

She, Zhen; Wang, Chunxia; Li, Jun; Sukhorukov, Gleb B; Antipina, Maria N

2012-07-09

Basic fibroblast growth factor (FGF2) is an important protein for cellular activity and highly vulnerable to environmental conditions. FGF2 protected by heparin and bovine serum albumin was loaded into the microcapsules by a coprecipitation-based layer-by-layer encapsulation method. Low cytotoxic and biodegradable polyelectrolytes dextran sulfate and poly-L-arginine were used for capsule shell assembly. The shell thickness-dependent encapsulation efficiency was measured by enzyme-linked immunosorbent assay. A maximum encapsulation efficiency of 42% could be achieved by microcapsules with a shell thickness of 14 layers. The effects of microcapsule concentration and shell thickness on cytotoxicity, FGF2 release kinetics, and L929 cell proliferation were evaluated in vitro. The advantage of using microcapsules as the carrier for FGF2 controlled release for enhancing L929 cell proliferation was analyzed.

Encapsulation of photosensitizer into multilayer microcapsules by combination of spontaneous deposition and heat-induced shrinkage for photodynamic therapy.

PubMed

Han, Yuanyuan; Bu, Jing; Zhang, Yuying; Tong, Weijun; Gao, Changyou

2012-10-01

Annealing of PDADMAC/PSS multilayer microcapsules assembled on PSS-doped CaCO(3) particles at 80 °C for 30 min reduces their size dramatically from 6.9 ± 0.3 to 3.1 ± 0.5 µm. Methylene blue molecules are encapsulated by spontaneous deposition and post-annealing with a concentration of 22 mg · mL(-1), which is 1000 times higher than the feeding value. The unreleased MB molecules are retained stably for a long time, which are then protected by the capsules against reductive enzymes and keep their photodynamic activity. The viability of HeLa cells incubated with the MB-loaded capsules decreases sharply from ≈ 75 (dark cytotoxicity) to ≈ 20% after irradiation with a laser at 671 nm and 60 J · cm(-2) for 75 s. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Underwater Facilities Inspections and Assessments at Philadelphia Naval Shipyard, Philadelphia, Pennsylvania. Volume 2.

DTIC Science & Technology

1983-10-01

broken piles in a concentrated area . As a result, the relieving platform is unsupported and in this are loading should be restricted to 50 psf until...concentration of damaged piles occurs on Section A near Drydock No. 4 and until repairs are made, loading should be restricted to 100 psf in this area . The...a concentrated area . As a result, the relieving platform is unsup- * ported and in this area loading should be restricted to 50 psf until repairs

The Effects of Ibogaine on Uterine Smooth Muscle Contractions: Relation to the Activity of Antioxidant Enzymes.

PubMed

Oreščanin-Dušić, Zorana; Tatalović, Nikola; Vidonja-Uzelac, Teodora; Nestorov, Jelena; Nikolić-Kokić, Aleksandra; Mijušković, Ana; Spasić, Mihajlo; Paškulin, Roman; Bresjanac, Mara; Blagojević, Duško

2018-01-01

Ibogaine is an indole alkaloid originally extracted from the root bark of the African rainforest shrub Tabernanthe iboga . It has been explored as a treatment for substance abuse because it interrupts drug addiction and relieves withdrawal symptoms. However, it has been shown that ibogaine treatment leads to a sharp and transient fall in cellular ATP level followed by an increase of cellular respiration and ROS production. Since contractile tissues are sensitive to changes in the levels of ATP and ROS, here we investigated an ibogaine-mediated link between altered redox homeostasis and uterine contractile activity. We found that low concentrations of ibogaine stimulated contractile activity in spontaneously active uteri, but incremental increase of doses inhibited it. Inhibitory concentrations of ibogaine led to decreased SOD1 and elevated GSH-Px activity, but doses that completely inhibited contractions increased CAT activity. Western blot analyses showed that changes in enzyme activities were not due to elevated enzyme protein concentrations but posttranslational modifications. Changes in antioxidant enzyme activities point to a vast concentration-dependent increase in H 2 O 2 level. Knowing that extracellular ATP stimulates isolated uterus contractility, while H 2 O 2 has an inhibitory effect, this concentration-dependent stimulation/inhibition could be linked to ibogaine-related alterations in ATP level and redox homeostasis.

The Effects of Ibogaine on Uterine Smooth Muscle Contractions: Relation to the Activity of Antioxidant Enzymes

PubMed Central

Paškulin, Roman

2018-01-01

Ibogaine is an indole alkaloid originally extracted from the root bark of the African rainforest shrub Tabernanthe iboga. It has been explored as a treatment for substance abuse because it interrupts drug addiction and relieves withdrawal symptoms. However, it has been shown that ibogaine treatment leads to a sharp and transient fall in cellular ATP level followed by an increase of cellular respiration and ROS production. Since contractile tissues are sensitive to changes in the levels of ATP and ROS, here we investigated an ibogaine-mediated link between altered redox homeostasis and uterine contractile activity. We found that low concentrations of ibogaine stimulated contractile activity in spontaneously active uteri, but incremental increase of doses inhibited it. Inhibitory concentrations of ibogaine led to decreased SOD1 and elevated GSH-Px activity, but doses that completely inhibited contractions increased CAT activity. Western blot analyses showed that changes in enzyme activities were not due to elevated enzyme protein concentrations but posttranslational modifications. Changes in antioxidant enzyme activities point to a vast concentration-dependent increase in H2O2 level. Knowing that extracellular ATP stimulates isolated uterus contractility, while H2O2 has an inhibitory effect, this concentration-dependent stimulation/inhibition could be linked to ibogaine-related alterations in ATP level and redox homeostasis. PMID:29599898

Invariance and optimality in the regulation of an enzyme

PubMed Central

2013-01-01

Background The Michaelis-Menten equation, proposed a century ago, describes the kinetics of enzyme-catalyzed biochemical reactions. Since then, this equation has been used in countless, increasingly complex models of cellular metabolism, often including time-dependent enzyme levels. However, even for a single reaction, there remains a fundamental disconnect between our understanding of the reaction kinetics, and the regulation of that reaction through changes in the abundance of active enzyme. Results We revisit the Michaelis-Menten equation under the assumption of a time-dependent enzyme concentration. We show that all temporal enzyme profiles with the same average enzyme level yield identical substrate degradation– a simple analytical conclusion that can be thought of as an invariance principle, and which we validate experimentally using a β-galactosidase assay. The ensemble of all time-dependent enzyme trajectories with the same average concentration constitutes a space of functions. We develop a simple model of biological fitness which assigns a cost to each of these trajectories (in the form of a function of functions, i.e. a functional). We then show how one can use variational calculus to analytically infer temporal enzyme profiles that minimize the overall enzyme cost. In particular, by separately treating the static costs of amino acid sequestration and the dynamic costs of protein production, we identify a fundamental cellular tradeoff. Conclusions The overall metabolic outcome of a reaction described by Michaelis-Menten kinetics is ultimately determined by the average concentration of the enzyme during a given time interval. This invariance in analogy to path-independent phenomena in physics, suggests a new way in which variational calculus can be employed to address biological questions. Together, our results point to possible avenues for a unified approach to studying metabolism and its regulation. Reviewers This article was reviewed by Sergei Maslov, William Hlavacek and Daniel Kahn. PMID:23522082

Enzymatic added extraction and clarification of fruit juices-A review.

PubMed

Sharma, Harsh P; Patel, Hiral; Sugandha

2017-04-13

Enzymatic treatment for juice extraction is most commonly used now a days. The enzymatic process is claimed to offer a number of advantages over mechanical-thermal comminution of several fruit pulps. Enzymes are an integral component of modern fruit juice manufacturing and are highly suitable for optimizing processes. Their main purposes are: increase extraction of juice from raw material, increase processing efficiency (pressing, solid settling or removal), and generate a final product that is clear and visually attractive. Juice extraction can be done by using various mechanical processes, which may be achieved through diffusion extraction, decanter centrifuge, screw type juice extractor, fruit pulper and by different types of presses. Enzymatic treatment prior to mechanical extraction significantly improves juice recovery compared to any other extraction process. Enzymatic hydrolysis of the cell walls increases the extraction yield, reducing sugars, soluble dry matter content and galacturonic acid content and titrable acidity of the products. Enzymatic degradation of the biomaterial depends upon the type of enzyme, incubation time, incubation temperature, enzyme concentration, agitation, pH and use of different enzyme combinations. We can conclude from the technical literature that use of the enzymes i.e. cellulases, pectinases, amylases and combination of these enzymes can give better juice yield with superior quality of the fruit juice. Pectinase enzyme can give maximum juice yield i.e. 92.4% at 360 minutes incubation time, 37°C incubation temperature and 5 mg/100 g of enzyme concentration. Whereas the combination of two enzymes i.e. pectin methyl esterase (PME) and polygalacturonase (PG) at 120 minutes of incubation time, 50°C of incubation temperature and 0.05 mg/100 gm of enzymatic concentration can give the maximum yield of 96.8% for plum fruits. This paper discusses the use of enzymes in fruit juice production focusing on the juice recovery, clarity and effect of the particular enzyme on the biochemical properties of the fruit juices.

The effects of xylanase supplementation on growth, digestion, circulating hormone and metabolite levels, immunity and gut microflora in cockerels fed on wheat-based diets.

PubMed

Gao, F; Jiang, Y; Zhou, G H; Han, Z K

2007-08-01

1. The xylanase product used in this study was derived from a genetically modified isolate of Aspergillus niger. Two trials were conducted to investigate the effects of xylanase supplementation on growth, digestion, circulating hormone and metabolite levels, immune parameters and composition of the gut microflora in cockerels fed on wheat-based diets. 2. The experimental diets consisted of a wheat-based control diet supplemented with 0 or 0.1% enzyme preparation. The diets were fed between 7 and 21 d of age. 3. Enzyme supplementation improved growth and feed conversion efficiency. The addition of enzyme to wheat-based diet increased the apparent total digestibility of dry matter (DM), crude protein and fat. 4. Enzyme supplementation reduced the relative weight of digestive organs to a certain extent, but there was no significant difference. Enzyme supplementation reduced digesta viscosity in the jejunum. There was no significant difference between the two experimental groups in counts of lactobacillus and coliform bacteria in the caeca. 5. Enzyme supplementation increased the concentration of blood thyroxine (T(4)), insulin-like growth factor I (IGF-I) and insulin, reduced the concentrations of blood uric acid, but had no significant effect on the concentrations of blood glucose and triiodothyronine (T(3)). 6. Enzyme supplementation increased the relative weight of spleen of cockerels, serum antibody titres to Newcastle disease virus (NDV), lymphocyte proliferation in response to phytohaemagglutinin (PHA) and the natural killer (NK) cell activity. 7. It is concluded that supplementation with an enzyme preparation (xylanase), which hydrolyses non-starch polysaccharides can improve growth in cockerels fed on wheat-based diets. This improvement is achieved through enzyme effects on digestion, absorption, metabolism and immunity of cockerels.

Impact of wastewater derived dissolved interfering compounds on growth, enzymatic activity and trace organic contaminant removal of white rot fungi - A critical review.

PubMed

Asif, Muhammad B; Hai, Faisal I; Hou, Jingwei; Price, William E; Nghiem, Long D

2017-10-01

White-rot fungi (WRF) and their ligninolytic enzymes have been investigated for the removal of a broad spectrum of trace organic contaminants (TrOCs) mostly from synthetic wastewater in lab-scale experiments. Only a few studies have reported the efficiency of such systems for the removal of TrOCs from real wastewater. Wastewater derived organic and inorganic compounds can inhibit: (i) WRF growth and their enzyme production capacity; (ii) enzymatic activity of ligninolytic enzymes; and (iii) catalytic efficiency of both WRF and enzymes. It is observed that essential metals such as Cu, Mn and Co at trace concertation (up to 1 mM) can improve the growth of WRF species, whereas non-essential metal such as Pb, Cd and Hg at 1 mM concentration can inhibit WRF growth and their enzyme production. In the case of purified enzymes, most of the tested metals at 1-5 mM concentration do not significantly inhibit the activity of laccases. Organic interfering compounds such as oxalic acid and ethylenediaminetetraacetic acid (EDTA) at 1 mM concentration are potent inhibitors of WRF and their extracellular enzymes. However, inhibitory effects induced by interfering compounds are strongly influenced by the type of WRF species as well as experimental conditions (e.g., incubation time and TrOC type). In this review, mechanisms and factors governing the interactions of interfering compounds with WRF and their ligninolytic enzymes are reviewed and elucidated. In addition, the performance of WRF and their ligninolytic enzymes for the removal of TrOCs from synthetic and real wastewater is critically summarized. Copyright © 2017 Elsevier Ltd. All rights reserved.

Storm loads of culturable and molecular fecal indicators in an inland urban stream.

PubMed

Liao, Hehuan; Krometis, Leigh-Anne H; Cully Hession, W; Benitez, Romina; Sawyer, Richard; Schaberg, Erin; von Wagoner, Emily; Badgley, Brian D

2015-10-15

Elevated concentrations of fecal indicator bacteria in receiving waters during wet-weather flows are a considerable public health concern that is likely to be exacerbated by future climate change and urbanization. Knowledge of factors driving the fate and transport of fecal indicator bacteria in stormwater is limited, and even less is known about molecular fecal indicators, which may eventually supplant traditional culturable indicators. In this study, concentrations and loading rates of both culturable and molecular fecal indicators were quantified throughout six storm events in an instrumented inland urban stream. While both concentrations and loading rates of each fecal indicator increased rapidly during the rising limb of the storm hydrographs, it is the loading rates rather than instantaneous concentrations that provide a better estimate of transport through the stream during the entire storm. Concentrations of general fecal indicators (both culturable and molecular) correlated most highly with each other during storm events but not with the human-associated HF183 Bacteroides marker. Event loads of general fecal indicators most strongly correlated with total runoff volume, maximum discharge, and maximum turbidity, while event loads of HF183 most strongly correlated with the time to peak flow in a hydrograph. These observations suggest that collection of multiple samples during a storm event is critical for accurate predictions of fecal indicator loading rates and total loads during wet-weather flows, which are required for effective watershed management. In addition, existing predictive models based on general fecal indicators may not be sufficient to predict source-specific genetic markers of fecal contamination. Copyright © 2015 Elsevier B.V. All rights reserved.

Mean Velocity vs. Mean Propulsive Velocity vs. Peak Velocity: Which Variable Determines Bench Press Relative Load With Higher Reliability?

PubMed

García-Ramos, Amador; Pestaña-Melero, Francisco L; Pérez-Castilla, Alejandro; Rojas, Francisco J; Gregory Haff, G

2018-05-01

García-Ramos, A, Pestaña-Melero, FL, Pérez-Castilla, A, Rojas, FJ, and Haff, GG. Mean velocity vs. mean propulsive velocity vs. peak velocity: which variable determines bench press relative load with higher reliability? J Strength Cond Res 32(5): 1273-1279, 2018-This study aimed to compare between 3 velocity variables (mean velocity [MV], mean propulsive velocity [MPV], and peak velocity [PV]): (a) the linearity of the load-velocity relationship, (b) the accuracy of general regression equations to predict relative load (%1RM), and (c) the between-session reliability of the velocity attained at each percentage of the 1-repetition maximum (%1RM). The full load-velocity relationship of 30 men was evaluated by means of linear regression models in the concentric-only and eccentric-concentric bench press throw (BPT) variants performed with a Smith machine. The 2 sessions of each BPT variant were performed within the same week separated by 48-72 hours. The main findings were as follows: (a) the MV showed the strongest linearity of the load-velocity relationship (median r = 0.989 for concentric-only BPT and 0.993 for eccentric-concentric BPT), followed by MPV (median r = 0.983 for concentric-only BPT and 0.980 for eccentric-concentric BPT), and finally PV (median r = 0.974 for concentric-only BPT and 0.969 for eccentric-concentric BPT); (b) the accuracy of the general regression equations to predict relative load (%1RM) from movement velocity was higher for MV (SEE = 3.80-4.76%1RM) than for MPV (SEE = 4.91-5.56%1RM) and PV (SEE = 5.36-5.77%1RM); and (c) the PV showed the lowest within-subjects coefficient of variation (3.50%-3.87%), followed by MV (4.05%-4.93%), and finally MPV (5.11%-6.03%). Taken together, these results suggest that the MV could be the most appropriate variable for monitoring the relative load (%1RM) in the BPT exercise performed in a Smith machine.

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.

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.

Complementary Split-Ring Resonator-Loaded Microfluidic Ethanol Chemical Sensor.

PubMed

Salim, Ahmed; Lim, Sungjoon

2016-10-28

In this paper, a complementary split-ring resonator (CSRR)-loaded patch is proposed as a microfluidic ethanol chemical sensor. The primary objective of this chemical sensor is to detect ethanol's concentration. First, two tightly coupled concentric CSRRs loaded on a patch are realized on a Rogers RT/Duroid 5870 substrate, and then a microfluidic channel engraved on polydimethylsiloxane (PDMS) is integrated for ethanol chemical sensor applications. The resonant frequency of the structure before loading the microfluidic channel is 4.72 GHz. After loading the microfluidic channel, the 550 MHz shift in the resonant frequency is ascribed to the dielectric perturbation phenomenon when the ethanol concentration is varied from 0% to 100%. In order to assess the sensitivity range of our proposed sensor, various concentrations of ethanol are tested and analyzed. Our proposed sensor exhibits repeatability and successfully detects 10% ethanol as verified by the measurement set-up. It has created headway to a miniaturized, non-contact, low-cost, reliable, reusable, and easily fabricated design using extremely small liquid volumes.

Complementary Split-Ring Resonator-Loaded Microfluidic Ethanol Chemical Sensor

PubMed Central

Salim, Ahmed; Lim, Sungjoon

2016-01-01

In this paper, a complementary split-ring resonator (CSRR)-loaded patch is proposed as a microfluidic ethanol chemical sensor. The primary objective of this chemical sensor is to detect ethanol’s concentration. First, two tightly coupled concentric CSRRs loaded on a patch are realized on a Rogers RT/Duroid 5870 substrate, and then a microfluidic channel engraved on polydimethylsiloxane (PDMS) is integrated for ethanol chemical sensor applications. The resonant frequency of the structure before loading the microfluidic channel is 4.72 GHz. After loading the microfluidic channel, the 550 MHz shift in the resonant frequency is ascribed to the dielectric perturbation phenomenon when the ethanol concentration is varied from 0% to 100%. In order to assess the sensitivity range of our proposed sensor, various concentrations of ethanol are tested and analyzed. Our proposed sensor exhibits repeatability and successfully detects 10% ethanol as verified by the measurement set-up. It has created headway to a miniaturized, non-contact, low-cost, reliable, reusable, and easily fabricated design using extremely small liquid volumes. PMID:27801842

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.

Stratification and loading of fecal indicator bacteria (FIB) in a tidally muted urban salt marsh.

PubMed

Johnston, Karina K; Dorsey, John H; Saez, Jose A

2015-03-01

Stratification and loading of fecal indicator bacteria (FIB) were assessed in the main tidal channel of the Ballona Wetlands, an urban salt marsh receiving muted tidal flows, to (1) determine FIB concentration versus loading within the water column at differing tidal flows, (2) identify associations of FIB with other water quality parameters, and (3) compare wetland FIB concentrations to the adjacent estuary. Sampling was conducted four times during spring-tide events; samples were analyzed for FIB and turbidity (NTU) four times over a tidal cycle at pre-allocated depths, depending on the water level. Additional water quality parameters measured included temperature, salinity, oxygen, and pH. Loadings were calculated by integrating the stratified FIB concentrations with water column cross-sectional volumes corresponding to each depth. Enterococci and Escherichia coli were stratified both by concentration and loading, although these variables portrayed different patterns over a tidal cycle. Greatest concentrations occurred in surface to mid-strata levels, during flood tides when contaminated water flowed in from the estuary, and during ebb flows when sediments were suspended. Loading was greatest during flood flows and diminished during low tide periods. FIB concentrations within the estuary often were significantly greater than those within the wetland tide channel, supporting previous studies that the wetlands act as a sink for FIB. For public health water quality monitoring, these results indicate that more accurate estimates of FIB concentrations would be obtained by sampling a number of points within a water column rather than relying only on single surface samples.

In vitro metabolic engineering for the salvage synthesis of NAD(.).

PubMed

Honda, Kohsuke; Hara, Naoya; Cheng, Maria; Nakamura, Anna; Mandai, Komako; Okano, Kenji; Ohtake, Hisao

2016-05-01

Excellent thermal and operational stabilities of thermophilic enzymes can greatly increase the applicability of biocatalysis in various industrial fields. However, thermophilic enzymes are generally incompatible with thermo-labile substrates, products, and cofactors, since they show the maximal activities at high temperatures. Despite their pivotal roles in a wide range of enzymatic redox reactions, NAD(P)(+) and NAD(P)H exhibit relatively low stabilities at high temperatures, tending to be a major obstacle in the long-term operation of biocatalytic chemical manufacturing with thermophilic enzymes. In this study, we constructed an in vitro artificial metabolic pathway for the salvage synthesis of NAD(+) from its degradation products by the combination of eight thermophilic enzymes. The enzymes were heterologously produced in recombinant Escherichia coli and the heat-treated crude extracts of the recombinant cells were directly used as enzyme solutions. When incubated with experimentally optimized concentrations of the enzymes at 60°C, the NAD(+) concentration could be kept almost constant for 15h. Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Construction of a high-performance magnetic enzyme nanosystem for rapid tryptic digestion

NASA Astrophysics Data System (ADS)

Cheng, Gong; Zheng, Si-Yang

2014-11-01

A magnetic enzyme nanosystem have been designed and constructed by a polydopamine (PDA)-modification strategy. The magnetic enzyme nanosystem has well defined core-shell structure and a relatively high saturation magnetization (Ms) value of 48.3 emu g-1. The magnetic enzyme system can realize rapid, efficient and reusable tryptic digestion of proteins by taking advantage of its magnetic core and biofunctional shell. Various standard proteins (e.g. cytochrome C (Cyt-C), myoglobin (MYO) and bovine serum albumin (BSA)) have been used to evaluate the effectiveness of the magnetic enzyme nanosystem. The results show that the magnetic enzyme nanosystem can digest the proteins in 30 minutes, and the results are comparable to conventional 12 hours in-solution digestion. Furthermore, the magnetic enzyme nanosystem is also effective in the digestion of low-concentration proteins, even at as low as 5 ng μL-1 substrate concentration. Importantly, the system can be reused several times, and has excellent stability for storage. Therefore, this work will be highly beneficial for the rapid digestion and identification of proteins in future proteomics.

Towards cell-free isobutanol production: Development of a novel immobilized enzyme system.

PubMed

Grimaldi, Joseph; Collins, Cynthia H; Belfort, Georges

2016-01-01

Producing fuels and chemical intermediates with cell cultures is severely limited by low product concentrations (≤0.2%(v/v)) due to feedback inhibition, cell instability, and lack of economical product recovery processes. We have developed an alternate simplified production scheme based on a cell-free immobilized enzyme system. Two immobilized enzymes (keto-acid decarboxylase (KdcA) and alcohol dehydrogenase (ADH)) and one enzyme in solution (formate dehydrogenase (FDH) for NADH recycle) produced isobutanol titers 8 to 20 times higher than the highest reported titers with S. cerevisiae on a mol/mol basis. These high conversion rates and low protein leaching were achieved by covalent immobilization of enzymes (ADH) and enzyme fusions (fKdcA) on methacrylate resin. The new enzyme system without in situ removal of isobutanol achieved a 55% conversion of ketoisovaleric acid to isobutanol at a concentration of 0.135 (mole isobutanol produced for each mole ketoisovaleric acid consumed). Further increasing titer will require continuous removal of the isobutanol using an in situ recovery system. © 2015 American Institute of Chemical Engineers.

Screening of Enzyme Biomarker for Nanotoxicity of Zinc Oxide in OREOCHROMIS MOSSAMBICUS

NASA Astrophysics Data System (ADS)

Subramanian, Periasamy; Bupesh, Giridharan

2011-06-01

Experiments were conducted to determine the effects of Zinc oxide (ZnO) nanoparticles (NPs) on fish models. Oreochromis mossambicus was orally administered with ZnO NPs (50-100 nm) once and its effects at five different concentrations (60 ppm-100 ppm) were observed for 12 days. Enzymatic assays were performed at every three days interval in the vital tissues of liver, gill, muscle and kidney. The defense enzymes, ethoxyresorufin O-deethylase (EROD) and glutathione S transferase (GST) exerted a dose dependent elevation up to 6 days. This hike then declines in higher concentrations and extended duration. Whereas the tissue damaging enzymes, glutamate oxaloacetic transaminase (GOT), glutamate pyruvic transaminase (GPT) and alkaline phosphatase (ALP) as well as the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) exhibited a dose and duration dependent increase until the end of the experiment. Among these enzymes, the antioxidant enzymes response to ZnO NP toxicity on fish showed notable continuous induction. This study demonstrates that antioxidant enzymes responses in O. mossambicus could be used as a biomarker for the early detection of nanotoxicity.

Purification and Characterization of Glucose 6-Phosphate Dehydrogenase, 6-Phosphogluconate Dehydrogenase, and Glutathione Reductase from Rat Heart and Inhibition Effects of Furosemide, Digoxin, and Dopamine on the Enzymes Activities.

PubMed

Adem, Sevki; Ciftci, Mehmet

2016-06-01

The present study was aimed to investigate characterization and purification of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and glutathione reductase from rat heart and the inhibitory effect of three drugs. The purification of the enzymes was performed using 2',5'-ADP sepharose 4B affinity material. The subunit and the natural molecular weights were analyzed by SDS-PAGE and gel filtration. Biochemical characteristics such as the optimum temperature, pH, stable pH, and salt concentration were examined for each enzyme. Types of product inhibition and Ki values with Km and Vmax values of the substrates and coenzymes were determined. According to the obtained Ki and IC50 values, furosemide, digoxin, and dopamine showed inhibitory effect on the enzyme activities at low millimolar concentrations in vitro conditions. Dopamine inhibited the activity of these enzymes as competitive, whereas furosemide and digoxin inhibited the activity of the enzyme as noncompetitive. © 2016 Wiley Periodicals, Inc.

Modulation of digestive physiology and biochemistry in Mytilus californianus in response to feeding level acclimation and microhabitat

PubMed Central

Sung, Aaron; Garcia, Nathan S.; Gracey, Andrew Y.; German, Donovan P.

2016-01-01

ABSTRACT The intertidal mussel Mytilus californianus is a critical foundation species that is exposed to fluctuations in the environment along tidal- and wave-exposure gradients. We investigated feeding and digestion in mussels under laboratory conditions and across environmental gradients in the field. We assessed whether mussels adopt a rate-maximization (higher ingestion and lower assimilation) or a yield-maximization acquisition (lower ingestion and higher assimilation) strategy under laboratory conditions by measuring feeding physiology and digestive enzyme activities. We used digestive enzyme activity to define resource acquisition strategies in laboratory studies, then measured digestive enzyme activities in three microhabitats at the extreme ends of the tidal- and wave-exposure gradients within a stretch of shore (<20 m) projected sea-ward. Our laboratory results indicated that mussels benefit from a high assimilation efficiency when food concentration is low and have a low assimilation efficiency when food concentration is high. Additionally, enzyme activities of carbohydrases amylase, laminarinase and cellulase were elevated when food concentration was high. The protease trypsin, however, did not increase with increasing food concentration. In field conditions, low-shore mussels surprisingly did not have high enzyme activities. Rather, high-shore mussels exhibited higher cellulase activities than low-shore mussels. Similarly, trypsin activity in the high-shore-wave-sheltered microhabitat was higher than that in high-shore-wave-exposed. As expected, mussels experienced increasing thermal stress as a function of reduced submergence from low to high shore and shelter from wave-splash. Our findings suggest that mussels compensate for limited feeding opportunities and thermal stress by modulating digestive enzyme activities. PMID:27402963

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.