Immobilization of Enzymes in Polymer Supports.

Science.gov (United States)

Conlon, Hugh D.; Walt, David R.

1986-01-01

Two experiments in which an enzyme is immobilized onto a polymeric support are described. The experiments (which also demonstrate two different polymer preparations) involve: (1) entrapping an enzyme in an acrylamide polymer; and (2) reacting the amino groups on the enzyme's (esterase) lysine residues with an activated polymer. (JN)

Studies of the immobilization of enzymes and microorganism pt.1

International Nuclear Information System (INIS)

Kim, S.K.

1979-01-01

A new method of immobilization of glucose oxidase by the aerobic gamma radiation of synthetic monomers was developed. The radiocopolymerization was conducted aerobically at -70 to-80 degC with the mixture of several polyfunctional esters, acrylates and native enzyme. The retained activity of immobilized glucoseoxidase was about 50 to 55% when a NK 23G ester, acrylamide-bis and water mixture (1:1:2) in cold toluene treated with 450 Krad of gamma radiation. The radiation dose did not influence significantly to the enzyme activity. The solvents used to prepare the beads of glucose oxidase and monomers were toluene, n-hexane, petoleum ether and chloroform. 0.05M tris-gycerol(pH 7.0) was a more suitable buffer solution for immobilizing the enzyme than was 0.02M phosphate. Immobilization of glucose oxidase shifted the optimum pH for its reaction from 6.0 to 6.5. The pH profile for the immobilized enzyme showed a broad range of optimum activity while the native enzyme gave a sharp pick for its optimum pH value. The immobilized enzyme reaction temperature was at the range of 30-40 degreesC. (Author)

Biotechnological production of vanillin using immobilized enzymes.

Science.gov (United States)

Furuya, Toshiki; Kuroiwa, Mari; Kino, Kuniki

2017-02-10

Vanillin is an important and popular plant flavor, but the amount of this compound available from plant sources is very limited. Biotechnological methods have high potential for vanillin production as an alternative to extraction from plant sources. Here, we report a new approach using immobilized enzymes for the production of vanillin. The recently discovered oxygenase Cso2 has coenzyme-independent catalytic activity for the conversion of isoeugenol and 4-vinylguaiacol to vanillin. Immobilization of Cso2 on Sepabeads EC-EA anion-exchange carrier conferred enhanced operational stability enabling repetitive use. This immobilized Cso2 catalyst allowed 6.8mg yield of vanillin from isoeugenol through ten reaction cycles at a 1mL scale. The coenzyme-independent decarboxylase Fdc, which has catalytic activity for the conversion of ferulic acid to 4-vinylguaiacol, was also immobilized on Sepabeads EC-EA. We demonstrated that the immobilized Fdc and Cso2 enabled the cascade synthesis of vanillin from ferulic acid via 4-vinylguaiacol with repetitive use of the catalysts. This study is the first example of biotechnological production of vanillin using immobilized enzymes, a process that provides new possibilities for vanillin production. Copyright © 2016 Elsevier B.V. All rights reserved.

Hierarchically Nanoporous Bioactive Glasses for High Efficiency Immobilization of Enzymes

DEFF Research Database (Denmark)

He, W.; Min, D.D.; Zhang, X.D.

2014-01-01

Bioactive glasses with hierarchical nanoporosity and structures have been heavily involved in immobilization of enzymes. Because of meticulous design and ingenious hierarchical nanostructuration of porosities from yeast cell biotemplates, hierarchically nanostructured porous bioactive glasses can...... and products of catalytic reactions can freely diffuse through open mesopores (2–40 nm). The formation mechanism of hierarchically structured porous bioactive glasses, the immobilization mechanism of enzyme and the catalysis mechanism of immobilized enzyme are then discussed. The novel nanostructure...

An easily regenerable enzyme reactor prepared from polymerized high internal phase emulsions

International Nuclear Information System (INIS)

Ruan, Guihua; Wu, Zhenwei; Huang, Yipeng; Wei, Meiping; Su, Rihui; Du, Fuyou

2016-01-01

A large-scale high-efficient enzyme reactor based on polymerized high internal phase emulsion monolith (polyHIPE) was prepared. First, a porous cross-linked polyHIPE monolith was prepared by in-situ thermal polymerization of a high internal phase emulsion containing styrene, divinylbenzene and polyglutaraldehyde. The enzyme of TPCK-Trypsin was then immobilized on the monolithic polyHIPE. The performance of the resultant enzyme reactor was assessed according to the conversion ability of N_α-benzoyl-L-arginine ethyl ester to N_α-benzoyl-L-arginine, and the protein digestibility of bovine serum albumin (BSA) and cytochrome (Cyt-C). The results showed that the prepared enzyme reactor exhibited high enzyme immobilization efficiency and fast and easy-control protein digestibility. BSA and Cyt-C could be digested in 10 min with sequence coverage of 59% and 78%, respectively. The peptides and residual protein could be easily rinsed out from reactor and the reactor could be regenerated easily with 4 M HCl without any structure destruction. Properties of multiple interconnected chambers with good permeability, fast digestion facility and easily reproducibility indicated that the polyHIPE enzyme reactor was a good selector potentially applied in proteomics and catalysis areas. - Graphical abstract: Schematic illustration of preparation of hypercrosslinking polyHIPE immobilized enzyme reactor for on-column protein digestion. - Highlights: • A reactor was prepared and used for enzyme immobilization and continuous on-column protein digestion. • The new polyHIPE IMER was quite suit for protein digestion with good properties. • On-column digestion revealed that the IMER was easy regenerated by HCl without any structure destruction.

An easily regenerable enzyme reactor prepared from polymerized high internal phase emulsions

Energy Technology Data Exchange (ETDEWEB)

Ruan, Guihua, E-mail: guihuaruan@hotmail.com [Guangxi Key Laboratory of Electrochemical and Magnetochemical Functional Materials, College of Chemistry and Bioengineering, Guilin University of Technology, Guangxi 541004 (China); Guangxi Collaborative Innovation Center for Water Pollution Control and Water Safety in Karst Area, Guilin University of Technology, Guilin 541004 (China); Wu, Zhenwei; Huang, Yipeng; Wei, Meiping; Su, Rihui [Guangxi Key Laboratory of Electrochemical and Magnetochemical Functional Materials, College of Chemistry and Bioengineering, Guilin University of Technology, Guangxi 541004 (China); Du, Fuyou, E-mail: dufu2005@126.com [Guangxi Key Laboratory of Electrochemical and Magnetochemical Functional Materials, College of Chemistry and Bioengineering, Guilin University of Technology, Guangxi 541004 (China); Guangxi Collaborative Innovation Center for Water Pollution Control and Water Safety in Karst Area, Guilin University of Technology, Guilin 541004 (China)

2016-04-22

A large-scale high-efficient enzyme reactor based on polymerized high internal phase emulsion monolith (polyHIPE) was prepared. First, a porous cross-linked polyHIPE monolith was prepared by in-situ thermal polymerization of a high internal phase emulsion containing styrene, divinylbenzene and polyglutaraldehyde. The enzyme of TPCK-Trypsin was then immobilized on the monolithic polyHIPE. The performance of the resultant enzyme reactor was assessed according to the conversion ability of N{sub α}-benzoyl-L-arginine ethyl ester to N{sub α}-benzoyl-L-arginine, and the protein digestibility of bovine serum albumin (BSA) and cytochrome (Cyt-C). The results showed that the prepared enzyme reactor exhibited high enzyme immobilization efficiency and fast and easy-control protein digestibility. BSA and Cyt-C could be digested in 10 min with sequence coverage of 59% and 78%, respectively. The peptides and residual protein could be easily rinsed out from reactor and the reactor could be regenerated easily with 4 M HCl without any structure destruction. Properties of multiple interconnected chambers with good permeability, fast digestion facility and easily reproducibility indicated that the polyHIPE enzyme reactor was a good selector potentially applied in proteomics and catalysis areas. - Graphical abstract: Schematic illustration of preparation of hypercrosslinking polyHIPE immobilized enzyme reactor for on-column protein digestion. - Highlights: • A reactor was prepared and used for enzyme immobilization and continuous on-column protein digestion. • The new polyHIPE IMER was quite suit for protein digestion with good properties. • On-column digestion revealed that the IMER was easy regenerated by HCl without any structure destruction.

Studies on the preparation of immobilized enzymes by radio-polymerization, 10

International Nuclear Information System (INIS)

Amarakone, S.P.; Hayashi, Toru; Kawashima, Koji.

1983-01-01

β-Galactosidase of E. coli origin was immobilized in the form of beads by the radiopolymerization of different combinations of monomers using a gamma irradiation technique. With the dialysed enzyme, recoveries of over 300 % could be obtained on suitable monomer combinations containing magnesium and sodium acrylates. The recovery of the enzyme also depended on the irradiation time. The immobilized enzyme had better pH and temperature stability and was less affected by the presence of metal ions in the medium, compared to the native enzyme. The optimum pH and temperatures of the immobilized enzyme were different from those of the native enzyme and were 7.0 to 7.5 and 50 deg C respectively. The immobilized enzyme was used in a column for the continuous determination of lactose with a standard type autoanalyser. Good linearity could be observed even up to 3 % lactose in the sample. (author)

Phospholipid-sepiolite biomimetic interfaces for the immobilization of enzymes.

Science.gov (United States)

Wicklein, Bernd; Darder, Margarita; Aranda, Pilar; Ruiz-Hitzky, Eduardo

2011-11-01

Biomimetic interfaces based on phosphatidylcholine (PC) assembled to the natural silicate sepiolite were prepared for the stable immobilization of the urease and cholesterol oxidase enzymes. This is an important issue in practical advanced applications such as biocatalysis or biosensing. The supported lipid bilayer (BL-PC), prepared from PC adsorption, was used for immobilization of enzymes and the resulting biomimetic systems were compared to several other supported layers including a lipid monolayer (ML-PC), a mixed phosphatidylcholine/octyl-galactoside layer (PC-OGal), a cetyltrimethylammonium monolayer (CTA), and also to the bare sepiolite surface. Interfacial characteristics of these layers were investigated with a focus on layer packing density, hydrophilicity/hydrophobicity, and surface charge, which are being considered as key points for enzyme immobilization and stabilization of their biological activity. Cytoplasmic urease and membrane-bound cholesterol oxidase, which served as model enzymes, were immobilized on the different PC-based hybrid materials to probe their biomimetic character. Enzymatic activity was assessed by cyclic voltammetry and UV-vis spectrophotometry. The resulting enzyme/bio-organoclay hybrids were applied as active phase of a voltammetric urea biosensor and cholesterol bioreactor, respectively. Urease supported on sepiolite/BL-PC proved to maintain its enzymatic activity over several months while immobilized cholesterol oxidase demonstrated high reusability as biocatalyst. The results emphasize the good preservation of bioactivity due to the accommodation of the enzymatic system within the biomimetic lipid interface on sepiolite.

The development, characterization, and application of biomimetic nanoscale enzyme immobilization

Science.gov (United States)

Haase, Nicholas R.

The utilization of enzymes is of interest for applications such as biosensors and biofuel cells. Immobilizing enzymes provides a means to develop these applications. Previous immobilization efforts have been accomplished by exposing surfaces on which silica-forming molecules are present to solutions containing an enzyme and a silica precursor. This approach leads to the enzyme being entrapped in a matrix three orders of magnitude larger than the enzyme itself, resulting in low retention of enzyme activity. The research herein introduces a method for the immobilization of enzymes during the layer-by-layer buildup of Si-O and Ti-O coatings which are nanoscale in thickness. This approach is an application of a peptide-induced mineral deposition method developed in the Sandhage and Kroger groups, and it involves the alternating exposure of a surface to solutions containing the peptide protamine and then an aqueous precursor solution of silicon- or titanium-oxide at near-neutral pH. A method has been developed that enables in situ immobilization of enzymes in the protamine/mineral oxide coatings. Depending on the layer and mineral (silica or titania) within which the enzyme is incorporated, the resulting multilayer biocatalytic hybrid materials retain 20 -- 100% of the enzyme activity. Analyses of kinetic properties of the immobilized enzyme, coupled with characterization of physical properties of the mineral-bearing layers (thickness, porosity, pore size distribution), indicates that the catalytic activities of the enzymes immobilized in the different layers are largely determined by substrate diffusion. The enzyme was also found to be substantially stabilized against heat-induced denaturation and largely protected from proteolytic attack. These functional coatings are then developed for use as antimicrobial materials. Glucose oxidase, which catalyzes production of the cytotoxic agent hydrogen peroxide, was immobilized with silver nanoparticles, can release

Immobilized enzymes in blood plasma exchangers via radiation grafting

Science.gov (United States)

Gombotz, Wayne; Hoffman, Allan; Schmer, Gottfried; Uenoyama, Satoshi

The enzyme asparaginase was immobilized onto a porous hollow polypropylene (PP) fiber blood plasma exchange device for the treatment of acute lymphocytic leukemia. The devices were first radiation grafted with polymethacrylic acid (poly(MAAc)). This introduces carboxyl groups onto the surface of the fibers. Several variables were studied in the grafting reaction including the effects of solvent type and monomer concentration. The carboxyl groups were activated with N-hydroxy succinimide (NHS) using carbodiimide chemistry. Asparaginase was then covalently immobilized on the activated surfaces. Quantitative relationships were found relating the percent graft to the amount of immobilized enzyme which was active. The enzyme reactor was tested both in vitro and in vivo using a sheep as an animal model.

Design and Properties of an Immobilization Enzyme System for Inulin Conversion.

Science.gov (United States)

Hang, Hua; Wang, Changbao; Cheng, Yiqun; Li, Ning; Song, Liuli

2018-02-01

A commercial inulinase could convert inulin into fructose, which was optimized to be entrapped in the calcium alginate-gelatin beads with the immobilization yield of 86% for free inulinase activities. The optimum pH values and temperatures were 4.5 and 40 °C for the free enzyme and 5.0-5.5 and 45-50 °C for the immobilized enzyme. The kinetic parameters of V max and K m were 5.24 μmol/min and 57.6 mg/mL for the free inulinase and 4.32 μmol/min and 65.8 mg/mL for the immobilized inulinase, respectively. The immobilized enzyme retained 80% of its initial activities at 45 °C for 4 days, which could exhibit better thermal stability. The reuse of immobilized inulinase throughout the continuous batch operations was explored, which had better reusability of the immobilized biocatalyst. At the same time, the stability of immobilized enzyme in the continuous packed-bed bioreactor was estimated, which showed the better results and had its potential scale-up fructose production for inulin conversion.

Directing filtration to optimize enzyme immobilization in reactive membranes

DEFF Research Database (Denmark)

Luo, Jianquan; Marpani, Fauziah; Brites, Rita

2014-01-01

enzymatic reaction efficiency were evaluated in terms of enzyme loading, conversion rate and biocatalytic stability. Alcohol dehydrogenase (ADH) was selected as a model enzyme. Lower pressure, higher enzyme concentration and lower pH resulted in higher irreversible fouling resistance and lower permeate flux....... High pH during immobilization produced increased permeate flux but declines in conversion rates, likely because of the weak immobilization resulting from strong electrostatic repulsion between enzymes and membrane. The results showed that pore blocking as a fouling mechanism permitted a higher enzyme...... loading but generated more permeability loss, while cake layer formation increased enzyme stability but resulted in low loading rate. Low pH (near isoelectric point) favored hydrophobic and electrostatic adsorption of enzymes on the membrane, which reduced the enzyme stability. Neutral pH, however...

Substrate Specificity and Enzyme Recycling Using Chitosan Immobilized Laccase

Directory of Open Access Journals (Sweden)

Everton Skoronski

2014-10-01

Full Text Available The immobilization of laccase (Aspergillus sp. on chitosan by cross-linking and its application in bioconversion of phenolic compounds in batch reactors were studied. Investigation was performed using laccase immobilized via chemical cross-linking due to the higher enzymatic operational stability of this method as compared to immobilization via physical adsorption. To assess the influence of different substrate functional groups on the enzyme’s catalytic efficiency, substrate specificity was investigated using chitosan-immobilized laccase and eighteen different phenol derivatives. It was observed that 4-nitrophenol was not oxidized, while 2,5-xylenol, 2,6-xylenol, 2,3,5-trimethylphenol, syringaldazine, 2,6-dimetoxyphenol and ethylphenol showed reaction yields up 90% at 40 °C. The kinetic of process, enzyme recyclability and operational stability were studied. In batch reactors, it was not possible to reuse the enzyme when it was applied to syringaldazne bioconversion. However, when the enzyme was applied to bioconversion of 2,6-DMP, the activity was stable for eight reaction batches.

Microbial production of raw starch digesting enzymes | Sun | African ...

African Journals Online (AJOL)

Raw starch digesting enzymes refer to enzymes that can act directly on raw starch granules below the gelatinization temperature of starch. With the view of energy-saving, a worldwide interest has been focused on raw starch digesting enzymes in recent years, especially since the oil crisis of 1973. Raw starch digesting ...

Electron beam technology for production of preparations of immobilized enzymes

International Nuclear Information System (INIS)

Gonchar, A.M.; Auslender, V.L.; Polyakov, V.A.

1995-01-01

Possibility of electron beam usage for proteases immobilization on 1,4-polyalkylene oxide (1,4-PAO) was studied to obtain biologically active complex for multi-purpose usage. It is shown that immobilization of Bacillus Subtilis protease is done due to free-radical linking of enzyme and carrier with formation of mycelium-like structures. Immobilization improves heat resistance of enzyme up to 60 centigrade without substrate and up to 80 centigrade in presence of substrate, widens range pH activity in comparison with non-immobilized forms. Immobilized proteases does not contain peroxides and long-live radicals. Our results permitted to create technologies for production of medical and veterinary preparations, active components for wool washing agents and leather fabrication technology

Approaching Immobilization of Enzymes onto Open Porous Basotect®

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Peter J. Allertz

2017-11-01

Full Text Available For the first time, commercial macroporous melamine formaldehyde foam Basotect® (BT was used as a basic carrier material for both adsorptive and covalent enzyme immobilization. In order to access inherent amino groups, the Basotect® surface was pretreated with hydrochloric acid. The resulting material revealed 6 nmol of superficial amino groups per milligram Basotect®. Different optimized strategies for tethering the laccase from Trametes versicolor and the lipase from Thermomyces lanuginosus onto the pre-treated Basotect® surface were studied. Particularly, for covalent immobilization, two different strategies were pursued: lipase was tethered via a cross-linking method using 1-ethyl-3-(3-dimethylaminopropylcarbodiimide, and laccase was bound after functionalizing Basotect® with hydrophilic copolymer poly(ethylene-alt-maleic anhydride (PEMA. Prior to laccase immobilization, the PEMA coating of Basotect® was verified by ATR-FTIR analysis. Subsequent quantification of available high-reactive PEMA anhydride moieties revealed an amount of 1028 ± 73 nmol per mg Basotect®. The surface-bound enzyme amounts were quantified as 4.1–5.8 μg per mg Basotect®. A theoretical surface-covered enzyme mass for the ideal case that an enzyme monolayer was immobilized onto the Basotect® surface was calculated and compared to the amount of adsorptive and covalently bound enzymes before and after treatment with SDS. Furthermore, the enzyme activities were determined for the different immobilization approaches, and the stability during storage over time and against sodium dodecyl sulfate treatment was monitored. Additionally, PEMA-BT-bound laccase was tested for the elimination of anthropogenic micropollutant bisphenol A from contaminated water in a cost-effective and environmentally-friendly way and resulted in a degradation rate higher than 80%.

Immobilization of antibodies and enzyme-labeled antibodies by radiation polymerization

International Nuclear Information System (INIS)

Kumakura, M.; Kaetsu, I.; Suzuki, M.; Adachi, S.

1983-01-01

Immobilization of antibodies and enzyme-labeled antibodies by radiation polymerization at low temperatures was studied. The antibody activity of antibody was not affected by irradiation at an irradiation dose of below 8 MR and low temperatures. Immobilization of peroxidase-labeled anti-rabbit IgG goat IgG, anti-peroxidase, peroxidase, and anti-alpha-fetoprotein was carried out with hydrophilic and hydrophobic monomers. The activity of the immobilized enzyme-labeled antibody membranes varied with the thickness of the membranes and increased with decreasing membrane thickness. The activity of the immobilized antibody particles was varied by particle size. Immobilized anti-alpha-fetoprotein particles and membranes can be used for the assay of alpha-fetoprotein by the antigen-antibody reaction, such as a solid-phase sandwich method with high sensitivity

Optimization of Enzyme Co-Immobilization with Sodium Alginate and Glutaraldehyde-Activated Chitosan Beads.

Science.gov (United States)

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.

Catalytic properties of immobilized tannase produced from Aspergillus aculeatus compared with the free enzyme

Directory of Open Access Journals (Sweden)

A. B El-Tanash

2011-09-01

Full Text Available Aspergillus aculeatus tannase was immobilized on several carriers by entrapment and covalent binding with cross - linking. Tannase immobilized on gelatin with cross - linking agent showed the highest activity and immobilization yield. The optimum pH of the immobilized enzyme was shifted to a more acidic range compared with the free enzyme (from pH 5.5 to pH 5.0. The optimum temperature of the reaction was determined to be 50ºC for the free enzyme and 60ºC for the immobilized form. The thermal stability, as well as stability over a wide range of pH, was significantly improved by the immobilization process. The calculated Km of the immobilized tannase (11.8 mg ml-1 is higher than that of the free tannase (6.5 mg ml-1, while Vmax of the immobilized enzyme (0.32 U (µg protein-1 is lower than that of the free tannase (2.7 U (µg protein-1. The immobilized enzyme was able to retain 84 % of the initial catalytic activity after 5.0 cycles.

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

Science.gov (United States)

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

2018-04-01

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

Immobilization of enzyme and antibody by low energy electron beam polymerization

International Nuclear Information System (INIS)

Kaetsu, Isao; Kumakura, Minoru

1987-01-01

Immobilization of glucoamylase and AFP-antibody was studied using an electron beam of relatively low energy. A thin polymer membrane formed by irradiation of monomer enzyme mixture in a buffer, which had a considerable enzymatic activity. A membrane of almost the same thickness and activity was obtained by repeated irradiation. The effect of irradiation conditions on the immobilization and the variations of irradiation method for immobilization were investigated. The immobilization of antibody was carried out in similar ways as for enzyme, and the product also showed a considerable activity. (author)

Acetylcholinesterase immobilization and characterization, and comparison of the activity of the porous silicon-immobilized enzyme with its free counterpart.

Science.gov (United States)

Saleem, Muhammad; Rafiq, Muhammad; Seo, Sung-Yum; Lee, Ki Hwan

2016-02-02

A successful prescription is presented for acetylcholinesterase physically adsorbed on to a mesoporous silicon surface, with a promising hydrolytic response towards acetylthiocholine iodide. The catalytic behaviour of the immobilized enzyme was assessed by spectrophotometric bioassay using neostigmine methyl sulfate as a standard acetycholinesterase inhibitor. The surface modification was studied through field emission SEM, Fourier transform IR spectroscopy, energy-dispersive X-ray spectroscopy, cathode luminescence and X-ray photoelectron spectroscopy analysis, photoluminescence measurement and spectrophotometric bioassay. The porous silicon-immobilized enzyme not only yielded greater enzyme stability, but also significantly improved the native photoluminescence at room temperature of the bare porous silicon architecture. The results indicated the promising catalytic behaviour of immobilized enzyme compared with that of its free counterpart, with a greater stability, and that it aided reusability and easy separation from the reaction mixture. The porous silicon-immobilized enzyme was found to retain 50% of its activity, promising thermal stability up to 90°C, reusability for up to three cycles, pH stability over a broad pH of 4-9 and a shelf-life of 44 days, with an optimal hydrolytic response towards acetylthiocholine iodide at variable drug concentrations. On the basis of these findings, it was believed that the porous silicon-immobilized enzyme could be exploited as a reusable biocatalyst and for screening of acetylcholinesterase inhibitors from crude plant extracts and synthesized organic compounds. Moreover, the immobilized enzyme could offer a great deal as a viable biocatalyst in bioprocessing for the chemical and pharmaceutical industries, and bioremediation to enhance productivity and robustness. © 2016 Authors.

Application of radiopolymerization for immobilization of enzymes

International Nuclear Information System (INIS)

Higa, O.Z.; Mastro, N.L. del; Castagnet, A.C.G.

1986-01-01

Hydrophilic glass-forming monomers were used in an application of irradiation technology for the immobilization of cellulase and cellobiase. Experiments to observe the effect of additives such as silicates and polyethylene glycol in the enzyme entrapment are reported on. In all cases, enzymatic activity was maintained for more than fifteen batch enzyme reactions. (Author) [pt

Co-immobilized Coupled Enzyme Systems in Biotechnology

Science.gov (United States)

2010-01-01

coimmobilized by ~n­ capsulation in silica spheres that were formed by a polymer -templated silicificatiOn reaction (Betancor et al., 2006). Nitrobenzene...F. , FERNANDEZ-LAFUENTE, R. , GUISAN J. M. (2005). Stabilization of enzymes by multipoint immobilization of thiolated proteins on new epoxy-thiol... polymer monoliths in microftuidic devices for steady- state kinetic analysis and spatially separated multi-enzyme reactions. Analytical Chemistry, 79

Enzyme Immobilization on Inorganic Surfaces for Membrane Reactor Applications: Mass Transfer Challenges, Enzyme Leakage and Reuse of Materials

DEFF Research Database (Denmark)

Sigurdardóttir, Sigyn Björk; Lehmann, Jonas; Ovtar, Simona

2018-01-01

Enzyme immobilization is an established method for the enhancement of enzyme stability and reusability, two factors that are of great importance for industrial biocatalytic applications. Immobilization can be achieved by different methods and on a variety of carrier materials, both organic and in...

Effect of exogenous cellulase enzyme on feed digestibility in lamb

International Nuclear Information System (INIS)

Boonek, Lerchat; Shinkoi, Henrry S; Piadang, Nattayana

2006-09-01

The aim of this study was to determine the effect of exogenous enzyme on digestibility and N retention in lamb. Eight lambs were randomly allocated to 2 experiment group in group comparison design trial. Experimental treatments were: 1) CTL (No enzyme) and 2 50NZ (Mixed enzyme with high cellulase at 50g/100kg.feed). The digestibility study showed that Exogenous enzyme increased (P<0.05) dry matter and crude protein digestibility of treated lamb compared to those of control. A similar trend (P=0.11) was observed for the NDF digestibility. Mean values for dry matter digestibility were 57.86 and 69.83% and for protein digestibility were 64.76 and 73.38%, for CTL and 50NZ, respectively). The N intake was similar among treatment, averaging 22.57g/head/day. Percent N retained of 50 NZ treated lambs was higher (P<.05) than those of CTL group (mean value were 47.74 and 59.07 for CTC and 50NZ, respectively). Feed efficiency or feed conversion ratio was numerically improved for enzyme-treated groups. Overall, the results of this study provide evidence that mixed cellulase enzyme can be used to improver performance of lambs as compare to non-enzyme diet.

From Protein Engineering to Immobilization: Promising Strategies for the Upgrade of Industrial Enzymes

Science.gov (United States)

Singh, Raushan Kumar; Tiwari, Manish Kumar; Singh, Ranjitha; Lee, Jung-Kul

2013-01-01

Enzymes found in nature have been exploited in industry due to their inherent catalytic properties in complex chemical processes under mild experimental and environmental conditions. The desired industrial goal is often difficult to achieve using the native form of the enzyme. Recent developments in protein engineering have revolutionized the development of commercially available enzymes into better industrial catalysts. Protein engineering aims at modifying the sequence of a protein, and hence its structure, to create enzymes with improved functional properties such as stability, specific activity, inhibition by reaction products, and selectivity towards non-natural substrates. Soluble enzymes are often immobilized onto solid insoluble supports to be reused in continuous processes and to facilitate the economical recovery of the enzyme after the reaction without any significant loss to its biochemical properties. Immobilization confers considerable stability towards temperature variations and organic solvents. Multipoint and multisubunit covalent attachments of enzymes on appropriately functionalized supports via linkers provide rigidity to the immobilized enzyme structure, ultimately resulting in improved enzyme stability. Protein engineering and immobilization techniques are sequential and compatible approaches for the improvement of enzyme properties. The present review highlights and summarizes various studies that have aimed to improve the biochemical properties of industrially significant enzymes. PMID:23306150

From protein engineering to immobilization: promising strategies for the upgrade of industrial enzymes.

Science.gov (United States)

Singh, Raushan Kumar; Tiwari, Manish Kumar; Singh, Ranjitha; Lee, Jung-Kul

2013-01-10

Enzymes found in nature have been exploited in industry due to their inherent catalytic properties in complex chemical processes under mild experimental and environmental conditions. The desired industrial goal is often difficult to achieve using the native form of the enzyme. Recent developments in protein engineering have revolutionized the development of commercially available enzymes into better industrial catalysts. Protein engineering aims at modifying the sequence of a protein, and hence its structure, to create enzymes with improved functional properties such as stability, specific activity, inhibition by reaction products, and selectivity towards non-natural substrates. Soluble enzymes are often immobilized onto solid insoluble supports to be reused in continuous processes and to facilitate the economical recovery of the enzyme after the reaction without any significant loss to its biochemical properties. Immobilization confers considerable stability towards temperature variations and organic solvents. Multipoint and multisubunit covalent attachments of enzymes on appropriately functionalized supports via linkers provide rigidity to the immobilized enzyme structure, ultimately resulting in improved enzyme stability. Protein engineering and immobilization techniques are sequential and compatible approaches for the improvement of enzyme properties. The present review highlights and summarizes various studies that have aimed to improve the biochemical properties of industrially significant enzymes.

Enhancing the functional properties of thermophilic enzymes by chemical modification and immobilization.

Science.gov (United States)

Cowan, Don A; Fernandez-Lafuente, Roberto

2011-09-10

The immobilization of proteins (mostly typically enzymes) onto solid supports is mature technology and has been used successfully to enhance biocatalytic processes in a wide range of industrial applications. However, continued developments in immobilization technology have led to more sophisticated and specialized applications of the process. A combination of targeted chemistries, for both the support and the protein, sometimes in combination with additional chemical and/or genetic engineering, has led to the development of methods for the modification of protein functional properties, for enhancing protein stability and for the recovery of specific proteins from complex mixtures. In particular, the development of effective methods for immobilizing large multi-subunit proteins with multiple covalent linkages (multi-point immobilization) has been effective in stabilizing proteins where subunit dissociation is the initial step in enzyme inactivation. In some instances, multiple benefits are achievable in a single process. Here we comprehensively review the literature pertaining to immobilization and chemical modification of different enzyme classes from thermophiles, with emphasis on the chemistries involved and their implications for modification of the enzyme functional properties. We also highlight the potential for synergies in the combined use of immobilization and other chemical modifications. Copyright © 2011 Elsevier Inc. All rights reserved.

Immobilized enzyme studies in a microscale bioreactor.

Science.gov (United States)

Jones, Francis; Forrest, Scott; Palmer, Jim; Lu, Zonghuan; Elmore, John; Elmore, Bill B

2004-01-01

Novel microreactors with immobilized enzymes were fabricated using both silicon and polymer-based microfabrication techniques. The effectiveness of these reactors was examined along with their behavior over time. Urease enzyme was successfully incorporated into microchannels of a polymeric matrix of polydimethylsiloxane and through layer-bylayer self-assembly techniques onto silicon. The fabricated microchannels had cross-sectional dimensions ranging from tens to hundreds of micrometers in width and height. The experimental results for continuous-flow microreactors are reported for the conversion of urea to ammonia by urease enzyme. Urea conversions of >90% were observed.

Development Of Enzyme Digestive Activity Of Blue Crab Portunus Pelagicus Larvae

OpenAIRE

Nikhlani, Andi; Sukarti, Komsanah

2017-01-01

Seed production continuity of Portunus pelagicus larvae had been conducted but the results were still un-consistent Digestive activity was known to be associated with the type of feed consumed by larvae. Amylase, lipase, and trypsin enzymes were used as a biological indicators to measure the digestion of feed. The aim of this study was to describe the activity of digestive enzymes in blue swimming crab larvae. Digestive enzyme activity data obtained was presented in graphical form and anal...

Polyketone polymer: a new support for direct enzyme immobilization.

Science.gov (United States)

Agostinelli, E; Belli, F; Tempera, G; Mura, A; Floris, G; Toniolo, L; Vavasori, A; Fabris, S; Momo, F; Stevanato, R

2007-01-20

Polyketone polymer -[-CO-CH(2)-CH(2)-](n)-, obtained by copolymerization of ethene and carbon monoxide, is utilized for immobilization of three different enzymes, one peroxidase from horseradish (HRP) and two amine oxidases, from bovine serum (BSAO) and lentil seedlings (LSAO). The easy immobilization procedure is carried out in diluted buffer, at pH 7.0 and 3 degrees C, gently mixing the proteins with the polymer. No bifunctional reagents and spacer arms are required for the immobilization, which occurs exclusively via a large number of hydrogen bonds between the carbonyl groups of the polymer and the -NH groups of the polypeptidic chain. Experiments demonstrate a high linking capacity of polymer for BSAO and an extraordinary strong linkage for LSAO. Moreover, activity measurements demonstrate that immobilized LSAO totally retains the catalytic characteristics of the free enzyme, where only a limited increase of K(M) value is observed. Finally, the HRP-activated polymer is successfully used as active packed bed of an enzymatic reactor for continuous flow conversion and flow injection analysis of hydrogen peroxide containing solutions.

Development of a thiol-ene based screening platform for enzyme immobilization demonstrated using horseradish peroxidase

DEFF Research Database (Denmark)

Hoffmann, Christian; Pinelo, Manuel; Woodley, John

2017-01-01

Efficient immobilization of enzymes on support surfaces requires an exact match between the surface chemistry and the specific enzyme. A successful match would normally be identified through time consuming screening of conventional resins in multiple experiments testing individual immobilization...... strategies. In this study we present a versatile strategy that largely expands the number of possible surface functionalities for enzyme immobilization in a single, generic platform. The combination of many individual surface chemistries and thus immobilization methods in one modular system permits faster...... functionalization by thiol-ene chemistry (TEC) resulted in the formation of a functional monolayer in each well, whereas, polymer surface grafts were introduced through surface chain transfer free radical polymerization (SCT-FRP). Enzyme immobilization on the modified surfaces was evaluated by using a rhodamine...

Immobilization of anaerobic bacteria on rubberized-coir for psychrophilic digestion of night soil.

Science.gov (United States)

Dhaked, Ram Kumar; Ramana, Karna Venkat; Tomar, Arvind; Waghmare, Chandrakant; Kamboj, Dev Vrat; Singh, Lokendra

2005-08-01

Low-ambient temperatures, biodigesters due to low-growth rate of the constituent bacterial consortium. Immobilization of anaerobic bacteria has been attempted in the biodigester operating at 10 degrees C. Various matrices were screened and evaluated for the immobilization of bacteria in digesters. Anaerobic digestion of night soil was carried out with hydraulic retention time in the range of 9-18 days. Among the tested matrices, rubberized-coir was found to be the most useful at 10 degrees C with optimum hydraulic retention time of 15 days. Optimum amount of coir was found as 25 g/L of the working volume of biodigesters. Immobilization of bacteria on the coir was observed by scanning electron microscopy and fluorescent microscopy. The study indicates that rubberized-coir can be utilized to increase biodegradation of night soil at higher organic loading. Another advantage of using this matrix is that it is renewable and easily available in comparison to other synthetic polymeric matrices.

Enzyme immobilization and biocatalysis of polysiloxanes

Science.gov (United States)

Poojari, Yadagiri

Lipases have been proven to be versatile and efficient biocatalysts which can be used in a broad variety of esterification, transesterification, and ester hydrolysis reactions. Due to the high chemo-, regio-, and stereo-selectivity and the mild conditions of lipase-catalyzed reactions, the vast potential of these biocatalysts for use in industrial applications has been increasingly recognized. Polysiloxanes (silicones) are well known for their unique physico-chemical properties and can be prepared in the form of fluids, elastomers, gels and resins for a wide variety of applications. However, the enzymatic synthesis of silicone polyesters and copolymers is largely unexplored. In the present investigations, an immobilized Candida antarctica lipase B (CALB) on macroporous acrylic resin beads (Novozym-435 RTM) has been successfully employed as a catalyst to synthesize silicone polyesters and copolymers under mild reaction conditions. The silicone aliphatic polyesters and the poly(dimethylsiloxane)--poly(ethylene glycol) (PDMS-PEG) copolymers were synthesized in the bulk (without using a solvent), while the silicone aromatic polyesters, the silicone aromatic polyamides and the poly(epsilon-caprolactone)--poly(dimethylsiloxane)--poly(epsilon-caprolactone) (PCL-PDMS-PCL) triblock copolymers were synthesized in toluene. The synthesized silicone polyesters and copolymers were characterized by Gel Permeation Chromatography (GPC), Fourier Transform Infrared Spectroscopy (FTIR), Thermogravimetric Analysis (TGA), Differential Scanning Calorimetry (DSC) and Wide Angle X-ray Diffraction (WAXD). This dissertation also describes a methodology for physical immobilization of the enzyme pepsin from Porcine stomach mucosa in silicone elastomers utilizing condensation-cure room temperature vulcanization (RTV) of silanol-terminated poly(dimethylsiloxane) (PDMS). The activity and the stability of free pepsin and pepsin immobilized in silicone elastomers were studied with respect to p

Immobilization of leachable toxic soil pollutants by using oxidative enzymes

International Nuclear Information System (INIS)

Shannon, M.J.R.; Bartha, R.

1988-01-01

Screening of leachable toxic chemicals in a horseradish peroxidase-H 2 O 2 immobilization system established that immobilization was promising for most phenolic pollutants but not for benzoic acid, 2,6-dinitrocresol, or dibutyl phthalate. The treatment did not mobilize inherently nonmobile pollutants such as anilines and benzo[a]pyrene. In a separate study, an extracellular laccase in the culture filtrate of Geotrichum candidum was selected from five fungal enzymes evaluated as a cost-effective substitute for horseradish peroxidase. This enzyme was used in demonstrating the immobilization and subsequent fate of 14 C-labeled 4-methylphenol and 2,4-dichlorophenol in soil columns. When applied to Lakewood sand, 98.1% of 4-methylpheno was leached through with distilled water. Two days after immobilization treatment with the G. candidum culture filtrate, only 9.1% of the added 4-methylphenol was leached with the same volume of water. Of the more refractory test pollutant 2,4-dichlorophenol, 91.6% had leached at time zero and 48.5% had leached 1 day after the immobilization treatment. However, 2 weeks after immobilization, only 12.0% of the 2,4-dichlorophenol was leached compared with 61.7% from the control column that received no immobilization treatment. No remobilization of the bound pollutants was detected during 3- and 4-week incubation periods

Integrating enzyme immobilization and protein engineering: An alternative path for the development of novel and improved industrial biocatalysts.

Science.gov (United States)

Bernal, Claudia; Rodríguez, Karen; Martínez, Ronny

2018-06-09

Enzyme immobilization often achieves reusable biocatalysts with improved operational stability and solvent resistance. However, these modifications are generally associated with a decrease in activity or detrimental modifications in catalytic properties. On the other hand, protein engineering aims to generate enzymes with increased performance at specific conditions by means of genetic manipulation, directed evolution and rational design. However, the achieved biocatalysts are generally generated as soluble enzymes, -thus not reusable- and their performance under real operational conditions is uncertain. Combined protein engineering and enzyme immobilization approaches have been employed as parallel or consecutive strategies for improving an enzyme of interest. Recent reports show efforts on simultaneously improving both enzymatic and immobilization components through genetic modification of enzymes and optimizing binding chemistry for site-specific and oriented immobilization. Nonetheless, enzyme engineering and immobilization are usually performed as separate workflows to achieve improved biocatalysts. In this review, we summarize and discuss recent research aiming to integrate enzyme immobilization and protein engineering and propose strategies to further converge protein engineering and enzyme immobilization efforts into a novel "immobilized biocatalyst engineering" research field. We believe that through the integration of both enzyme engineering and enzyme immobilization strategies, novel biocatalysts can be obtained, not only as the sum of independently improved intrinsic and operational properties of enzymes, but ultimately tailored specifically for increased performance as immobilized biocatalysts, potentially paving the way for a qualitative jump in the development of efficient, stable biocatalysts with greater real-world potential in challenging bioprocess applications. Copyright © 2018. Published by Elsevier Inc.

Enzyme-immobilized SiO2-Si electrode: Fast interfacial electron transfer with preserved enzymatic activity

Science.gov (United States)

Wang, Gang; Yau, Siu-Tung

2005-12-01

The enzyme, glucose oxidase (GOx), is immobilized using electrostatic interaction on the native oxide of heavily doped n-type silicon. Voltammetric measurement shows that the immobilized GOx gives rise to a very fast enzyme-silicon interfacial electron transfer rate constant of 7.9s-1. The measurement also suggests that the enzyme retains its native conformation when immobilized on the silicon surface. The preserved native conformation of GOx is further confirmed by testing the enzymatic activity of the immobilized GOx using glucose. The GOx-immobilized silicon is shown to behave as a glucose sensor that detects glucose with concentrations as low as 50μM.

Highly Stable Trypsin-Aggregate Coatings on Polymer Nanofibers for Repeated Protein Digestion

Energy Technology Data Exchange (ETDEWEB)

Kim, Byoung Chan; Lopez-Ferrer, Daniel; Lee, Sang-mok; Ahn, Hye-kyung; Nair, Sujith; Kim, Seong H.; Kim, Beom S.; Petritis, Konstantinos; Camp, David G.; Grate, Jay W.; Smith, Richard D.; Koo, Yoon-mo; Gu, Man Bock; Kim, Jungbae

2009-04-01

A stable and robust trypsin-based biocatalytic system was developed and demonstrated for proteomic applications. The system utilizes polymer nanofibers coated with trypsin aggregates for immobilized protease digestions. After covalently attaching an initial layer of trypsin to the polymer nanofibers, highly concentrated trypsin molecules are crosslinked to the layered trypsin by way of a glutaraldehyde treatment. This new process produced a 300-fold increase in trypsin activity compared with a conventional method for covalent trypsin immobilization and proved to be robust in that it still maintained a high level of activity after a year of repeated recycling. This highly stable form of immobilized trypsin was also resistant to autolysis, enabling repeated digestions of bovine serum albumin over 40 days and successful peptide identification by LC-MS/MS. Finally, the immobilized trypsin was resistant to proteolysis when exposed to other enzymes (i.e. chymotrypsin), which makes it suitable for use in “real-world” proteomic applications. Overall, the biocatalytic nanofibers with enzyme aggregate coatings proved to be an effective approach for repeated and automated protein digestion in proteomic analyses.

Chronic ethanol feeding modulates the synthesis of digestive enzymes

International Nuclear Information System (INIS)

Ponnappa, B.C.; Hoek, J.B.; Rubin, E.

1987-01-01

The effects of chronic ethanol feeding on pancreatic protein synthesis were investigated. Protein synthesis was assessed by studying the rate of incorporation of 3 H-leucine into TCA-precipitable proteins in isolated pancreatic acini from rats. Chronic ethanol ingestion increased the rate of pancreatic protein synthesis by 2-4 fold. The onset of the increase in protein synthesis was detectable two days after ethanol feeding, reached a maximum after 7 days and remained unchanged after 4 months on the ethanol-containing diet. The rate of synthesis of individual digestive enzymes was studied by SDS-PAGE on extracts obtained from purified zymogen granules. Ethanol feeding induced an increase in the rate of synthesis of most of the digestive enzymes; chymotrypsinogen, trypsinogen and an unidentified protein were increased to a greater extent than other digestive enzymes. By contrast, the synthesis of amylase was selectively decreased after ethanol feeding. These results suggest that chronic ethanol ingestion has specific effects on the rate of synthesis of individual digestive enzymes in the exocrine pancreas

Immobilization of trypsin on miniature incandescent bulbs for infrared-assisted proteolysis

Energy Technology Data Exchange (ETDEWEB)

Ge, Huimin; Bao, Huimin; Zhang, Luyan; Chen, Gang, E-mail: gangchen@fudan.edu.cn

2014-10-03

Highlights: • Trypsin was immobilized on miniature incandescent bulbs via chitosan coating. • The bulbs acted as enzymatic reactors and the generators of infrared radiation. • The bulb bioreactors were successfully employed in infrared-assisted proteolysis. • The proteolysis could accomplish within 5 min with high sequence coverages. - Abstract: A novel efficient proteolysis approach was developed based on trypsin-immobilized miniature incandescent bulbs and infrared (IR) radiation. Trypsin was covalently immobilized in the chitosan coating on the outer surface of miniature incandescent bulbs with the aid of glutaraldehyde. When an illuminated enzyme-immobilized bulb was immersed in protein solution, the emitted IR radiation could trigger and accelerate heterogeneous protein digestion. The feasibility and performance of the novel proteolysis approach were demonstrated by the digestion of hemoglobin (HEM), cytochrome c (Cyt-c), lysozyme (LYS), and ovalbumin (OVA) and the digestion time was significantly reduced to 5 min. The obtained digests were identified by MALDI-TOF-MS with the sequence coverages of 91%, 77%, 80%, and 52% for HEM, Cyt-c, LYS, and OVA (200 ng μL{sup −1} each), respectively. The suitability of the prepared bulb bioreactors to complex proteins was demonstrated by digesting human serum.

Strategies for an enzyme immobilization on electrodes: Structural and electrochemical characterizations

Science.gov (United States)

Ganesh, V.; Muthurasu, A.

2012-04-01

In this paper, we propose various strategies for an enzyme immobilization on electrodes (both metal and semiconductor electrodes). In general, the proposed methodology involves two critical steps viz., (1) chemical modification of substrates using functional monolayers [Langmuir - Blodgett (LB) films and/or self-assembled monolayers (SAMs)] and (2) anchoring of a target enzyme using specific chemical and physical interactions by attacking the terminal functionality of the modified films. Basically there are three ways to immobilize an enzyme on chemically modified electrodes. First method consists of an electrostatic interaction between the enzyme and terminal functional groups present within the chemically modified films. Second and third methods involve the introduction of nanomaterials followed by an enzyme immobilization using both the physical and chemical adsorption processes. As a proof of principle, in this work we demonstrate the sensing and catalytic activity of horseradish peroxidase (HRP) anchored onto SAM modified indium tin oxide (ITO) electrodes towards hydrogen peroxide (H2O2). Structural characterization of such modified electrodes is performed using X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM) and contact angle measurements. The binding events and the enzymatic reactions are monitored using electrochemical techniques mainly cyclic voltammetry (CV).

Preparation of immobilized enzyme membrane by radiation-cast-polymerization

International Nuclear Information System (INIS)

Kumakura, M.; Kaetsu, I.

1989-01-01

The preparation of immobilized enzyme membranes was studied by radiation cast-polymerization at low temperatures using cellulase enzyme, hydrophilic and hydrophobic monomers. The enzyme activity of the membranes was affected by monomer concentration, membrane thickness, and hydrophilicity of monomer, in which the membranes with 100 μm thickness from high monomer concentration (80%) had high enzyme activity, which was similar to that of the membranes with 1.0 mm thickness from low monomer concentration (20%). (author)

Enzyme Technology of Peroxidases: Immobilization, Chemical and Genetic Modification

Science.gov (United States)

Longoria, Adriana; Tinoco, Raunel; Torres, Eduardo

An overview of enzyme technology applied to peroxidases is made. Immobilization on organic, inorganic, and hybrid supports; chemical modification of amino acids and heme group; and genetic modification by site-directed and random mutagenesis are included. Different strategies that were carried out to improve peroxidase performance in terms of stability, selectivity, and catalytic activity are analyzed. Immobilization of peroxidases on inorganic and organic materials enhances the tolerance of peroxidases toward the conditions normally found in many industrial processes, such as the presence of an organic solvent and high temperature. In addition, it is shown that immobilization helps to increase the Total Turnover Number at levels high enough to justify the use of a peroxidase-based biocatalyst in a synthesis process. Chemical modification of peroxidases produces modified enzymes with higher thermostability and wider substrate variability. Finally, through mutagenesis approaches, it is possible to produce modified peroxidases capable of oxidizing nonnatural substrates with high catalytic activity and affinity.

Preparation of polymer brushes grafted graphene oxide by atom transfer radical polymerization as a new support for trypsin immobilization and efficient proteome digestion.

Science.gov (United States)

Guo, Cong; Zhao, Xinyuan; Zhang, Wanjun; Bai, Haihong; Qin, Weijie; Song, Haifeng; Qian, Xiaohong

2017-08-01

Highly efficient protein digestion is one of the key issues in the "bottom-up" strategy-based proteomic studies. Compared with the time-consuming solution-based free protease digestion, immobilized protease digestion offers a promising alternative with obviously improved sample processing throughput. In this study, we proposed a new immobilized protease digestion strategy using two kinds of polymer-grafted graphene oxide (GO) conjugated trypsin. The polymer brush grafted GO was prepared using in situ polymer growth on initiator-functionalized GO using surface-initiated atom transfer radical polymerization (SI-ATRP) and characterized by AFM, TEM, TGA, and XPS. The polymer brush grafted GO supports three-dimensional trypsin immobilization, which not only increases the loading amount but also improves accessibility towards protein substrates. Both of the two types of immobilized trypsin provide 700 times shorter digestion time, while maintaining comparable protein/peptide identification scale compared with that of free trypsin digestion. More interestingly, combined application of the two types of immobilized trypsin with different surface-grafted polymers leads to at least 18.3/31.3% enhancement in protein/peptide identification compared with that obtained by digestion using a single type, indicating the potential of this digestion strategy for deeper proteome coverage using limited mass spectrometer machine hour. We expect these advantages may find valuable application in high throughput clinical proteomic studies, which often involve processing of a large number of samples. Graphical abstract Preparation of polymer brushes grafted and trypsin immobilized graphene oxide and its application in proteome digestion and mass spectrometry identification.

Immobilization of cross-linked tannase enzyme on multiwalled carbon nanotubes and its catalytic behavior.

Science.gov (United States)

Ong, Chong-Boon; Annuar, Mohamad S M

2018-02-07

Immobilization of cross-linked tannase on pristine multiwalled carbon nanotubes (MWCNT) was successfully performed. Cross-linking of tannase molecules was made through glutaraldehyde. The immobilized tannase exhibited significantly improved pH, thermal, and recycling stability. The optimal pH for both free and immobilized tannase was observed at pH 5.0 with optimal operating temperature at 30°C. Moreover, immobilized enzyme retained greater biocatalytic activities upon 10 repeated uses compared to free enzyme in solution. Immobilization of tannase was accomplished by strong hydrophobic interaction most likely between hydrophobic amino acid moieties of the glutaraldehyde-cross-linked tannase to the MWCNT.

Investigation of the Effect of Plasma Polymerized Siloxane Coating for Enzyme Immobilization and Microfluidic Device Conception

Directory of Open Access Journals (Sweden)

Kalim Belhacene

2016-12-01

Full Text Available This paper describes the impact of a physical immobilization methodology, using plasma polymerized 1,1,3,3, tetramethyldisiloxane, on the catalytic performance of β-galactosidase from Aspergillus oryzae in a microfluidic device. The β-galactosidase was immobilized by a polymer coating grown by Plasma Enhanced Chemical Vapor Deposition (PEVCD. Combined with a microchannel patterned in the silicone, a microreactor was obtained with which the diffusion through the plasma polymerized layer and the hydrolysis of a synthetic substrate, the resorufin-β-d-galactopyranoside, were studied. A study of the efficiency of the immobilization procedure was investigated after several uses and kinetic parameters of immobilized β-galactosidase were calculated and compared with those of soluble enzyme. Simulation and a modelling approach were also initiated to understand phenomena that influenced enzyme behavior in the physical immobilization method. Thus, the catalytic performances of immobilized enzymes were directly influenced by immobilization conditions and particularly by the diffusion behavior and availability of substrate molecules in the enzyme microenvironment.

Effect of enzyme supplements on macronutrient digestibility by healthy adult dogs.

Science.gov (United States)

Villaverde, Cecilia; Manzanilla, Edgar G; Molina, Jenifer; Larsen, Jennifer A

2017-01-01

Some enzyme supplement products claim benefits for healthy dogs to compensate for alleged suboptimal production of endogenous enzymes and the loss of enzymes in commercial pet foods secondary to processing. The objective of the current study was to determine macronutrient and energy digestibility by healthy adult dogs fed a commercial maintenance diet with or without supplementation with plant- and animal-origin enzyme products at the dosage recommended by their respective manufacturers. A group of fourteen healthy neutered adult Beagle dogs (average age 8 years) was divided into two equal groups and fed the basal diet alone and then with either the plant- or animal-origin enzyme supplement in three consecutive 10-d periods; the treatment groups received the opposite enzyme supplement in the third period. Digestibility in each period was performed by the total faecal collection method. Serum trypsin-like immunoreactivity (TLI) was measured at the end of each trial. Data were analysed by repeated measures and the α level of significance was set at 0·05. There were no differences in energy and nutrient digestibility between enzyme treatments. When comparing basal with enzyme supplementation, fat digestibility was higher for the basal diet compared with the animal-origin enzyme treatment, which could be a period effect and was not biologically significant (94·7 v . 93·5 %). Serum TLI was not affected by supplementation with either enzyme product. Exogenous enzyme supplementation did not significantly increase digestibility of a typical commercial dry diet in healthy adult dogs and routine use of such products is not recommended.

Inorganic Materials as Supports for Covalent Enzyme Immobilization: Methods and Mechanisms

Directory of Open Access Journals (Sweden)

Paolo Zucca

2014-09-01

Full Text Available Several inorganic materials are potentially suitable for enzymatic covalent immobilization, by means of several different techniques. Such materials must meet stringent criteria to be suitable as solid matrices: complete insolubility in water, reasonable mechanical strength and chemical resistance under the operational conditions, the capability to form manageable particles with high surface area, reactivity towards derivatizing/functionalizing agents. Non-specific protein adsorption should be always considered when planning covalent immobilization on inorganic solids. A huge mass of experimental work has shown that silica, silicates, borosilicates and aluminosilicates, alumina, titania, and other oxides, are the materials of choice when attempting enzyme immobilizations on inorganic supports. More recently, some forms of elemental carbon, silicon, and certain metals have been also proposed for certain applications. With regard to the derivatization/functionalization techniques, the use of organosilanes through silanization is undoubtedly the most studied and the most applied, although inorganic bridge formation and acylation with selected acyl halides have been deeply studied. In the present article, the most common inorganic supports for covalent immobilization of the enzymes are reviewed, with particular focus on their advantages and disadvantages in terms of enzyme loadings, operational stability, undesired adsorption, and costs. Mechanisms and methods for covalent immobilization are also discussed, focusing on the most widespread activating approaches (such as glutaraldehyde, cyanogen bromide, divinylsulfone, carbodiimides, carbonyldiimidazole, sulfonyl chlorides, chlorocarbonates, N-hydroxysuccinimides.

Preliminary characterization of digestive enzymes in freshwater mussels

Science.gov (United States)

Sauey, Blake W.; Amberg, Jon J.; Cooper, Scott T.; Grunwald, Sandra K.; Newton, Teresa J.; Haro, Roger J.

2015-01-01

Resource managers lack an effective chemical tool to control the invasive zebra mussel Dreissena polymorpha. Zebra mussels clog water intakes for hydroelectric companies, harm unionid mussel species, and are believed to be a reservoir of avian botulism. Little is known about the digestive physiology of zebra mussels and unionid mussels. The enzymatic profile of the digestive glands of zebra mussels and native threeridge (Amblema plicata) and plain pocketbook mussels (Lampsilis cardium) are characterized using a commercial enzyme kit, api ZYM, and validated the kit with reagent-grade enzymes. A linear correlation was shown for only one of nineteen enzymes, tested between the api ZYM kit and a specific enzyme kit. Thus, the api ZYM kit should only be used to make general comparisons of enzyme presence and to observe trends in enzyme activities. Enzymatic trends were seen in the unionid mussel species, but not in zebra mussels sampled 32 days apart from the same location. Enzymatic classes, based on substrate, showed different trends, with proteolytic and phospholytic enzymes having the most change in relative enzyme activity.

Effect of temperature and mixing speed on immobilization of crude enzyme from Aspergillus niger on chitosan for hydrolyzing cellulose

Science.gov (United States)

Hamzah, Afan; Gek Ela Kumala, P.; Ramadhani, Dwi; Maziyah, Nurul; Rahmah, Laila Nur; Soeprijanto, Widjaja, Arief

2017-05-01

Conversion of cellulose into reducing sugar through enzymatic hydrolysis has advantageous because it produces greater product yield, higher selectivity, require less energy, more moderate operating conditions and environment friendly. However, the nature of the enzyme that is difficult to separate and its expensive price become an obstacle. These obstacles can be overcome by immobilizing the enzyme on chitosan material so that the enzyme can be reused. Chitosan is chosen because it is cheap, inert, hydrophilic, and biocompatible. In this research, we use covalent attachment and combination between covalent attachment and cross-linking method for immobilizing crude enzyme. This research was focusing in study of Effect of temperature and mixing speed on Immobilization Enzyme From Aspergillus Niger on Chitosan For Hydrolyzing both soluble (Carboxymethylcellulose) and insoluble Cellulose (coconut husk). This Research was carried out by three main step. First, coconut husk was pre-treated mechanically and chemically, Second, Crude enzyme from Aspergillus niger strain was immobilized on chitosan in various immobilization condition. At last, the pre-treated coconut husk and Carboxymetylcellulose (CMC) were hydrolyzed by immobilized cellulose on chitosan for reducing sugar production. The result revealed that the most reducing sugar produced by immobilized enzyme on chitosan+GDA with immobilization condition at 30 °C and 125 rpm. Enzyme immobilized on chitosan cross-linked with GDA produced more reducing sugar from preteated coconut husk than enzyme immobilized on chitosan.

Drying of enzyme immobilized on eco-friendly supports | Costa-Silva ...

African Journals Online (AJOL)

Immobilized derivatives obtained had decreased enzyme activity (≈ 30.0%) during a storage period of six months; and retained an average of 50.0% of the initial activity after five reuse cycles. Water content in immobilized derivatives varied between 4.2 and 6.1% and the water activities ranged from 0.14 to 0.30. Key words: ...

Magnetic cross-linked enzyme aggregates (CLEAs): a novel concept towards carrier free immobilization of lignocellulolytic enzymes.

Science.gov (United States)

Bhattacharya, Abhishek; Pletschke, Brett I

2014-01-01

The enzymatic conversion of lignocellulosic biomass into biofuels has been identified as an excellent strategy to generate clean energy. However, the current process is cost-intensive as an effective immobilization approach to reuse the enzyme(s) has been a major challenge. The present study introduces the concept and application of novel magnetic cross-linked enzyme aggregates (mag-CLEAs). Both mag-CLEAs and calcium-mag-CLEAs (Ca-mag-CLEAs) exhibited a 1.35 fold higher xylanase activity compared to the free enzyme and retained more than 80.0% and 90.0% activity, respectively, after 136h of incubation at 50°C, compared to 50% activity retained by CLEAs. A 7.4 and 9.0 fold higher sugar release from lime-pretreated and NH4OH pre-treated sugar bagasse, respectively, was achieved with Ca-mag-CLEAs compared to the free enzymes. The present study promotes the successful application of mag-CLEAs and Ca-mag-CLEAs as carrier free immobilized enzymes for the effective hydrolysis of lignocellulolytic biomass and associated biofuel feedstocks. Copyright © 2014 Elsevier Inc. All rights reserved.

Synthesis of magnetic thermosensitive microcontainers for enzyme immobilization

Energy Technology Data Exchange (ETDEWEB)

Wang, Jianzhi; Zhao, Guanghui, E-mail: zhaogh@lzu.edu.cn; Wang, Xinyu, E-mail: wangxy08@lzu.cn; Peng, Xiaomen; Li, Yanfeng, E-mail: liyf@lzu.edu.cn [Lanzhou University, State Key Laboratory of Applied Organic Chemistry, Institute of Biochemical Engineering & Environmental Technology, College of Chemistry and Chemical Engineering (China)

2015-05-15

We present a new approach for the fabrication of magnetic thermoresponsive polymer microcapsules with mobile magnetic spherical cores. The microcontainers form fried-egg-like structures with a polymer shell layer of 50 nm due to the existence of hollow cavities. The microcontainers undergo a temperature-induced volume phase transition upon changing the temperature and present an impressive magnetic response. The magnetic saturation of these smart microcontainers (42 emu/g) is high enough to meet most requirements of bioapplications. To further investigate the potential application of these smart microcontainers in biotechnology, Candida rugosa lipase was selected for the enzyme immobilization process. The immobilized lipase exhibited excellent thermal stability and reusability in comparison with the free enzyme. The adsorption/release of the lipase from the microcontainers can be controlled by the environmental temperature and magnetic force, thus, offering new potential applications such as in controlled drug delivery, bioseparation, and catalysis.

Synthesis of magnetic thermosensitive microcontainers for enzyme immobilization

International Nuclear Information System (INIS)

Wang, Jianzhi; Zhao, Guanghui; Wang, Xinyu; Peng, Xiaomen; Li, Yanfeng

2015-01-01

We present a new approach for the fabrication of magnetic thermoresponsive polymer microcapsules with mobile magnetic spherical cores. The microcontainers form fried-egg-like structures with a polymer shell layer of 50 nm due to the existence of hollow cavities. The microcontainers undergo a temperature-induced volume phase transition upon changing the temperature and present an impressive magnetic response. The magnetic saturation of these smart microcontainers (42 emu/g) is high enough to meet most requirements of bioapplications. To further investigate the potential application of these smart microcontainers in biotechnology, Candida rugosa lipase was selected for the enzyme immobilization process. The immobilized lipase exhibited excellent thermal stability and reusability in comparison with the free enzyme. The adsorption/release of the lipase from the microcontainers can be controlled by the environmental temperature and magnetic force, thus, offering new potential applications such as in controlled drug delivery, bioseparation, and catalysis

Enzyme production in immobilized Trichoderma reesei cells with hydrophobic polymers prepared by radiation polymerization method

International Nuclear Information System (INIS)

Luzhao Xin; Kumakura, Minoru; Kaetsu, Isao

1993-01-01

Trichoderma reesei cells were immobilized on paper covered with hydrophobic monomer, trimethylpropane triacrylate by radiation polymerization. The effect of immobilization condition on enzyme productivity was studied by measuring filter paper and cellobiose activity. The cells were adhered and grew on the surface of the carrier with the polymer giving high enzyme productivity in the immobilized cells in comparison with the free cells. Optimum concentration and volume of the coating monomer for the preparation of the immobilized cells were obtained. (author)

Immobilization of cellulases on magnetic particles to enable enzyme recycling during hydrolysis of lignocellulose

DEFF Research Database (Denmark)

Alftrén, Johan

feedstocks containing insolubles. This could potentially be overcome by immobilizing the cellulases on magnetically susceptible particles. Consequently, the immobilized cellulases could be magnetically recovered and recycled for a new cycle of enzymatic hydrolysis of cellulose. The main objective...... of this thesis was to examine the possibility of immobilizing cellulases on magnetic particles in order to enable enzyme re-use. Studies at lab and pilot scale (20 L) were conducted using model and real substrates. In paper I and III beta-glucosidase or a whole cellulase mixture was covalently immobilized...... on commercial, but expensive, magnetic particles activated with different chemistries. It was observed that the highest immobilized enzyme activities were obtained using magnetic particles activated with cyanuric chloride. In paper II biotinylated recombinant beta-glucosidase was produced and immobilized...

activity of enzyme trypsin immobilized onto macroporous poly(epoxy

African Journals Online (AJOL)

dell

consequential effects of covalent immobilization. EXPERIMENTAL. Materials .... immersed into water bath. ... storage stability of the enzyme was studied ... pore size range of about 10 to 150 µm. ... figures, the differences in activities (slopes.

Silica-Immobilized Enzyme Reactors; Application to Cholinesterase-Inhibition Studies

National Research Council Canada - National Science Library

Luckarift, Heather R; Johnson, Glenn R; Spain, Jim C

2006-01-01

...) using silica-encapsulated equine butyrycholinestearse (BuChE) as a model system. Peptide-mediated silica formation was used to encapsulate BuChE, directly immobilizing the enzyme within a commercial pre-packed column...

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

Energy Technology Data Exchange (ETDEWEB)

Kim, Moon Il; Kim, Jungbae; Lee, Jinwoo; Jia, Hongfei; Na, Hyon Bin; Youn, Jongkyu; Kwak, Ja Hun; Dohnalkova, Alice; Grate, Jay W.; Wang, Ping; Hyeon, Taeghwan; Park, Hyun-Gyu; Chang, Ho Nam

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 shaking 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.

Effect of immobile isolated enzymes from rumen liquid by using alginate matrices on the bay leaf extraction

Science.gov (United States)

Paramita, Vita; Yulianto, Mohammad Endy; Yohana, Eflita; Arifan, Fahmi; Hanifah, Amjad, Muhammad Taqiyuddin

2015-12-01

This research aims to develop the enzymatically of bay leaves phytochemical extraction process. The novelty and the main innovations of this research is the development of extraction process by using enzymatic extractor and isolate the enzymes from rumen liquid to shift the equilibrium phase, increase the extraction rate and increase the extraction yield. The activity of rumen liquid enzyme was represented by the activity of cellulase and protease. The analyze of total flavonoid content was performed by using UV-Vis Spectrofometry. The activity of immobilized enzyme of cellulase (0.08±0.00 U/ml) was lower than the un-immobilized one (0.23±0.00 U/ml). However, there was no difference activity of the immobilized (0.75±0.00 U/ml) and un-immobilized (0.76±0.01 U/ml) of protease. The model of mass transfer of un-immobilized enzyme can be fitted on the experimental data, however the model of mass transfer of immobilized enzyme did not match with the experimental data. The mass transfer coefficient of enzymatic extraction flavonoids bay leaf without immobilization was 0.17167 s-1 which greater than the reported value of obtained KLa from extraction by using electric heating.

Ineffective Degradation of Immunogenic Gluten Epitopes by Currently Available Digestive Enzyme Supplements

Science.gov (United States)

Janssen, George; Christis, Chantal; Kooy-Winkelaar, Yvonne; Edens, Luppo; Smith, Drew

2015-01-01

Background Due to the high proline content of gluten molecules, gastrointestinal proteases are unable to fully degrade them leaving large proline-rich gluten fragments intact, including an immunogenic 33-mer from α-gliadin and a 26-mer from γ-gliadin. These latter peptides can trigger pro-inflammatory T cell responses resulting in tissue remodeling, malnutrition and a variety of other complications. A strict lifelong gluten-free diet is currently the only available treatment to cope with gluten intolerance. Post-proline cutting enzymes have been shown to effectively degrade the immunogenic gluten peptides and have been proposed as oral supplements. Several existing digestive enzyme supplements also claim to aid in gluten degradation. Here we investigate the effectiveness of such existing enzyme supplements in comparison with a well characterized post-proline cutting enzyme, Prolyl EndoPeptidase from Aspergillus niger (AN-PEP). Methods Five commercially available digestive enzyme supplements along with purified digestive enzymes were subjected to 1) enzyme assays and 2) mass spectrometric identification. Gluten epitope degradation was monitored by 1) R5 ELISA, 2) mass spectrometric analysis of the degradation products and 3) T cell proliferation assays. Findings The digestive enzyme supplements showed comparable proteolytic activities with near neutral pH optima and modest gluten detoxification properties as determined by ELISA. Mass spectrometric analysis revealed the presence of many different enzymes including amylases and a variety of different proteases with aminopeptidase and carboxypeptidase activity. The enzyme supplements leave the nine immunogenic epitopes of the 26-mer and 33-mer gliadin fragments largely intact. In contrast, the pure enzyme AN-PEP effectively degraded all nine epitopes in the pH range of the stomach at much lower dose. T cell proliferation assays confirmed the mass spectrometric data. Conclusion Currently available digestive enzyme

Ineffective degradation of immunogenic gluten epitopes by currently available digestive enzyme supplements.

Directory of Open Access Journals (Sweden)

George Janssen

Full Text Available Due to the high proline content of gluten molecules, gastrointestinal proteases are unable to fully degrade them leaving large proline-rich gluten fragments intact, including an immunogenic 33-mer from α-gliadin and a 26-mer from γ-gliadin. These latter peptides can trigger pro-inflammatory T cell responses resulting in tissue remodeling, malnutrition and a variety of other complications. A strict lifelong gluten-free diet is currently the only available treatment to cope with gluten intolerance. Post-proline cutting enzymes have been shown to effectively degrade the immunogenic gluten peptides and have been proposed as oral supplements. Several existing digestive enzyme supplements also claim to aid in gluten degradation. Here we investigate the effectiveness of such existing enzyme supplements in comparison with a well characterized post-proline cutting enzyme, Prolyl EndoPeptidase from Aspergillus niger (AN-PEP.Five commercially available digestive enzyme supplements along with purified digestive enzymes were subjected to 1 enzyme assays and 2 mass spectrometric identification. Gluten epitope degradation was monitored by 1 R5 ELISA, 2 mass spectrometric analysis of the degradation products and 3 T cell proliferation assays.The digestive enzyme supplements showed comparable proteolytic activities with near neutral pH optima and modest gluten detoxification properties as determined by ELISA. Mass spectrometric analysis revealed the presence of many different enzymes including amylases and a variety of different proteases with aminopeptidase and carboxypeptidase activity. The enzyme supplements leave the nine immunogenic epitopes of the 26-mer and 33-mer gliadin fragments largely intact. In contrast, the pure enzyme AN-PEP effectively degraded all nine epitopes in the pH range of the stomach at much lower dose. T cell proliferation assays confirmed the mass spectrometric data.Currently available digestive enzyme supplements are ineffective in

A facile and efficient method of enzyme immobilization on silica particles via Michael acceptor film coatings: immobilized catalase in a plug flow reactor.

Science.gov (United States)

Bayramoglu, Gulay; Arica, M Yakup; Genc, Aysenur; Ozalp, V Cengiz; Ince, Ahmet; Bicak, Niyazi

2016-06-01

A novel method was developed for facile immobilization of enzymes on silica surfaces. Herein, we describe a single-step strategy for generating of reactive double bonds capable of Michael addition on the surfaces of silica particles. This method was based on reactive thin film generation on the surfaces by heating of impregnated self-curable polymer, alpha-morpholine substituted poly(vinyl methyl ketone) p(VMK). The generated double bonds were demonstrated to be an efficient way for rapid incorporation of enzymes via Michael addition. Catalase was used as model enzyme in order to test the effect of immobilization methodology by the reactive film surface through Michael addition reaction. Finally, a plug flow type immobilized enzyme reactor was employed to estimate decomposition rate of hydrogen peroxide. The highly stable enzyme reactor could operate continuously for 120 h at 30 °C with only a loss of about 36 % of its initial activity.

Profiles of digestive enzymes of two competing planktivores, silver carp and gizzard shad, differ

Science.gov (United States)

Amberg, Jon J.; Jensen, Nathan R.; Erickson, Richard A.; Sauey, Blake W.; Jackson, Craig

2018-01-01

Typically, studies in digestive physiology in fish focus on a few enzymes and provide insight into the specific processes of the enzyme in a targeted species. Comparative studies assessing a wide number of digestive enzymes on fishes that compete for food resources are lacking, especially in the context of an introduced species. It is generally thought that the invasive silver carp (SVC; Hypophthalmichthys molitrix) directly compete for food resources with the native gizzard shad (GZS; Dorosoma cepedianum) in waters where they coexist. We compared 19 digestive enzymes between SVC and GZS throughout a year and in two rivers in the Midwestern United States: Illinois River and Wabash River. All digestive enzymes analyzed were detected in both SVC and GZS in both rivers. However, the profiles of the digestive enzymes varied by species. Alkaline phosphatase, valine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase and N-acetyl-β-glucosaminidase were all much higher in SVC than in GZS. Differences between digestive enzyme profiles were also observed between rivers and months. This study demonstrates the utility of using an ecological approach to compare physiological features in fishes.

Efficient immobilization of AGE and NAL enzymes onto functional amino resin as recyclable and high-performance biocatalyst.

Science.gov (United States)

Cheng, Jian; Zhuang, Wei; Tang, Chenglun; Chen, Yong; Wu, Jinglan; Guo, Ting; Ying, Hanjie

2017-03-01

N-Acetylglucosamine-2-epimerase (AGE) and N-acetylneuraminic acid lyase (NAL) were immobilized for synthesis of N-acetylneuraminic acid (Neu5Ac) on three resins: Amberzyme oxirane resin (AOR), poly (styrene-co-DVB)-Br resin (PBR) and amino resin (AR). The loading capacity and immobilized enzyme activity showed that AR was the best carrier. Three methods of glutaraldehyde cross-linking were tested and simultaneous cross-linking and immobilization was demonstrated to be the best method. The functional properties of immobilized AGE and NAL were studied and compared to those of the free enzyme. The highest enzyme activities of free and immobilized AGE were obtained in 0.1 M potassium phosphate buffer at pH 7.5 and a temperature of 37 °C. Comparatively, the highest NAL activities were at pH 8.5. Meanwhile, an increase in K m (from 1.14 to 1.31 mg·mL -1 for AGE and from 1.05 to 1.25 mg·mL -1 for NAL) and a decrease in V max (from 177.53 to 106.37 µg·min -1 mL -1 for AGE and from 126.41 to 95.96 µg·min -1 mL -1 for NAL) were recorded after immobilization. The AR-glutaraldehyde-enzyme system exhibited better thermal stability than the free enzyme, and retained 72% of its initial activity even after eight repeated runs. The apparent activation energy (E a ) of the free and immobilized AGE (NAL) was 117.14 kJ·mol -1 (124.21 kJ·mol -1 ) and 78.45 kJ·mol -1 (66.64 kJ·mol -1 ), respectively, implying that the catalytic efficiency of the immobilized enzyme was restricted by mass-transfer rather than kinetic limit. Subsequently, Neu5Ac production from GlcNAc using immobilized enzymes in one reactor was carried out resulting 101.45 g·L -1 of Neu5Ac and the highest conversion ratio of 82%. This method of enzyme immobilization may have a promising future for Neu5Ac production in industry.

Monoterpenes as inhibitors of digestive enzymes and counter-adaptations in a specialist avian herbivore.

Science.gov (United States)

Kohl, Kevin D; Pitman, Elizabeth; Robb, Brecken C; Connelly, John W; Dearing, M Denise; Forbey, Jennifer Sorensen

2015-05-01

Many plants produce plant secondary metabolites (PSM) that inhibit digestive enzymes of herbivores, thus limiting nutrient availability. In response, some specialist herbivores have evolved digestive enzymes that are resistant to inhibition. Monoterpenes, a class of PSMs, have not been investigated with respect to the interference of specific digestive enzymes, nor have such interactions been studied in avian herbivores. We investigated this interaction in the Greater Sage-Grouse (Phasianidae: Centrocercus urophasianus), which specializes on monoterpene-rich sagebrush species (Artemisia spp.). We first measured the monoterpene concentrations in gut contents of free-ranging sage-grouse. Next, we compared the ability of seven individual monoterpenes present in sagebrush to inhibit a protein-digesting enzyme, aminopeptidase-N. We also measured the inhibitory effects of PSM extracts from two sagebrush species. Inhibition of aminopeptidase-N in sage-grouse was compared to inhibition in chickens (Gallus gallus). We predicted that sage-grouse enzymes would retain higher activity when incubated with isolated monoterpenes or sagebrush extracts than chicken enzymes. We detected unchanged monoterpenes in the gut contents of free-ranging sage-grouse. We found that three isolated oxygenated monoterpenes (borneol, camphor, and 1,8-cineole) inhibited digestive enzymes of both bird species. Camphor and 1,8-cineole inhibited enzymes from chickens more than from sage-grouse. Extracts from both species of sagebrush had similar inhibition of chicken enzymes, but did not inhibit sage-grouse enzymes. These results suggest that specific monoterpenes may limit the protein digestibility of plant material by avian herbivores. Further, this work presents additional evidence that adaptations of digestive enzymes to plant defensive compounds may be a trait of specialist herbivores.

Optimizing Immobilized Enzyme Performance in Cell-Free Environments to Produce Liquid Fuels

Energy Technology Data Exchange (ETDEWEB)

Belfort, Georges [Rensselaer Polytechnic Inst., Troy, NY (United States). Dept. of Chemical and Biological Engineering; Grimaldi, Joseph J. [Rensselaer Polytechnic Inst., Troy, NY (United States). Dept. of Chemical and Biological Engineering

2015-01-27

Limitations on biofuel production using cell culture (Escherichia coli, Clostridium, Saccharomyces cerevisiae, brown microalgae, blue-green algae and others) include low product (alcohol) concentrations (≤0.2 vol%) due to feedback inhibition, instability of cells, and lack of economical product recovery processes. To overcome these challenges, an alternate simplified biofuel production scheme was tested based on a cell-free immobilized enzyme system. Using this cell free system, we were able to obtain about 2.6 times higher concentrations of iso-butanol using our non-optimized system as compared with live cell systems. This process involved two steps: (i) converts acid to aldehyde using keto-acid decarboxylase (KdcA), and (ii) produces alcohol from aldehyde using alcohol dehydrogenase (ADH) with a cofactor (NADH) conversion from inexpensive formate using a third enzyme, formate dehydrogenase (FDH). To increase stability and conversion efficiency with easy separations, the first two enzymes were immobilized onto methacrylate resin. Fusion proteins of labile KdcA (fKdcA) were expressed to stabilize the covalently immobilized KdcA. Covalently immobilized ADH exhibited long-term stability and efficient conversion of aldehyde to alcohol over multiple batch cycles without fusions. High conversion rates and low protein leaching were achieved by covalent immobilization of enzymes on methacrylate resin. The complete reaction scheme was demonstrated by immobilizing both ADH and fKdcA and using FDH free in solution. The new system without in situ removal of isobutanol achieved a 55% conversion of ketoisovaleric acid to isobutanol at a concentration of 0.5 % (v/v). Further increases in titer will require continuous removal of the isobutanol using our novel brush membrane system that exhibits a 1.5 fold increase in the separation factor of isobutanol from water versus that obtained for commercial silicone rubber membranes. These bio-inspired brush membranes are based on the

Effect of enzymes on anaerobic digestion of primary sludge and septic tank performance.

Science.gov (United States)

Diak, James; Örmeci, Banu; Kennedy, Kevin J

2012-11-01

Enzyme additives are believed to improve septic tank performance by increasing the hydrolysis and digestion rates and maintaining a healthy microbial population. Previous studies reported mixed results on the effectiveness of enzymes on mesophilic and thermophilic digestion, and it is not clear whether enzymes would be effective under septic tank conditions where there is no heating or mixing, quantities of enzymes added are small, and they can be washed out quickly. In this study, batch reactors and continuous-flow reactors designed and operated as septic tanks were used to evaluate whether enzymatic treatment would increase the hydrolysis and digestion rates in primary sludge. Total solids, volatile solids, total suspended solids, total and soluble chemical oxygen demand, concentrations of protein, carbohydrate, ammonia and volatile acids in sludge and effluent samples were measured to determine the differences in digestion rates in the presence and absence of enzymes. Overall, no significant improvement was observed in enzyme-treated reactors compared with the control reactors.

Bioethanol from lignocellulose - pretreatment, enzyme immobilization and hydrolysis kinetics

DEFF Research Database (Denmark)

Tsai, Chien Tai

, the cost of enzyme is still the bottle neck, re-using the enzyme is apossible way to reduce the input of enzyme in the process. In the point view of engineering, the prediction of enzymatic hydrolysis kinetics under different substrate loading, enzyme combination is usful for process design. Therefore...... lignocellulose is the required high cellulase enzyme dosages that increase the processing costs. One method to decrease the enzyme dosage is to re-use BG, which hydrolyze the soluble substrate cellobiose. Based on the hypothesis that immobilized BG can be re-used, how many times the enzyme could be recycled...... liquid and pretreatment time can be reduced, the influence of substrate concentration, pretreatment time and temperature were investigated and optimized. Pretreatment of barley straw by [EMIM]Ac, correlative models were constructed using 3 different pretreatment parameters (temperature, time...

Digestive enzyme activities and gastrointestinal fermentation in wood-eating catfishes.

Science.gov (United States)

German, Donovan P; Bittong, Rosalie A

2009-11-01

To determine what capabilities wood-eating and detritivorous catfishes have for the digestion of refractory polysaccharides with the aid of an endosymbiotic microbial community, the pH, redox potentials, concentrations of short-chain fatty acids (SCFAs), and the activity levels of 14 digestive enzymes were measured along the gastrointestinal (GI) tracts of three wood-eating taxa (Panaque cf. nigrolineatus "Marañon", Panaque nocturnus, and Hypostomus pyrineusi) and one detritivorous species (Pterygoplichthys disjunctivus) from the family Loricariidae. Negative redox potentials (-600 mV) were observed in the intestinal fluids of the fish, suggesting that fermentative digestion was possible. However, SCFA concentrations were low (<3 mM in any intestinal region), indicating that little GI fermentation occurs in the fishes' GI tracts. Cellulase and xylanase activities were low (<0.03 U g(-1)), and generally decreased distally in the intestine, whereas amylolytic and laminarinase activities were five and two orders of magnitude greater, respectively, than cellulase and xylanase activities, suggesting that the fish more readily digest soluble polysaccharides. Furthermore, the Michaelis-Menten constants (K(m)) of the fishes' beta-glucosidase and N-acetyl-beta-D-glucosaminidase enzymes were significantly lower than the K(m) values of microbial enzymes ingested with their food, further suggesting that the fish efficiently digest soluble components of their detrital diet rather than refractory polysaccharides. Coupled with rapid gut transit and poor cellulose digestibility, the wood-eating catfishes appear to be detritivores reliant on endogenous digestive mechanisms, as are other loricariid catfishes. This stands in contrast to truly "xylivorous" taxa (e.g., beavers, termites), which are reliant on an endosymbiotic community of microorganisms to digest refractory polysaccharides.

Co-Immobilization of Enzymes and Magnetic Nanoparticles by Metal-Nucleotide Hydrogelnanofibers for Improving Stability and Recycling

Directory of Open Access Journals (Sweden)

Chunfang Li

2017-01-01

Full Text Available In this paper we report a facile method for preparing co-immobilized enzyme and magnetic nanoparticles (MNPs using metal coordinated hydrogel nanofibers. Candida rugosa lipase (CRL was selected as guest protein. For good aqueous dispersity, low price and other unique properties, citric acid-modified magnetic iron oxide nanoparticles (CA-Fe3O4 NPs have been widely used for immobilizing enzymes. As a result, the relative activity of CA-Fe3O4@Zn/AMP nanofiber-immobilized CRL increased by 8-fold at pH 10.0 and nearly 1-fold in a 50 °C water bath after 30 min, compared to free CRL. Moreover, the immobilized CRL had excellent long-term storage stability (nearly 80% releative activity after storage for 13 days. This work indicated that metal-nucleotide nanofibers could efficiently co-immobilize enzymes and MNPs simultaneously, and improve the stability of biocatalysts.

Graphene immobilized enzyme/polyethersulfone mixed matrix membrane: Enhanced antibacterial, permeable and mechanical properties

International Nuclear Information System (INIS)

Duan, Linlin; Wang, Yuanming; Zhang, Yatao; Liu, Jindun

2015-01-01

Graphical abstract: - Highlights: • Lysozyme was immobilized on the surface of graphene oxide (GO) and reduced GO (RGO). • The novel hybrid membranes based on lysozyme and graphene were fabricated firstly. • These membranes showed good antibacterial and mechanical performance. - Abstract: Enzyme immobilization has been developed to address lots of issues of free enzyme, such as instability, low activity and difficult to retain. In this study, graphene was used as an ideal carrier for lysozyme immobilization, including graphene oxide (GO) immobilized lysozyme (GO-Ly) and chemically reduced graphene oxide (CRGO) immobilized lysozyme (CRGO-Ly). Herein, lysozyme as a bio-antibacterial agent has excellent antibacterial performance and the products of its catalysis are safety and nontoxic. Then the immobilized lysozyme materials were blended into polyethersulfone (PES) casting solution to prepare PES ultrafiltration membrane via phase inversion method. GO and CRGO were characterized by Fourier transform infrared spectroscopy (FTIR), Ultraviolet–visible spectrum (UV), X-ray diffraction (XRD), and transmission electron microscopy (TEM) and the immobilized lysozyme composites were observed by fluorescent microscopy. The results revealed that GO and CRGO were successfully synthesized and lysozyme was immobilized on their surfaces. The morphology, hydrophilicity, mechanical properties, separation properties and antibacterial activity of the hybrid membranes were characterized in detail. The hydrophilicity, water flux and mechanical strength of the hybrid membranes were significantly enhanced after adding the immobilized lysozyme. In the antibacterial experiment, the hybrid membranes exhibited an effective antibacterial performance against Escherichia coli (E. coli).

Activity of an enzyme immobilized on superparamagnetic particles in a rotational magnetic field

Energy Technology Data Exchange (ETDEWEB)

Mizuki, Toru; Watanabe, Noriyuki; Nagaoka, Yutaka [Bio-Nano Electronics Research Centre, Toyo University, Saitama 350-8585 (Japan); Fukushima, Tadamasa [Shimadzu GLC Ltd., Phenomenex Support Centre, Tokyo 110-0016 (Japan); Morimoto, Hisao; Usami, Ron [Bio-Nano Electronics Research Centre, Toyo University, Saitama 350-8585 (Japan); Maekawa, Toru, E-mail: maekawa@toyonet.toyo.ac.jp [Bio-Nano Electronics Research Centre, Toyo University, Saitama 350-8585 (Japan)

2010-03-19

We immobilize {alpha}-amylase extracted from Bacillus Iicheniformis on the surfaces of superparamagnetic particles and investigate the effect of a rotational magnetic field on the enzyme's activity. We find that the activity of the enzyme molecules immobilized on superparamagnetic particles increases in the rotational magnetic field and reaches maximum at a certain frequency. We clarify the effect of the cluster structures formed by the superparamagnetic particles on the activity. Enzyme reactions are enhanced even in a tiny volume of solution using the present method, which is very important for the development of efficient micro reactors and micro total analysis systems ({mu}-TAS).

Chitin hydrolysis assisted by cell wall degrading enzymes immobilized of Thichoderma asperellum on totally cinnamoylated D-sorbitol beads

International Nuclear Information System (INIS)

Fernandes, Kátia F.; Cortijo-Triviño, David; Batista, Karla A.; Ulhoa, Cirano J.; García-Ruiz, Pedro A.

2013-01-01

In this study, cell wall degrading enzymes produced by Thrichoderma asperellum (TCWDE) were immobilized on totally cinnamoylated D-sorbitol (TCNSO) beads and used for chitin hydrolysis. In order to optimize immobilization efficiency, the reaction time was varied from 2 to 12 h and reactions were conducted in the presence or absence of Na 2 SO 4 . Immobilized enzymes were analysed concerning to thermal and operational stability. Immobilization in presence of Na 2 SO 4 was 54% more efficient than immobilization in absence of salt. After optimization, 32% of the total enzyme offered was immobilized, with 100% of bounding efficiency, measured as the relation between protein and enzyme immobilized. Free and TCNSO–TCWDE presented very similar kinetics with maximum hydrolysis reached at 90 min of reaction. Thermal stability of both free and TCNSO–TCWDE was similar, with losses in activity after 55 °C. Moreover, free and TCNSO–TCWDE retained 100% activity after 3 h incubation at 55 °C. TCNSO–TCWDE were used in a bath-wise reactor during 14 cycles, producing 1825 μg of N-acetylglucosamine (NAG) maintaining 83% of initial activity. - Highlights: • TCWDE immobilized on TCNSO, a support with highly hydrophobic character • New immobilization strategy for immobilization on a hydrophobic support • TCNSO–TCWDE were retained during washes and during incubation at 55 °C for 3 h

Chitin hydrolysis assisted by cell wall degrading enzymes immobilized of Thichoderma asperellum on totally cinnamoylated D-sorbitol beads

Energy Technology Data Exchange (ETDEWEB)

Fernandes, Kátia F., E-mail: katia@icb.ufg.br [Departamento de Bioquímica e Biologia Molecular, Instituo de Ciências Biológicas, Universidade Federal de Goiás, Cx. Postal 131, 74001-970 Goiânia, GO (Brazil); Grupo de Química de Carbohidratos y Biotecnología de Alimentos (QCBA), Departamento de Química Orgánica, Facultad de Química, Universidad de Murcia, E-30100 Espinardo, Murcia (Spain); Cortijo-Triviño, David [Grupo de Química de Carbohidratos y Biotecnología de Alimentos (QCBA), Departamento de Química Orgánica, Facultad de Química, Universidad de Murcia, E-30100 Espinardo, Murcia (Spain); Batista, Karla A.; Ulhoa, Cirano J. [Departamento de Bioquímica e Biologia Molecular, Instituo de Ciências Biológicas, Universidade Federal de Goiás, Cx. Postal 131, 74001-970 Goiânia, GO (Brazil); García-Ruiz, Pedro A. [Grupo de Química de Carbohidratos y Biotecnología de Alimentos (QCBA), Departamento de Química Orgánica, Facultad de Química, Universidad de Murcia, E-30100 Espinardo, Murcia (Spain)

2013-07-01

In this study, cell wall degrading enzymes produced by Thrichoderma asperellum (TCWDE) were immobilized on totally cinnamoylated D-sorbitol (TCNSO) beads and used for chitin hydrolysis. In order to optimize immobilization efficiency, the reaction time was varied from 2 to 12 h and reactions were conducted in the presence or absence of Na{sub 2}SO{sub 4}. Immobilized enzymes were analysed concerning to thermal and operational stability. Immobilization in presence of Na{sub 2}SO{sub 4} was 54% more efficient than immobilization in absence of salt. After optimization, 32% of the total enzyme offered was immobilized, with 100% of bounding efficiency, measured as the relation between protein and enzyme immobilized. Free and TCNSO–TCWDE presented very similar kinetics with maximum hydrolysis reached at 90 min of reaction. Thermal stability of both free and TCNSO–TCWDE was similar, with losses in activity after 55 °C. Moreover, free and TCNSO–TCWDE retained 100% activity after 3 h incubation at 55 °C. TCNSO–TCWDE were used in a bath-wise reactor during 14 cycles, producing 1825 μg of N-acetylglucosamine (NAG) maintaining 83% of initial activity. - Highlights: • TCWDE immobilized on TCNSO, a support with highly hydrophobic character • New immobilization strategy for immobilization on a hydrophobic support • TCNSO–TCWDE were retained during washes and during incubation at 55 °C for 3 h.

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

Science.gov (United States)

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

2016-01-01

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

Utilization of ionizing radiation to obtention of polymeric supports for the enzyme immobilization with clinical potential use

International Nuclear Information System (INIS)

Rodas, Andrea Cecilia Dorion

1997-01-01

In the development of polymers with biological activity, it was studied the grafting of acrylic acid monomer onto polyethylene and polypropylene pellets by mutual radiation grafting technique. The effect of dose rate, irradiation total dose, and monomer concentration were studied. With the Pp pellets the best grafting yield occurred at dose rate of 0.25 kGy/h and with the PE pellets the dose rate was lower. The irradiation dose from 8 to 10 kGy was sufficient to obtain the highest grafting degree, and the AA concentration of 40% v/v was suitable. The graft of poly (acrylic acid) was chemically modified for the immobilization of two enzymes, the glucose oxidase and the urease. For both enzymes the increasing of grafting degree onto the pellets, increased the enzyme immobilization yield. The immobilized glucose oxidase showed the best activity when immobilized onto Pp-A A supports with grafting degree around 2%. The optimum p H and temperature profiles, and the Km and Vmax for free and immobilized enzyme were determined. The supports grafted with A A were not suitable for the chemical immobilization of urease. (author)

Real-time monitoring of peptic and tryptic digestions of bovine {beta}-casein using quartz crystal microbalance

Energy Technology Data Exchange (ETDEWEB)

Huenerbein, Andreas [Institute of Pharmaceutics and Biopharmaceutics, Martin Luther University Halle-Wittenberg, Wolfgang-Langenbeck-Str. 4, 06120 Halle (Saale) (Germany)]. E-mail: andreas.huenerbein@pharmazie.uni-halle.de; Schmelzer, Christian E.H. [Institute of Pharmaceutics and Biopharmaceutics, Martin Luther University Halle-Wittenberg, Wolfgang-Langenbeck-Str. 4, 06120 Halle (Saale) (Germany); Neubert, Reinhard H.H. [Institute of Pharmaceutics and Biopharmaceutics, Martin Luther University Halle-Wittenberg, Wolfgang-Langenbeck-Str. 4, 06120 Halle (Saale) (Germany)

2007-02-12

In this study peptic and tryptic digestions of bovine {beta}-casein were investigated using quartz crystal microbalance (QCM). {beta}-Casein, which was used as a model protein, was immobilized on the surface of the QCM sensor where its degradation caused shifts in the resonant frequency. Atomic force microscopy was applied for the characterization of the protein layer. Different pH-values for peptic or tryptic digestions were chosen to visualize their effect on enzyme activity. Lower frequency shifts were observed at pH-values deviating from those at the maximum enzyme activity. In the case of the peptic digestion the frequency shift at pH 4 was more than 10 times smaller than those at pH 2. The frequency shifts for tryptic digestions at pH 5.4 and pH 6.4 were about two thirds compared to that obtained for the digestion at pH 7.4. The identification of peptides using MALDI-ToF mass spectrometry was used for verification of the proteolyses of the immobilized protein. Furthermore, it was shown that the QCM technique allows close observation of the effect of different pH-values on the immobilized casein layer. All in all, QCM facilitates the monitoring of the progress of enzymatic reactions in real-time.

Real-time monitoring of peptic and tryptic digestions of bovine β-casein using quartz crystal microbalance

International Nuclear Information System (INIS)

Huenerbein, Andreas; Schmelzer, Christian E.H.; Neubert, Reinhard H.H.

2007-01-01

In this study peptic and tryptic digestions of bovine β-casein were investigated using quartz crystal microbalance (QCM). β-Casein, which was used as a model protein, was immobilized on the surface of the QCM sensor where its degradation caused shifts in the resonant frequency. Atomic force microscopy was applied for the characterization of the protein layer. Different pH-values for peptic or tryptic digestions were chosen to visualize their effect on enzyme activity. Lower frequency shifts were observed at pH-values deviating from those at the maximum enzyme activity. In the case of the peptic digestion the frequency shift at pH 4 was more than 10 times smaller than those at pH 2. The frequency shifts for tryptic digestions at pH 5.4 and pH 6.4 were about two thirds compared to that obtained for the digestion at pH 7.4. The identification of peptides using MALDI-ToF mass spectrometry was used for verification of the proteolyses of the immobilized protein. Furthermore, it was shown that the QCM technique allows close observation of the effect of different pH-values on the immobilized casein layer. All in all, QCM facilitates the monitoring of the progress of enzymatic reactions in real-time

Development of Cellulose Nano fibre (CNF) Derived From Kenaf Bast Fibre and Its Potential in Enzyme Immobilization Support

International Nuclear Information System (INIS)

Safwan Sulaiman; Mohd Noriznan Mokhtar; Mohd Nazli Naim; Azhari Samsu Baharuddin

2016-01-01

This research mainly focuses on developing a natural cellulose nano fibre (CNF) from kenaf bast fibre and its potential for enzyme immobilization support. CNF was isolated by using a combination between chemical and mechanical treatments such as alkaline process and high-intensity ultrasonication process to increase the efficiency of hemicelluloses and lignin removal, and to reduce its size into nano-order. The morphological study was carried out by using scanning electron microscope (SEM), indicating most of CNF diameter in range of 50-90 nm was obtained. The result of chemical analysis shows that cellulose content of raw bast fibre, bleached pulp fibre and CNF are 66.4 %, 83.7 % and 90.0 %, respectively. By decreasing the size of cellulose fibre, it increases the number of (O-H) group on the surface that plays as important role in enzyme immobilization. Covalent immobilization of cyclodextrin glucanotransferase (CGTase) onto CNF support resulted in about 95.0 % of protein loading with 69.48 % of enzyme activity, indicating high immobilization yield of enzyme. The enzymatic reaction of immobilized CGTase was able to produce more than 40 % yield of α-CD. Reusability profile of immobilized CGTase resulted in more than 60 % of retained activity up to 7 cycles. Therefore, the CNF is highly potential to be applied as enzyme immobilization support. (author)

Thermostable trypsin conjugates immobilized to biogenic magnetite show a high operational stability and remarkable reusability for protein digestion

Science.gov (United States)

Pečová, M.; Šebela, M.; Marková, Z.; Poláková, K.; Čuda, J.; Šafářová, K.; Zbořil, R.

2013-03-01

In this work, magnetosomes produced by microorganisms were chosen as a suitable magnetic carrier for covalent immobilization of thermostable trypsin conjugates with an expected applicability for efficient and rapid digestion of proteins at elevated temperatures. First, a biogenic magnetite was isolated from Magnetospirillum gryphiswaldense and its free surface was coated with the natural polysaccharide chitosan containing free amino and hydroxy groups. Prior to covalent immobilization, bovine trypsin was modified by conjugating with α-, β- and γ-cyclodextrin. Modified trypsin was bound to the magnetic carriers via amino groups using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysulfosuccinimide as coupling reagents. The magnetic biomaterial was characterized by magnetometric analysis and electron microscopy. With regard to their biochemical properties, the immobilized trypsin conjugates showed an increased resistance to elevated temperatures, eliminated autolysis, had an unchanged pH optimum and a significant storage stability and reusability. Considering these parameters, the presented enzymatic system exhibits properties that are superior to those of trypsin forms obtained by other frequently used approaches. The proteolytic performance was demonstrated during in-solution digestion of model proteins (horseradish peroxidase, bovine serum albumin and hen egg white lysozyme) followed by mass spectrometry. It is shown that both magnetic immobilization and chemical modification enhance the characteristics of trypsin making it a promising tool for protein digestion.

Digestive enzymes in the alimentary canal of Clarias anguillaris ...

African Journals Online (AJOL)

Three groups of digestive enzymes were observed in the alimentary canals of the fingerlings and adults of Clarias anguillaris. The enzymes were carbohydrases including amylase, sucrase, lactase and maltase, proteases including peptase, tryptase and peptidase, and lipase. The activities of the protease were found to be ...

Anaerobic digestion of palm oil mill effluent with lampung natural zeolite as microbe immobilization medium and digested cow manure as starter

Science.gov (United States)

Halim, Lenny; Mellyanawaty, Melly; Cahyono, Rochim Bakti; Sudibyo, Hanifrahmawan; Budhijanto, Wiratni

2017-05-01

Indonesia is well-known as the world's biggest palm oil producer with 32.5 million tons of annual production. Palm oil processing contributes to 60% wastewater, leading to environmental problem caused by excessive production of wastewater. This wastewater, i.e. Palm Oil Mill Effluent (POME), has high organic content (40,000-60,000 mg COD/L) which is potential for biogas production. However, its low pH value and long chain fatty acid content likely inhibit the anaerobic digestion. Porous media might reduce the inhibitory effect during POME digestion since the media act as both immobilization media for bacteria and as inhibitor adsorbent. Excessive amount of porous media might interfere with the nutrient consumption by microbes. There will be an optimum amount of porous media added, which depends on the wastewater characteristics. This research studied Lampung natural zeolite as immobilization media in digesting POME. The batch experiment was conducted for 40 days with different amount of natural zeolite, i.e. 0; 45; 100; and 200 g/g COD. Digested cow manure was used as the starter inoculum, considering the abundance of anaerobic bacteria therein. Zeolite addition was proven to accelerate COD reduction and stabilized the volatile fatty acid as the intermediate product of anaerobic digestion. The addition of natural zeolite up to 45 g/g COD is considered enough to increase the COD removal (85.695 %), maintain the methane content up to 50%, and enhance the bacteria activity. However, larger amount of natural zeolite lowered the methane production and COD reduction, which indicated nutrient adsorption on to the media and hence caused decreasing nutrient access by the microbes.

Digestive enzyme activities in the guts of bonnethead sharks (Sphyrna tiburo) provide insight into their digestive strategy and evidence for microbial digestion in their hindguts.

Science.gov (United States)

Jhaveri, Parth; Papastamatiou, Yannis P; German, Donovan P

2015-11-01

Few investigations have studied digestive enzyme activities in the alimentary tracts of sharks to gain insight into how these organisms digest their meals. In this study, we examined the activity levels of proteases, carbohydrases, and lipase in the pancreas, and along the anterior intestine, spiral intestine, and colon of the bonnethead shark, Sphyrna tiburo. We then interpreted our data in the context of a rate-yield continuum to discern this shark's digestive strategy. Our data show anticipated decreasing patterns in the activities of pancreatic enzymes moving posteriorly along the gut, but also show mid spiral intestine peaks in aminopeptidase and lipase activities, which support the spiral intestine as the main site of absorption in bonnetheads. Interestingly, we observed spikes in the activity levels of N-acetyl-β-D-glucosaminidase and β-glucosidase in the bonnethead colon, and these chitin- and cellulose-degrading enzymes, respectively, are likely of microbial origin in this distal gut region. Taken in the context of intake and relatively long transit times of food through the gut, the colonic spikes in N-acetyl-β-D-glucosaminidase and β-glucosidase activities suggest that bonnetheads take a yield-maximizing strategy to the digestive process, with some reliance on microbial digestion in their hindguts. This is one of the first studies to examine digestive enzyme activities along the gut of any shark, and importantly, the data match with previous observations that sharks take an extended time to digest their meals (consistent with a yield-maximizing digestive strategy) and that the spiral intestine is the primary site of absorption in sharks. Copyright © 2015 Elsevier Inc. All rights reserved.

Immobilized enzyme reactor chromatography: Optimization of protein retention and enzyme activity in monolithic silica stationary phases

International Nuclear Information System (INIS)

Besanger, Travis R.; Hodgson, Richard J.; Green, James R.A.; Brennan, John D.

2006-01-01

Our group recently reported on the application of protein-doped monolithic silica columns for immobilized enzyme reactor chromatography, which allowed screening of enzyme inhibitors present in mixtures using mass spectrometry for detection. The enzyme was immobilized by entrapment within a bimodal meso/macroporous silica material prepared by a biocompatible sol-gel processing route. While such columns proved to be useful for applications such as screening of protein-ligand interactions, significant amounts of entrapped proteins leached from the columns owing to the high proportion of macropores within the materials. Herein, we describe a detailed study of factors affecting the morphology of protein-doped bioaffinity columns and demonstrate that specific pH values and concentrations of poly(ethylene glycol) can be used to prepare essentially mesoporous columns that retain over 80% of initially loaded enzyme in an active and accessible form and yet still retain sufficient porosity to allow pressure-driven flow in the low μL/min range. Using the enzyme γ-glutamyl transpeptidase (γ-GT), we further evaluated the catalytic constants of the enzyme entrapped in capillary columns with different silica morphologies as a function of flowrate and backpressure using the enzyme reactor assay mode. It was found that the apparent activity of the enzyme was highest in mesoporous columns that retained high levels of enzyme. In such columns, enzyme activity increased by ∼2-fold with increases in both flowrate (from 250 to 1000 nL/min) and backpressure generated (from 500 to 2100 psi) during the chromatographic activity assay owing to increases in k cat and decreases in K M , switching from diffusion controlled to reaction controlled conditions at ca. 2000 psi. These results suggest that columns with minimal macropore volumes (<5%) are advantageous for the entrapment of soluble proteins for bioaffinity and bioreactor chromatography

Study on immobilization enzyme using radiation grafting and condensation covalent

International Nuclear Information System (INIS)

Cao Jin; Su Zongxian; Gao Jianfeng

1989-01-01

The immobilization of gluecose oxidase (GOD) on polyethylene and F 46 is described by radiation grafting and condensation covalent. The GOD on polyethylene film is characterized with IR-spectrum. The results show that the enzyme activity on F 46 film is high when dose rate and covalent yield are low. When covalent yield is 4.3% the enzyme relative activity achieves the greatest value for F 46 film. The experiment also demonstrates that acrylic acid affects the relative activity of enzyme and the method of IR-pectrum character is convenient and efficient for GOD on polyethylene film

Biogenic porous silica and silicon sourced from Mexican Giant Horsetail (Equisetum myriochaetum) and their application as supports for enzyme immobilization.

Science.gov (United States)

Sola-Rabada, Anna; Sahare, Padma; Hickman, Graham J; Vasquez, Marco; Canham, Leigh T; Perry, Carole C; Agarwal, Vivechana

2018-06-01

Porous silica-based materials are attractive for biomedical applications due to their biocompatibility and biodegradable character. In addition, inorganic supports such as porous silicon are being developed due to integrated circuit chip compatibility and tunable properties leading to a wide range of multidisciplinary applications. In this contribution, biosilica extracted from a rarely studied plant material (Equisetum Myriochaetum), its conversion to silicon and the potential for both materials to be used as supports for enzyme immobilization are investigated. E. myriochaetum was subject to conventional acid digestion to extract biogenic silica with a% yield remarkably higher (up to 3 times) than for other Equisetum sp. (i.e. E. Arvense). The surface area of the isolated silica was ∼400 m 2 /g, suitable for biotechnological applications. Biogenic silicon was obtained by magnesiothermic reduction. The materials were characterized by SEM-EDX, XRD, FT-IR, ICP-OES, TGA and BET analysis and did not contain significant levels of class 1 heavy elements (such as Pb, Cd, Hg and As). Two commercial peroxidases, horseradish peroxidase (HRP) and Coprinus cinereus peroxidase (CiP) were immobilized onto the biogenic materials using three different functionalization routes: (A) carbodiimide, (B) amine + glutaraldehyde and (C) amine + carbodiimide. Although both biogenic silica and porous silicon could be used as supports differences in behaviour were observed for the two enzymes. For HRP, loading onto biogenic silica via the glutaraldehyde immobilization technique (route B) was most effective. The loading of CiP showed a much higher peroxidase activity onto porous silicon than silica functionalized by the carbodiimide method (route A). From the properties of the extracted materials obtained from Equisetum Myriochaetum and the immobilization results observed, these materials appear to be promising for industrial and biomedical applications. Copyright © 2018 Elsevier

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

Directory of Open Access Journals (Sweden)

Lin Yuan

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

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

Science.gov (United States)

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.

{sup 125}I Labelling of Protein Using Immobilized Enzyme

Energy Technology Data Exchange (ETDEWEB)

Kim, Jae Rok; Park, Kyung Bae; Awh, Ok Doo [Korea Advanced Energy Research Institute, Daejeon (Korea, Republic of)

1984-03-15

For an effective solid-phase labelling of protein with {sup 125}I, studies on the immobilization of lactoperoxidase (LPO) on the inner wall of polystyrene tubes were carried out. Labelling of bovine serum albumin (BSA) and insulin was also practiced using the LPO immobilized tubes. The immobilized enzyme of about 2.5 mu g/tube was sufficient for small scale labelling since the results of radio-paper chromatography of the labelling mixture of insulin indicated that the yields were sufficiently high (80%) even in the reactions conducted at room temperature for 60 sec. The results of the Sephadex column chromatography indicated that the labelled products were not contaminated with LPO-{sup 125}I, and the radiochemical purity of the products was more than 90%. In considering the general trend that the {sup 125}I labelled protein obtained by using LPO maintains its intactness better than those obtained by using chloramine-T, together with the tendency of yield enhancing with increase of reactants-concentration, the LPO immobilized tube method is estimated to be one of the simple methods of labelling. The product might be applicable without further purification.

Electron paramagnetic resonance spin label titration: a novel method to investigate random and site-specific immobilization of enzymes onto polymeric membranes with different properties

International Nuclear Information System (INIS)

Butterfield, D. Allan; Colvin, Joshua; Liu Jiangling; Wang Jianquan; Bachas, Leonidas; Bhattacharrya, Dibakar

2002-01-01

The immobilization of biological molecules onto polymeric membranes to produce biofunctional membranes is used for selective catalysis, separation, analysis, and artificial organs. Normally, random immobilization of enzymes onto polymeric membranes leads to dramatic reduction in activity due to chemical reactions involved in enzyme immobilization, multiple-point binding, etc., and the extent of activity reduction is a function of membrane hydrophilicity (e.g. activity in cellulosic membrane >> polysulfone membrane). We have used molecular biology to effect site-specific immobilization of enzymes in a manner that orients the active site away from the polymeric membrane surface, thus resulting in higher enzyme activity that approaches that in solution and in increased stability of the enzyme relative to the enzyme in solution. A prediction of this site-specific method of enzyme immobilization, which in this study with subtilisin and organophosphorus hydrolase consists of a fusion tag genetically added to these enzymes and subsequent immobilization via the anti-tag antibody and membrane-bound protein A, is that the active site conformation will more closely resemble that of the enzyme in solution than is the case for random immobilization. This hypothesis was confirmed using a new electron paramagnetic resonance (EPR) spin label active site titration method that determines the amount of spin label bound to the active site of the immobilized enzyme. This value nearly perfectly matched the enzyme activity, and the results suggested: (a) a spectroscopic method for measuring activity and thus the extent of active enzyme immobilization in membrane, which may have advantages in cases where optical methods can not be used due to light scattering interference; (b) higher spin label incorporation (and hence activity) in enzymes that had been site-specifically immobilized versus random immobilization; (c) higher spin label incorporation in enzymes immobilized onto hydrophilic

Enzyme immobilization by fouling in ultrafiltration membranes: Impact of membrane configuration and type on flux behavior and biocatalytic conversion efficacy

DEFF Research Database (Denmark)

Luo, Jianquan; Meyer, Anne S.; Jonsson, Gunnar Eigil

2014-01-01

Enzyme-immobilization in membranes accomplished by fostering membrane fouling was evaluated. Four different membrane configurations and five membranes were compared for immobilization of alcohol dehydrogenase (ADH) in terms of enzyme loading, permeate flux and final biocatalytic conversion...... and PLGC regenerated cellulose membranes. With these two highly hydrophilic membranes, the ADH enzyme activity was fully retained even after 24h of storage of the membrane. Filtration blocking and resistance models were used to analyze the fouling/immobilization mechanisms and give explanations...... for the different results. The work confirms that fouling-induced enzyme immobilization is a promising option for enhancing biocatalytic productivity, and highlights the significance of the membrane type and configuration for optimal performance....

The effect of natural zeolite as microbial immobilization media in anaerobic digestion at various concentrations of palm oil mill effluent (POME)

Science.gov (United States)

Ayu, Erika Dyah; Halim, Lenny; Mellyanawaty, Melly; Sudibyo, Hanifrahmawan; Budhijanto, Wiratni

2017-05-01

Anaerobic digestion is one of the solutions in POME treatment because it generates energy in the form of biogas. Nevertheless, high concentration of POME fed into the digester will cause high acidification level and eventually cause process failure. The failure can also be affected by high inhibitor concentration contained in POME, one of which is long chain fatty acid. The addition of immobilization media is commonly used to increase the bacteria resistance to inhibitor. Natural zeolite is considered as appropriate immobilization media for waste treatment purpose since it is cheap, has high mechanical strength, high immobilization capacity, and its ability as cation exchanger. The digested biodiesel waste was used as starter inoculum for its good capability in digesting oil-containing feed such as POME. This research was conducted to evaluate critical concentration of POME fed to digester where the natural zeolite could not reduce the inhibitory effect. The concentrations of POME evaluated were 10,000 mg sCOD/L and 17,000 mg sCOD/L, which were higher than previous studies(5,000 to 8,000 mg sCOD/L). The research showed that at such high sCOD concentrations, the addition of zeolite did not significantly increase COD reduction nor biogas production rate, for both POME concentrations to be compared to control. The biogas produced by digester fed by 10,000 mg/L POME and 17,000 mg/L POME was 751 and 100 mL/g sCOD, respectively, indicating higher inhibitor effect in the digester with 17,000 mg SCOD/L initial concentration.

Angiotensin converting enzyme immobilized on magnetic beads as a tool for ligand fishing.

Science.gov (United States)

de Almeida, Fernando G; Vanzolini, Kenia L; Cass, Quezia B

2017-01-05

Angiotensin converting enzyme (ACE) presents an important role in blood pressure regulation, since that converts angiotensin I to the vasoconstrictor angiotensin II. Some commercially available ACE inhibitors are captopril, lisinopril and enalapril; due to their side effects, naturally occurring inhibitors have been prospected. In order to endorse this research field we have developed a new tool for ACE ligand screening. To this end, ACE was extracted from bovine lung, purified and chemically immobilized in modified ferrite magnetic beads (ACE-MBs). The ACE-MBs have shown a Michaelian kinetic behavior towards hippuryl-histidyl-leucine. Moreover, as proof of concept, the ACE-MBs was inhibited by lisinopril with a half maximal inhibitory concentration (IC 50 ) of 10nM. At the fishing assay, ACE-MBs were able not only to fish out the reference inhibitor, but also one peptide from a pool of tryptic digested BSA. In conclusion, ACE-MBs emerge as new straightforward tool for ACE kinetics determination, inhibition and binder screening. Copyright © 2016 Elsevier B.V. All rights reserved.

Immobilization of lipases on alkyl silane modified magnetic nanoparticles: effect of alkyl chain length on enzyme activity.

Directory of Open Access Journals (Sweden)

Jiqian Wang

Full Text Available BACKGROUND: Biocatalytic processes often require a full recycling of biocatalysts to optimize economic benefits and minimize waste disposal. Immobilization of biocatalysts onto particulate carriers has been widely explored as an option to meet these requirements. However, surface properties often affect the amount of biocatalysts immobilized, their bioactivity and stability, hampering their wide applications. The aim of this work is to explore how immobilization of lipases onto magnetite nanoparticles affects their biocatalytic performance under carefully controlled surface modification. METHODOLOGY/PRINCIPAL FINDINGS: Magnetite nanoparticles, prepared through a co-precipitation method, were coated with alkyl silanes of different alkyl chain lengths to modulate their surface hydrophobicity. Candida rugosa lipase was then directly immobilized onto the modified nanoparticles through hydrophobic interaction. Enzyme activity was assessed by catalytic hydrolysis of p-nitrophenyl acetate. The activity of immobilized lipases was found to increase with increasing chain length of the alkyl silane. Furthermore, the catalytic activities of lipases immobilized on trimethoxyl octadecyl silane (C18 modified Fe(3O(4 were a factor of 2 or more than the values reported from other surface immobilized systems. After 7 recycles, the activities of the lipases immobilized on C18 modified nanoparticles retained 65%, indicating significant enhancement of stability as well through hydrophobic interaction. Lipase immobilized magnetic nanoparticles facilitated easy separation and recycling with high activity retaining. CONCLUSIONS/SIGNIFICANCE: The activity of immobilized lipases increased with increasing alkyl chain length of the alkyl trimethoxy silanes used in the surface modification of magnetite nanoparticles. Lipase stability was also improved through hydrophobic interaction. Alkyl silane modified magnetite nanoparticles are thus highly attractive carriers for

Immobilization of lipases on alkyl silane modified magnetic nanoparticles: effect of alkyl chain length on enzyme activity.

Science.gov (United States)

Wang, Jiqian; Meng, Gang; Tao, Kai; Feng, Min; Zhao, Xiubo; Li, Zhen; Xu, Hai; Xia, Daohong; Lu, Jian R

2012-01-01

Biocatalytic processes often require a full recycling of biocatalysts to optimize economic benefits and minimize waste disposal. Immobilization of biocatalysts onto particulate carriers has been widely explored as an option to meet these requirements. However, surface properties often affect the amount of biocatalysts immobilized, their bioactivity and stability, hampering their wide applications. The aim of this work is to explore how immobilization of lipases onto magnetite nanoparticles affects their biocatalytic performance under carefully controlled surface modification. Magnetite nanoparticles, prepared through a co-precipitation method, were coated with alkyl silanes of different alkyl chain lengths to modulate their surface hydrophobicity. Candida rugosa lipase was then directly immobilized onto the modified nanoparticles through hydrophobic interaction. Enzyme activity was assessed by catalytic hydrolysis of p-nitrophenyl acetate. The activity of immobilized lipases was found to increase with increasing chain length of the alkyl silane. Furthermore, the catalytic activities of lipases immobilized on trimethoxyl octadecyl silane (C18) modified Fe(3)O(4) were a factor of 2 or more than the values reported from other surface immobilized systems. After 7 recycles, the activities of the lipases immobilized on C18 modified nanoparticles retained 65%, indicating significant enhancement of stability as well through hydrophobic interaction. Lipase immobilized magnetic nanoparticles facilitated easy separation and recycling with high activity retaining. The activity of immobilized lipases increased with increasing alkyl chain length of the alkyl trimethoxy silanes used in the surface modification of magnetite nanoparticles. Lipase stability was also improved through hydrophobic interaction. Alkyl silane modified magnetite nanoparticles are thus highly attractive carriers for enzyme immobilization enabling efficient enzyme recovery and recycling.

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

Science.gov (United States)

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.

Immobilization of the Enzyme Glucose Oxidase on Both Bulk and Porous SiO2 Surfaces

Directory of Open Access Journals (Sweden)

Fulvia Sinatra

2008-09-01

Full Text Available Silicon dioxide surfaces, both bulk and porous, were used to anchor the enzyme glucose oxidase. The immobilization protocol was optimized and the samples characterized using X-ray Photoelectron Spectroscopy, Energy Dispersive X-rays coupled to scanning electron microscopy and enzymatic activity measurements. We show that a uniform layer was obtained by activating the oxide before immobilization. X-ray Photoelectron Spectroscopy measurements carried out on bulk oxide showed that the silicon substrate signal was fully screened after the enzyme deposition showing the absence of uncovered surface regions. The enzyme presence was detected monitoring both the C 1s and N 1s signals. Finally, enzymatic activity measurements confirmed that the glucose oxidase activity was preserved after immobilization and maintained after three months of shelf life if the sample was properly stored. The importance of using porous silicon oxide to maximize the surface area was also evidenced.

Depolymerization of chitosan by enzymes from the digestive tract of ...

African Journals Online (AJOL)

A complex of enzymes was isolated in a preparation derived from the digestive tract of sea cucumber, Stichopus japonicus. Hydrolysis of chitosan using this enzyme preparation decreased its molecular weight (Mw), increased its water solubility and produced water-soluble chitosan (WSC). The conditions for hydrolysis were ...

Digestive enzymes of the Californian two-spot octopus, Octopus bimaculoides (Pickford and McConnaughey, 1949).

Science.gov (United States)

Ibarra-García, Laura Elizabeth; Tovar-Ramírez, Dariel; Rosas, Carlos; Campa-Córdova, Ángel Isidro; Mazón-Suástegui, José Manuel

2018-01-01

Octopus bimaculoides is an important commercially fished species in the California Peninsula with aquaculture potential; however, to date limited information is available regarding its digestive physiology. The objective of this study was focused on biochemically characterizing the main enzymes involved in the digestion of O. bimaculoides. Optimum pH, temperature and thermostability were determined for amylases, lipases, trypsin and chymotrypsin; optimum pH and protease inhibitor effect were assessed for acidic and alkaline proteases, and the effect of divalent ions on trypsin and chymotrypsin activity was evaluated in enzymatic extracts from the digestive (DG) and salivary glands (SG) and crop gastric juices (GJ). High amylase activity was detected in GD and GJ whereas this activity is very low in other cephalopods. Salivary glands had the greatest activity in most of the enzyme groups, showing the importance of this organ in digestion. Optimum pH was different depending on the organ and enzyme analyzed. The optimum pH in DG was 3 showing the predominance of acidic proteases in the digestion process. All enzymes were resistant and stable at high temperatures in contrast with other marine species. Trypsin and chymotrypsin activity were highly incremented with the presence of Mg 2+ , Co 2+ , Cu 2+ and Zn 2+ in some tissues. The inhibitor assay showed the importance of serine proteases, metalloproteases and aspartic proteases in the digestive process of this species. This study is the first in assessing the main digestive enzymes of O. bimaculoides and in remarking the importance of other digestive enzyme groups besides proteases in octopuses. Copyright © 2017 Elsevier Inc. All rights reserved.

Synthesis and effect of modification on methacylate - acrylate microspheres for Trametes versicolor laccase enzyme immobilization

Science.gov (United States)

Mazlan, Siti Zulaikha; Hanifah, Sharina Abu

2014-09-01

Immobilization of laccase on the modified copolymer methacrylate-acrylate microspheres was studied. A poly (glycidyl methacrylate-co-n-butyl acrylate) microsphere consists of epoxy groups were synthesized using suspension photocuring technique. The epoxy group in poly (GMA-nBA) microspheres were converted into amino groups with aldehyde group. Laccase immobilization is based on having the amino groups on the enzyme surface and aldehyde group on the microspheres via covalent binding. Fourier transform infrared spectroscopy (FT-IR) analysis proved the successful surface modification on microspheres. The FTIR spectrum shows the characteristic peaks at 1646 cm-1 assigned to the conformation of the polymerization that took place between monomer GMA and nBA respectively. In addition, after modification, FTIR peaks that assigned to the epoxy ring (844 cm-1 and 904 cm-1) were decreased. The results obtained from FTIR method signify good agreement with the epoxy content method. Hence, the activity of the laccase-immobilized microspheres increased upon increasing the epoxy content. Furthermore, poly (GMA-nBA) exhibited uniform microspheres with below 2 μm surface. Immobilized enzyme showed a broader pH profile and higher temperature compared native enzyme.

Continuous glycerolysis in an immobilized enzyme packed reactor for industrial monoacylglycerol production

DEFF Research Database (Denmark)

. In spite of optimal reaction conditions a complex heterogeneous reactant mixture with a glycerol in oil emulsion occurs. Hence, the movement of material from phase to phase as well as through the catalyst pores becomes important since it can influence the performance of the immobilized enzyme reactor...... and sunflower oil dissolved in a binary tert-butanol:tert-pentanol medium. Practical design-related issues such as required reaction time, enzyme capacity, expansion of the enzyme during wetting, and the effect of different column length-to-diameter ratios, fluid velocities and particle sizes of the enzymes...

Vitellin- and hemoglobin-digesting enzymes in Rhipicephalus (Boophilus) microplus larvae and females.

Science.gov (United States)

Estrela, Andréia Bergamo; Seixas, Adriana; Teixeira, Vivian de Oliveira Nunes; Pinto, Antônio Frederico Michel; Termignoni, Carlos

2010-12-01

The aim of the present study was to address the involvement of Rhipicephalus microplus larval cysteine endopeptidase (RmLCE) in protein digestion in R. microplus larvae and adult females. In this work, an improved purification protocol for native RmLCE was developed. Partial amino acid sequence of the purified enzyme indicates that it is the same enzyme as Boophilus microplus cathepsin-L1 (BmCL1). When vitellin (Vt) degradation by egg and larval enzymes was analyzed, stage-specific differences for RmLCE activity in comparison to vitellin-degrading cysteine endopeptidase (VTDCE) were observed. RmLCE is also able to degrade host hemoglobin (Hb). In agreement, an acidic cysteine endopeptidase activity was detected in larval gut. It was shown that cysteine and aspartic endopeptidases are involved in Vt and Hb digestion in R. microplus larvae and females. Interestingly, we observed that the aspartic endopeptidase Boophilus yolk cathepsin (BYC) is associated with a cysteine endopeptidase activity, in larvae. Synergic hemoglobin digestion by BYC and RmLCE was observed and indicates the presence of an Hb-degrading enzymatic cascade involving these enzymes. Our results suggest that RmLCE/BmCL1 has a continued role in vitellin and hemoglobin digestion during tick development. Copyright © 2010 Elsevier Inc. All rights reserved.

Improved Starch Digestion of Sucrase-deficient Shrews Treated With Oral Glucoamylase Enzyme Supplements.

Science.gov (United States)

Nichols, Buford L; Avery, Stephen E; Quezada-Calvillo, Roberto; Kilani, Shadi B; Lin, Amy Hui-Mei; Burrin, Douglas G; Hodges, Benjamin E; Chacko, Shaji K; Opekun, Antone R; Hindawy, Marwa El; Hamaker, Bruce R; Oda, Sen-Ichi

2017-08-01

Although named because of its sucrose hydrolytic activity, this mucosal enzyme plays a leading role in starch digestion because of its maltase and glucoamylase activities. Sucrase-deficient mutant shrews, Suncus murinus, were used as a model to investigate starch digestion in patients with congenital sucrase-isomaltase deficiency.Starch digestion is much more complex than sucrose digestion. Six enzyme activities, 2 α-amylases (Amy), and 4 mucosal α-glucosidases (maltases), including maltase-glucoamylase (Mgam) and sucrase-isomaltase (Si) subunit activities, are needed to digest starch to absorbable free glucose. Amy breaks down insoluble starch to soluble dextrins; mucosal Mgam and Si can either directly digest starch to glucose or convert the post-α-amylolytic dextrins to glucose. Starch digestion is reduced because of sucrase deficiency and oral glucoamylase enzyme supplement can correct the starch maldigestion. The aim of the present study was to measure glucogenesis in suc/suc shrews after feeding of starch and improvement of glucogenesis by oral glucoamylase supplements. Sucrase mutant (suc/suc) and heterozygous (+/suc) shrews were fed with C-enriched starch diets. Glucogenesis derived from starch was measured as blood C-glucose enrichment and oral recombinant C-terminal Mgam glucoamylase (M20) was supplemented to improve starch digestion. After feedings, suc/suc and +/suc shrews had different starch digestions as shown by blood glucose enrichment and the suc/suc had lower total glucose concentrations. Oral supplements of glucoamylase increased suc/suc total blood glucose and quantitative starch digestion to glucose. Sucrase deficiency, in this model of congenital sucrase-isomaltase deficiency, reduces blood glucose response to starch feeding. Supplementing the diet with oral recombinant glucoamylase significantly improved starch digestion in the sucrase-deficient shrew.

Development of Digestive Enzyme of Patin Pangasius hypohthalmus Larvae

Directory of Open Access Journals (Sweden)

I. Effendi

2007-01-01

Full Text Available Culture of patin Pangasius hypophthalmus especially larval rearing  very depends on the supply of natural food as energy source.  Artemia is the main natural food for fish larvae as a starter food, but its price is high.  To reduce production cost, farmers tend to reduce the feeding frequency and shorten  the Artemia feeding period.  Altering feeding regime however may reduce fry quality. This relate to the availability of digestive enzymes.  The objective of this study was  to examine digestive enzymes activity in patin larvae fed with  different feeding regime.  By shorten feeding period with Artemia to 2-4 days and Tubifex,substitution, the enzymes activity of protease, lipase and amylase were revealed similar pattern   The enzymes activity tends to increase and reach the peak at day 7 , and decrease later on until day 15 after hatching.  Survival rate of fish were varied for each treatment, and the highest survival rate was obtained when larvae were fed by Artemia for 8 days.  Blood worm were not fully digested by patin larvae at early stage. Keywords: enzyme, digestion, patin, Pangasius hypophthalmus   ABSTRAK Proses budidaya ikan patin, Pangasius hypophthalmus terutama pembenihan sangat tergantung oleh ketersediaan pakan alami sebagai sumber energinya. Artemia merupakan pakan alami yang banyak diberikan pada saat larva ikan mulai makan, namun harganya relatif tinggi. Untuk menekan biaya produksi, petani ikan patin cenderung mengurangi frekuensi pemberian Artemia dan mempersingkat waktu pemberiannya. Penggeseran jadwal ini diduga mengakibatkan penurunan kualitas benih ikan patin yang dihasilkan yang berhubungan dengan kesiapan enzim pencernaannya. Penelitian ini dilakukan untuk mengetahui aktivitas enzim pada larva ikan patin dengan jadwal pemberian pakan yang berbeda. Dengan memotong waktu pemberian Artemia 2 - 4 hari dan disubstitusi dengan Tubifex, aktifitas enzim protease, lipase dan amilase pada larva ikan patin, memiliki

Low-cost, easy-to-prepare magnetic chitosan microparticles for enzymes immobilization

Czech Academy of Sciences Publication Activity Database

Pospišková, K.; Šafařík, Ivo

2013-01-01

Roč. 96, č. 2 (2013), s. 545-548 ISSN 0144-8617 R&D Projects: GA MŠk(CZ) LD13021 Institutional support: RVO:67179843 Keywords : Chitosan * Magnetite * Microwave irradiation * Enzymes immobilization Subject RIV: CE - Biochemistry Impact factor: 3.916, year: 2013

Immobilization of enzymes by radiation-induced polymerization of glass-forming monomers

International Nuclear Information System (INIS)

Yoshida, M.; Kumakura, M.; Kaetsu, I.

1979-01-01

The effect of cooling rate of a monomeric system on the porosity and activity of an immobilized enzyme prepared by radiation-induced polymerization of 2-hydroxyethyl methacrylate at low temperatures has been studied. Slow cooling gave the same effect on porosity of the polymer as decreasing the monomer concentration. A glass-forming solvent such as diethylene glycol was added to water to study the effect of the supercooling tendency of the solvent. Addition of diethylene glycol decreased porosity and also enzymic activity. Water was replaced by the miscible solvent p-dioxane and the immiscible solvent n-decane in order to clarify the effect of solvent. p-Dioxane had a similar effect to water on the relation between the monomer concentration, porosity and activity. On the other hand, polymer prepared from the system containing n-decane showed different immobilization properties owing to the presence of independent pores in the matrix. (author)

Effect of water quality and confounding factors on digestive enzyme activities in Gammarus fossarum.

Science.gov (United States)

Charron, L; Geffard, O; Chaumot, A; Coulaud, R; Queau, H; Geffard, A; Dedourge-Geffard, O

2013-12-01

The feeding activity and subsequent assimilation of the products resulting from food digestion allow organisms to obtain energy for growth, maintenance and reproduction. Among these biological parameters, we studied digestive enzymes (amylase, cellulase and trypsin) in Gammarus fossarum to assess the impact of contaminants on their access to energy resources. However, to enable objective assessment of a toxic effect of decreased water quality on an organisms' digestive capacity, it is necessary to establish reference values based on its natural variability as a function of changing biotic and abiotic factors. To limit the confounding influence of biotic factors, a caging approach with calibrated male organisms from the same population was used. This study applied an in situ deployment at 23 sites of the Rhone basin rivers, complemented by a laboratory experiment assessing the influence of two abiotic factors (temperature and conductivity). The results showed a small effect of conductivity on cellulase activity and a significant effect of temperature on digestive enzyme activity but only at the lowest temperature (7 °C). The experimental conditions allowed us to define an environmental reference value for digestive enzyme activities to select sites where the quality of the water impacted the digestive capacity of the organisms. In addition to the feeding rate, this study showed the relevance of digestive enzymes as biomarkers to be used as an early warning tool to reflect organisms' health and the chemical quality of aquatic ecosystems.

Nucleic acids digestion by enzymes in the stomach of snakehead (Channa argus) and banded grouper (Epinephelus awoara).

Science.gov (United States)

Liu, Yu; Zhang, Yanfang; Jiang, Wei; Wang, Jing; Pan, Xiaoming; Wu, Wei; Cao, Minjie; Dong, Ping; Liang, Xingguo

2017-02-01

Dietary nucleic acids (NAs) were important nutrients. However, the digestion of NAs in stomach has not been studied. In this study, the digestion of NAs by enzymes from fish stomach was investigated. The snakehead pepsins (SP) which were the main enzymes in stomach were extracted and purified. The purity of SP was evaluated by SDS-PAGE and HPLC. The snakehead pepsin 2 (SP2) which was the main component in the extracts was used for investigating the protein and NAs digestion activity. SP2 could digest NAs, including λ DNA and salmon sperm DNA. Interestingly, the digestion could be inhibited by treatment of alkaline solution at pH 8.0 and pepstatin A, and the digestion could happen either in the presence or absence of hemoglobin (Hb) and BSA as the protein substrates. Similarly, the stomach enzymes of banded grouper also showed the NAs digestion activity. NAs could be digested by the stomach enzymes of snakehead and banded grouper. It may be helpful for understanding both animal nutrition and NAs metabolic pathway.

Enzyme-Catalyzed Oxidation of 17β-Estradiol Using Immobilized Laccase from Trametes versicolor

Science.gov (United States)

Cardinal-Watkins, Chantale; Nicell, Jim A.

2011-01-01

Many natural and synthetic estrogens are amenable to oxidation through the catalytic action of oxidative enzymes such as the fungal laccase Trametes versicolor. This study focused on characterizing the conversion of estradiol (E2) using laccase that had been immobilized by covalent bonding onto silica beads contained in a bench-scale continuous-flow packed bed reactor. Conversion of E2 accomplished in the reactor declined when the temperature of the system was changed from room temperature to just above freezing at pH 5 as a result of a reduced rate of reaction rather than inactivation of the enzyme. Similarly, conversion increased when the system was brought to warmer temperatures. E2 conversion increased when the pH of the influent to the immobilized laccase reactor was changed from pH 7 to pH 5, but longer-term experiments showed that the enzyme is more stable at pH 7. Results also showed that the immobilized laccase maintained its activity when treating a constant supply of aqueous E2 at a low mean residence time over a 12-hour period and when treating a constant supply of aqueous E2 at a high mean residence time over a period of 9 days. PMID:21869925

Production of immobilized cellulase enzyme by some microorganisms from the rice straw agro-waste using γ-irradiation

International Nuclear Information System (INIS)

Mohamed, M.A.Z.

2014-01-01

Studies were carried out using 14 fungal cultures screened for their ability to produce cellulase enzymes. A .hortai was selected for the present research as a potent cellulase producer. Cultural and nutritional factors affecting cellulase production were also investigated in order to optimize the fermentation conditions for the maximization of production. The obtained results revealed that, the maximum cellulase production (0.23 U/ml) was achieved after 96 h in a liquid medium (Ph 7.0) inoculated with 10% v/v inoculum size, at temperature 37 ºC, containing (gL -1 ) CMC, 5.0; yeast extract, 0.1; (NH 4 ) 2 SO 4 , 0.5; KH 2 PO 4 , 10.0; MgSO 4 .7H 2 O, 0.1 and NaCl, 0.2. The activity remained almost stable between ph 6.0 and 7.0. The highest cellulase activity (1.18 U/ml) was obtained at a lactose concentration of (5.0 gL -1 ). Partial purification of the crude cellulase by ammonium sulphate 70% saturation showed the highest specific enzyme activity and purification fold (2.3 U/mg protein and 2.12 fold, respectively). Different carriers and methods were used to select the suitable one for cellulase immobilization. Poly (acrylamide-co-acrylic acid) prepared by diazotization method increase S.E.A and the amount of immobilized enzyme to be (2.3 U/mg protein and 2.8 mg), respectively. The immobilized cellulase shows better operational stability, including wider ph and thermal ranges. The immobilized cellulase remained fully active up to 60°C. The kinetic parameters Km and Vmax were determined. The increase of the apparent Km after immobilization clearly indicates an apparent lower affinity of the immobilized enzyme for its substrate than the free enzyme. The resulting immobilized cellulase exhibited good reusability on degradation of rice straw agricultural wastes and also show good storage stability, that it lost only 17 % of its initial activity after 6 weeks.

Changes in growth, survival and digestive enzyme activities of Asian ...

African Journals Online (AJOL)

A study was conducted to determine the effects of different dietary treatments on the growth, survival and digestive enzyme activities of Mystus nemurus larvae. Newly hatched larvae were reared for 14 days in twelve 15 L glass aquaria (for growth and survival) and eight 300 L fiberglass tanks (for enzyme samples) at a ...

FT-IR microspectroscopy characterization of supports for enzyme immobilization in biosensing applications

Science.gov (United States)

Portaccio, M.; Della Ventura, B.; Gabrovska, K.; Marinov, I.; Godjevargova, T.; Mita, D. G.; Lepore, M.

2010-04-01

The investigation of materials suitable for enzyme immobilization in biosensing applications has a widespread interest. There are many studies on physico-chemical properties of these materials at macroscopic level but few studies have been devoted to examine and correlate these properties at microscopic level. FT-IR spectroscopy with Micro-Attenuated Total Reflection (Micro-ATR) approach can be extremely useful for understanding a variety of aspects of materials which can be used for optimising immobilization procedures. Moreover, this experimental approach is particularly simple to use (no sample preparation is required) and minimally invasive. Using a Perkin Elmer Spectrum One FT-IR spectrometer equipped with a mercury-cadmium-telluride detector and a micro-ATR element we investigated different materials used for immobilization procedures with various enzymes widely used for biosensing in environmental and clinical applications. In particular, composite membranes constituted by a chemically modified poly-acrylonitrile (PAN) membrane plus layers of tethered chitosan of different molecular weight have been examined. Also silica gel matrices without and with glucose oxidase have been investigated. Spectra have been analysed and the contribution of principal functional groups has been evidenced.

Magnetically modified bacterial cellulose: A promising carrier for immobilization of affinity ligands, enzymes, and cells

Energy Technology Data Exchange (ETDEWEB)

Baldikova, Eva [Global Change Research Institute, CAS, Na Sadkach 7, 370 05 Ceske Budejovice (Czech Republic); Pospiskova, Kristyna [Regional Centre of Advanced Technologies and Materials, Palacky University, Slechtitelu 27, 783 71 Olomouc (Czech Republic); Ladakis, Dimitrios; Kookos, Ioannis K. [Department of Chemical Engineering, University of Patras, 26504 Patras, Rio (Greece); Koutinas, Apostolis A. [Department of Food Science and Human Nutrition, Agricultural University of Athens, Iera Odos 75, Athens 11855 (Greece); Safarikova, Mirka [Global Change Research Institute, CAS, Na Sadkach 7, 370 05 Ceske Budejovice (Czech Republic); Department of Nanobiotechnology, Biology Centre, ISB, CAS, Na Sadkach 7, 370 05 Ceske Budejovice (Czech Republic); Safarik, Ivo, E-mail: safarik@nh.cas.cz [Global Change Research Institute, CAS, Na Sadkach 7, 370 05 Ceske Budejovice (Czech Republic); Regional Centre of Advanced Technologies and Materials, Palacky University, Slechtitelu 27, 783 71 Olomouc (Czech Republic); Department of Nanobiotechnology, Biology Centre, ISB, CAS, Na Sadkach 7, 370 05 Ceske Budejovice (Czech Republic)

2017-02-01

Bacterial cellulose (BC) produced by Komagataeibacter sucrofermentans was magnetically modified using perchloric acid stabilized magnetic fluid. Magnetic bacterial cellulose (MBC) was used as a carrier for the immobilization of affinity ligands, enzymes and cells. MBC with immobilized reactive copper phthalocyanine dye was an efficient adsorbent for crystal violet removal; the maximum adsorption capacity was 388 mg/g. Kinetic and thermodynamic parameters were also determined. Model biocatalysts, namely bovine pancreas trypsin and Saccharomyces cerevisiae cells were immobilized on MBC using several strategies including adsorption with subsequent cross-linking with glutaraldehyde and covalent binding on previously activated MBC using sodium periodate or 1,4-butanediol diglycidyl ether. Immobilized yeast cells retained approximately 90% of their initial activity after 6 repeated cycles of sucrose solution hydrolysis. Trypsin covalently bound after MBC periodate activation was very stable during operational stability testing; it could be repeatedly used for ten cycles of low molecular weight substrate hydrolysis without loss of its initial activity. - Highlights: • Bacterial cellulose was magnetically modified with magnetic fluid. • Magnetic cellulose is an efficient carrier for affinity ligands. • Enzymes and cells can be efficiently immobilized to magnetic cellulose.

Immobilization of enzymes on radiation-modified gelatine gel by using a chemical cross-linking agent

International Nuclear Information System (INIS)

Bachmann, S.; Gebicka, L.; Galant, S.

1981-01-01

Investigations into the effect of ionizing radiation on the gelatine gels have shown that water-insoluble gel can be formed under suitable irradiation conditions. To establish the optimal conditions for the processing of the insoluble gel, the yield of cross-linking has been determined for gelatine solutions and its gels irradiated with various doses in the absence and in the presence of oxygen. Glucose isomerase (GI) was used as a test enzyme for immobilization on the gelatine gel. This enzyme which catalyses the isomerization of glucose to fructose has been used on the commercial-scale production of high fructose syrups. The support matrix chosen for the enzyme immobilization has been obtained by irradiating 4% wt/vol. de-aerated gelatine gel at a dose of 1.5 x 10 4 kGy at 15 0 C. Actinoplanes missouriensis cells containing GI were mixed with gelatine gel particles and cross-linked with glutaraldehyde. It was found that the immobilized GI can be successfully applied in the continuous isomerization of glucose to fructose. (author)

The complexities of hydrolytic enzymes from the termite digestive system.

Science.gov (United States)

Saadeddin, Anas

2014-06-01

The main challenge in second generation bioethanol production is the efficient breakdown of cellulose to sugar monomers (hydrolysis). Due to the recalcitrant character of cellulose, feedstock pretreatment and adapted hydrolysis steps are needed to obtain fermentable sugar monomers. The conventional industrial production process of second-generation bioethanol from biomass comprises several steps: thermochemical pretreatment, enzymatic hydrolysis and sugar fermentation. This process is undergoing continuous optimization in order to increase the bioethanol yield and reduce the economic cost. Therefore, the discovery of new enzymes with high lignocellulytic activity or new strategies is extremely important. In nature, wood-feeding termites have developed a sophisticated and efficient cellulose degrading system in terms of the rate and extent of cellulose hydrolysis and exploitation. This system, which represents a model for digestive symbiosis has attracted the attention of biofuel researchers. This review describes the termite digestive system, gut symbionts, termite enzyme resources, in vitro studies of isolated enzymes and lignin degradation in termites.

Cellulose digestion in Monochamus marmorator Kby. (coleoptera: Cerambycidae): role of acquired fungal enzymes

Energy Technology Data Exchange (ETDEWEB)

Kukol, J.J.; Martin, M.M.

1986-05-01

Larvae of the balsam fir sawyer, Monochamus marmorator Kby. (Coleoptera, Cerambycidae), contain midgut digestive enzymes active against hemicellulose and cellulose. Cellulases from larvae fed on balsam fir wood infected with the fungus, Trichoderma harzianum Rifai (Deuteromycetes, Moniliales, Moniliaceae), were found to be identical to those of the cellulase complex produced by this fungus when compared using chromatography, electrophoresis, and isofocusing. When larvae are maintained on a fungusfree diet, their midgut fluids lack cellulolytic activity, and they are unable to digest cellulose. Cellulolytic capacity can be restored by feeding the larvae wood permeated by fungi. We conclude that the enzymes which enable M. marmorator larvae to digest cellulose are not produced by the larvae. Instead, the larvae acquire the capacity to digest cellulose by ingesting active fungal cellulases while feeding in fungus-infected wood.

Cellulose digestion in Monochamus marmorator Kby. (coleoptera: Cerambycidae): role of acquired fungal enzymes

International Nuclear Information System (INIS)

Kukol, J.J.; Martin, M.M.

1986-01-01

Larvae of the balsam fir sawyer, Monochamus marmorator Kby. (Coleoptera, Cerambycidae), contain midgut digestive enzymes active against hemicellulose and cellulose. Cellulases from larvae fed on balsam fir wood infected with the fungus, Trichoderma harzianum Rifai (Deuteromycetes, Moniliales, Moniliaceae), were found to be identical to those of the cellulase complex produced by this fungus when compared using chromatography, electrophoresis, and isofocusing. When larvae are maintained on a fungusfree diet, their midgut fluids lack cellulolytic activity, and they are unable to digest cellulose. Cellulolytic capacity can be restored by feeding the larvae wood permeated by fungi. We conclude that the enzymes which enable M. marmorator larvae to digest cellulose are not produced by the larvae. Instead, the larvae acquire the capacity to digest cellulose by ingesting active fungal cellulases while feeding in fungus-infected wood

Self-assembled monolayers of 1-alkenes on oxidized platinum surfaces as platforms for immobilized enzymes for biosensing

International Nuclear Information System (INIS)

Alonso, Jose Maria; Bielen, Abraham A.M.; Olthuis, Wouter; Kengen, Servé W.M.; Zuilhof, Han; Franssen, Maurice C.R.

2016-01-01

Highlights: • Three different oxidases are covalently attached to alkene based SAMs on PtOx. • Attached enzymes remain active and their activity is assessed by chronoamperometry. • Functionalized PtOx allows electron mediator free chronoamperometry measurements. • The thus formed enzyme electrodes are useful as biosensors for glucose and lactate. • Immobilization of human HAOX foresees in vivo lactate monitoring in humans. - Abstract: Alkene-based self-assembled monolayers grafted on oxidized Pt surfaces were used as a scaffold to covalently immobilize oxidase enzymes, with the aim to develop an amperometric biosensor platform. NH_2-terminated organic layers were functionalized with either aldehyde (CHO) or N-hydroxysuccinimide (NHS) ester-derived groups, to provide anchoring points for enzyme immobilization. The functionalized Pt surfaces were characterized by X-ray photoelectron spectroscopy (XPS), static water contact angle (CA), infrared reflection absorption spectroscopy (IRRAS) and atomic force microscopy (AFM). Glucose oxidase (GOX) was covalently attached to the functionalized Pt electrodes, either with or without additional glutaraldehyde crosslinking. The responses of the acquired sensors to glucose concentrations ranging from 0.5 to 100 mM were monitored by chronoamperometry. Furthermore, lactate oxidase (LOX) and human hydroxyacid oxidase (HAOX) were successfully immobilized onto the PtOx surface platform. The performance of the resulting lactate sensors was investigated for lactate concentrations ranging from 0.05 to 20 mM. The successful attachment of active enzymes (GOX, LOX and HAOX) on Pt electrodes demonstrates that covalently functionalized PtOx surfaces provide a universal platform for the development of oxidase enzyme-based sensors.

Self-assembled monolayers of 1-alkenes on oxidized platinum surfaces as platforms for immobilized enzymes for biosensing

Energy Technology Data Exchange (ETDEWEB)

Alonso, Jose Maria; Bielen, Abraham A.M. [Laboratory of Organic Chemistry, Wageningen University, Dreijenplein 8, 6703 HB, Wageningen (Netherlands); Olthuis, Wouter [BIOS Lab on a Chip Group, MESA+ and MIRA Institutes, University of Twente, P.O. Box 217, 7500 AE Enschede (Netherlands); Kengen, Servé W.M. [Laboratory of Microbiology, Wageningen University, 6703HB Wageningen (Netherlands); Zuilhof, Han, E-mail: han.zuilhof@wur.nl [Laboratory of Organic Chemistry, Wageningen University, Dreijenplein 8, 6703 HB, Wageningen (Netherlands); Department of Chemical and Materials Engineering, King Abdulaziz University, Jeddah 22254 (Saudi Arabia); Franssen, Maurice C.R., E-mail: maurice.franssen@wur.nl [Laboratory of Organic Chemistry, Wageningen University, Dreijenplein 8, 6703 HB, Wageningen (Netherlands)

2016-10-15

Highlights: • Three different oxidases are covalently attached to alkene based SAMs on PtOx. • Attached enzymes remain active and their activity is assessed by chronoamperometry. • Functionalized PtOx allows electron mediator free chronoamperometry measurements. • The thus formed enzyme electrodes are useful as biosensors for glucose and lactate. • Immobilization of human HAOX foresees in vivo lactate monitoring in humans. - Abstract: Alkene-based self-assembled monolayers grafted on oxidized Pt surfaces were used as a scaffold to covalently immobilize oxidase enzymes, with the aim to develop an amperometric biosensor platform. NH{sub 2}-terminated organic layers were functionalized with either aldehyde (CHO) or N-hydroxysuccinimide (NHS) ester-derived groups, to provide anchoring points for enzyme immobilization. The functionalized Pt surfaces were characterized by X-ray photoelectron spectroscopy (XPS), static water contact angle (CA), infrared reflection absorption spectroscopy (IRRAS) and atomic force microscopy (AFM). Glucose oxidase (GOX) was covalently attached to the functionalized Pt electrodes, either with or without additional glutaraldehyde crosslinking. The responses of the acquired sensors to glucose concentrations ranging from 0.5 to 100 mM were monitored by chronoamperometry. Furthermore, lactate oxidase (LOX) and human hydroxyacid oxidase (HAOX) were successfully immobilized onto the PtOx surface platform. The performance of the resulting lactate sensors was investigated for lactate concentrations ranging from 0.05 to 20 mM. The successful attachment of active enzymes (GOX, LOX and HAOX) on Pt electrodes demonstrates that covalently functionalized PtOx surfaces provide a universal platform for the development of oxidase enzyme-based sensors.

Immobilization of β-glucosidase onto mesoporous silica support: Physical adsorption and covalent binding of enzyme

Directory of Open Access Journals (Sweden)

Ivetić Darjana Ž.

2014-01-01

Full Text Available This paper investigates β-glucosidase immobilization onto mesoporous silica support by physical adsorption and covalent binding. The immobilization was carried out onto micro-size silica aggregates with the average pore size of 29 nm. During physical adsorption the highest yield of immobilized β-glucosidase was obtained at initial protein concentration of 0.9 mg ml-1. Addition of NaCl increased 1.7-fold, while Triton X-100 addition decreased 6-fold yield of adsorption in comparison to the one obtained without any addition. Covalently bonded β-glucosidase, via glutaraldehyde previously bonded to silanized silica, had higher yield of immobilized enzyme as well as higher activity and substrate affinity in comparison to the one physically adsorbed. Covalent binding did not considerably changed pH and temperature stability of obtained biocatalyst in range of values that are commonly used in reactions in comparison to unbounded enzyme. Furthermore, covalent binding provided biocatalyst which retained over 70% of its activity after 10 cycles of reuse. [Projekat Ministarstva nauke Republike Srbije, br. III 45021

Quantitative analysis of immobilized penicillinase using enzyme-modified AlGaN/GaN field-effect transistors.

Science.gov (United States)

Müntze, Gesche Mareike; Baur, Barbara; Schäfer, Wladimir; Sasse, Alexander; Howgate, John; Röth, Kai; Eickhoff, Martin

2015-02-15

Penicillinase-modified AlGaN/GaN field-effect transistors (PenFETs) are utilized to systematically investigate the covalently immobilized enzyme penicillinase under different experimental conditions. We demonstrate quantitative evaluation of covalently immobilized penicillinase layers on pH-sensitive field-effect transistors (FETs) using an analytical kinetic PenFET model. This kinetic model is explicitly suited for devices with thin enzyme layers that are not diffusion-limited, as it is the case for the PenFETs discussed here. By means of the kinetic model it was possible to extract the Michaelis constant of covalently immobilized penicillinase as well as relative transport coefficients of the different species associated with the enzymatic reaction which, exempli gratia, give information about the permeability of the enzymatic layer. Based on this analysis we quantify the reproducibility and the stability of the analyzed PenFETs over the course of 33 days as well as the influence of pH and buffer concentration on the properties of the enzymatic layer. Thereby the stability measurements reveal a Michalis constant KM of (67 ± 13)μM while the chronological development of the relative transport coefficients suggests a detachment of physisorbed penicillinase during the first two weeks since production. Our results show that AlGaN/GaN PenFETs prepared by covalent immobilization of a penicillinase enzyme layer present a powerful tool for quantitative analysis of enzyme functionality. Copyright © 2014 Elsevier B.V. All rights reserved.

Transforming Growth Factor β/Activin Signaling Functions as a Sugar-Sensing Feedback Loop to Regulate Digestive Enzyme Expression

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Wen-bin Alfred Chng

2014-10-01

Full Text Available Summary: Organisms need to assess their nutritional state and adapt their digestive capacity to the demands for various nutrients. Modulation of digestive enzyme production represents a rational step to regulate nutriment uptake. However, the role of digestion in nutrient homeostasis has been largely neglected. In this study, we analyzed the mechanism underlying glucose repression of digestive enzymes in the adult Drosophila midgut. We demonstrate that glucose represses the expression of many carbohydrases and lipases. Our data reveal that the consumption of nutritious sugars stimulates the secretion of the transforming growth factor β (TGF-β ligand, Dawdle, from the fat body. Dawdle then acts via circulation to activate TGF-β/Activin signaling in the midgut, culminating in the repression of digestive enzymes that are highly expressed during starvation. Thus, our study not only identifies a mechanism that couples sugar sensing with digestive enzyme expression but points to an important role of TGF-β/Activin signaling in sugar metabolism. : Organisms modulate their digestive processes to reflect their nutritional state. In this study, Chng et al. demonstrate that the TGF-β/Activin pathway functions as a carbohydrate-sensing mechanism in the adult Drosophila midgut to regulate digestive enzyme expression. They show that the TGF-β ligand, Dawdle, and the canonical TGF-β/Activin signaling are essential to couple carbohydrate sensing with digestive enzyme expression. Thus, their study highlights an unexpected function of TGF-β/Activin signaling that is beyond their established roles in development and immunity.

Alginate Immobilization of Metabolic Enzymes (AIME) for High-Throughput Screening Assays (SOT)

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Alginate Immobilization of Metabolic Enzymes (AIME) for High-Throughput Screening Assays DE DeGroot, RS Thomas, and SO SimmonsNational Center for Computational Toxicology, US EPA, Research Triangle Park, NC USAThe EPA’s ToxCast program utilizes a wide variety of high-throughput s...

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

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

2015-03-01

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

Breakdown of mucin as barrier to digestive enzymes in the ischemic rat small intestine.

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Marisol Chang

Full Text Available Loss of integrity of the epithelial/mucosal barrier in the small intestine has been associated with different pathologies that originate and/or develop in the gastrointestinal tract. We showed recently that mucin, the main protein in the mucus layer, is disrupted during early periods of intestinal ischemia. This event is accompanied by entry of pancreatic digestive enzymes into the intestinal wall. We hypothesize that the mucin-containing mucus layer is the main barrier preventing digestive enzymes from contacting the epithelium. Mucin breakdown may render the epithelium accessible to pancreatic enzymes, causing its disruption and increased permeability. The objective of this study was to investigate the role of mucin as a protection for epithelial integrity and function. A rat model of 30 min splanchnic arterial occlusion (SAO was used to study the degradation of two mucin isoforms (mucin 2 and 13 and two epithelial membrane proteins (E-cadherin and toll-like receptor 4, TLR4. In addition, the role of digestive enzymes in mucin breakdown was assessed in this model by luminal inhibition with acarbose, tranexamic acid, or nafamostat mesilate. Furthermore, the protective effect of the mucin layer against trypsin-mediated disruption of the intestinal epithelium was studied in vitro. Rats after SAO showed degradation of mucin 2 and fragmentation of mucin 13, which was not prevented by protease inhibition. Mucin breakdown was accompanied by increased intestinal permeability to FITC-dextran as well as degradation of E-cadherin and TLR4. Addition of mucin to intestinal epithelial cells in vitro protected against trypsin-mediated degradation of E-cadherin and TLR4 and reduced permeability of FITC-dextran across the monolayer. These results indicate that mucin plays an important role in the preservation of the mucosal barrier and that ischemia but not digestive enzymes disturbs mucin integrity, while digestive enzymes actively mediate epithelial cell

Development of digestive enzymes in larvae of Mayan cichlid Cichlasoma urophthalmus.

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López-Ramírez, G; Cuenca-Soria, C A; Alvarez-González, C A; Tovar-Ramírez, D; Ortiz-Galindo, J L; Perales-García, N; Márquez-Couturier, G; Arias-Rodríguez, L; Indy, J R; Contreras-Sánchez, W M; Gisbert, E; Moyano, F J

2011-03-01

The development of digestive enzymes during the early ontogeny of the Mayan cichlid (Cichlasoma urophthalmus) was studied using biochemical and electrophoretic techniques. From yolk absorption (6 days after hatching: dah), larvae were fed Artemia nauplii until 15 dah, afterward they were fed with commercial microparticulated trout food (45% protein and 16% lipids) from 16 to 60 dah. Several samples were collected including yolk-sac larvae (considered as day 1 after hatching) and specimens up to 60 dah. Most digestive enzymes were present from yolk absorption (5-6 dah), except for the specific acid proteases activity (pepsin-like), which increase rapidly from 8 dah up to 20 dah. Three alkaline proteases isoforms (24.0, 24.8, 84.5 kDa) were detected at 8 dah using SDS-PAGE zymogram, corresponding to trypsin, chymotrypsin and probably leucine aminopeptidase enzymes, and only one isoform was detected (relative electromobility, Rf = 0.54) for acid proteases (pepsin-like) from 3 dah onwards using PAGE zymogram. We concluded that C. urophthamus is a precocious fish with a great capacity to digest all kinds of food items, including artificial diets provided from 13 dah.

A capillary monolithic trypsin reactor for efficient protein digestion in online and offline coupling to ESI and MALDI mass spectrometry.

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Spross, Jens; Sinz, Andrea

2010-02-15

We describe the preparation of a capillary trypsin immobilized monolithic enzyme reactor (IMER) for a rapid and efficient digestion of proteins down to the femtomole level. Trypsin was immobilized on a poly(glycidyl methacrylate-co-acrylamide-co-ethylene glycol dimethycrylate) monolith using the glutaraldehyde technique. Digestion efficiencies of the IMER were evaluated using model proteins and protein mixtures as well as chemically cross-linked lysozyme regarding the addition of denaturants and increasing digestion temperature. The trypsin IMER described herein is applicable for the digestion of protein mixtures. Even at a 1000-fold molar excess of one protein, low-abundance proteins are readily identified, in combination with MS/MS analysis. An online setup of the IMER with reversed phase nano-HPLC separation and nano-ESI-MS/MS analysis was established. The great potential of the trypsin IMER for proteomics applications comprise short digestion times in the range of seconds to minutes, in addition to improved digestion efficiencies, compared to in-solution digestion.

Conservation of the egg envelope digestion mechanism of hatching enzyme in euteleostean fishes.

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Kawaguchi, Mari; Yasumasu, Shigeki; Shimizu, Akio; Sano, Kaori; Iuchi, Ichiro; Nishida, Mutsumi

2010-12-01

We purified two hatching enzymes, namely high choriolytic enzyme (HCE; EC 3.4.24.67) and low choriolytic enzyme (LCE; EC 3.4.24.66), from the hatching liquid of Fundulus heteroclitus, which were named Fundulus HCE (FHCE) and Fundulus LCE (FLCE). FHCE swelled the inner layer of egg envelope, and FLCE completely digested the FHCE-swollen envelope. In addition, we cloned three Fundulus cDNAs orthologous to cDNAs for the medaka precursors of egg envelope subunit proteins (i.e. choriogenins H, H minor and L) from the female liver. Cleavage sites of FHCE and FLCE on egg envelope subunit proteins were determined by comparing the N-terminal amino acid sequences of digests with the sequences deduced from the cDNAs for egg envelope subunit proteins. FHCE and FLCE cleaved different sites of the subunit proteins. FHCE efficiently cleaved the Pro-X-Y repeat regions into tripeptides to dodecapeptides to swell the envelope, whereas FLCE cleaved the inside of the zona pellucida domain, the core structure of egg envelope subunit protein, to completely digest the FHCE-swollen envelope. A comparison showed that the positions of hatching enzyme cleavage sites on egg envelope subunit proteins were strictly conserved between Fundulus and medaka. Finally, we extended such a comparison to three other euteleosts (i.e. three-spined stickleback, spotted halibut and rainbow trout) and found that the egg envelope digestion mechanism was well conserved among them. During evolution, the egg envelope digestion by HCE and LCE orthologs was established in the lineage of euteleosts, and the mechanism is suggested to be conserved. © 2010 The Authors Journal compilation © 2010 FEBS.

Effects of enzyme Additive on Nutrient intake, Digestibility and ...

African Journals Online (AJOL)

Nutrient intake, digestibility and rumen metabolites were determined in sixteen yearling cattle fed Panicum maximum hay supplemented with concentrate diet in which an exogenous fibrolytic enzyme, ROXAZYME G2® (which consist of Cellulase, hemicellulase and beta glucanase) was included at 0, 50, 100 and 150mg/kg.

Improved starch digestion of sucrase deficient shrews treated with oral glucoamylase enzyme supplements

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Although named because of its sucrose hydrolytic activity, this mucosal enzyme plays a leading role in starch digestion because of its maltase and glucoamylase activities. Sucrase deficient mutant shrews, Suncus murinus, were used as a model to investigate starch digestion in patients with Congenita...

Effect of starvation and refeeding on digestive enzyme activities in sturgeon (Acipenser naccarii) and trout (Oncorhynchus mykiss).

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Furné, Miriam; García-Gallego, Manuel; Hidalgo, M Carmen; Morales, Amalia E; Domezain, Alberto; Domezain, Julio; Sanz, Ana

2008-04-01

The digestive enzyme activities were determined in Adriatic sturgeon and rainbow trout during starvation and refeeding period. Overall, the digestive enzyme activities are affected in the same sense in both species. The protease and lipase activities were decreased later than amylase activity. Even after 1 month of starvation, both species would be prepared to digest protein and lipids in an effective way. After 72 days of starvation, the digestive machinery of the sturgeon and of the trout shows an altered capacity to digest macronutrients. The capacity to digest proteins and lipids, after 60 days of refeeding, begins to become re-established in sturgeon and trout. In contrast, in this period, the capacity to digest carbohydrates remains depressed in both species.

Effect of Alchornea cordifolia leaf meal inclusion and enzyme supplementation on performance and digestibility of rabbits

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S.O. Ayodele

2016-09-01

Full Text Available A feeding trial was conducted to study the performance, digestibility and health status of weaner rabbits fed diets including Alchornea cordifolia leaf meal (ALM: 18% crude protein [CP] and 12.9% crude fibre and supplemented with a multi-enzyme additive (cellulase, xylanase, β-glucanase, α-amylase, protease, lipase. Six experimental diets were arranged factorially: 3 levels of ALM (0, 5 and 10% substituting palm kernel cake: 16.3% CP and 39.1% neutral detergent fibre combined with 2 levels of enzyme supplementation (0 and 0.35 g/kg. One hundred and eighty healthy, 5-wk-old weaner rabbits of cross-breeds were randomly allotted to 6 dietary treatments (30 rabbits/treatment, 3 rabbits/replicate. Growth rate was not affected (P>0.05 by the main factors (exogenous enzyme and ALM inclusion and their interactions (13.5 g/d on av.. Daily feed intake and feed conversion ratio decreased (P=0.01 with the ALM inclusion by 8%, but did not affect faecal digestibility. However, enzyme supplementation improved crude protein and crude fibre digestibility (P<0.001 by 6%. In conclusion, ALM inclusion and enzyme supplementation had no adverse effect on the performance and digestibility of rabbits.

Site-Specific, Covalent Immobilization of Dehalogenase ST2570 Catalyzed by Formylglycine-Generating Enzymes and Its Application in Batch and Semi-Continuous Flow Reactors

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Hui Jian

2016-07-01

Full Text Available Formylglycine-generating enzymes can selectively recognize and oxidize cysteine residues within the sulfatase sub motif at the terminus of proteins to form aldehyde-bearing formylglycine (FGly residues, and are normally used in protein labeling. In this study, an aldehyde tag was introduced to proteins using formylglycine-generating enzymes encoded by a reconstructed set of the pET28a plasmid system for enzyme immobilization. The haloacid dehalogenase ST2570 from Sulfolobus tokodaii was used as a model enzyme. The C-terminal aldehyde-tagged ST2570 (ST2570CQ exhibited significant enzymological properties, such as new free aldehyde groups, a high level of protein expression and improved enzyme activity. SBA-15 has widely been used as an immobilization support for its large surface and excellent thermal and chemical stability. It was functionalized with amino groups by aminopropyltriethoxysilane. The C-terminal aldehyde-tagged ST2570 was immobilized to SBA-15 by covalent binding. The site-specific immobilization of ST2570 avoided the chemical denaturation that occurs in general covalent immobilization and resulted in better fastening compared to physical adsorption. The site-specific immobilized ST2570 showed 3-fold higher thermal stability, 1.2-fold higher catalytic ability and improved operational stability than free ST2570. The site-specific immobilized ST2570 retained 60% of its original activity after seven cycles of batch operation, and it was superior to the ST2570 immobilized to SBA-15 by physical adsorption, which loses 40% of its original activity when used for the second time. It is remarkable that the site-specific immobilized ST2570 still retained 100% of its original activity after 10 cycles of reuse in the semi-continuous flow reactor. Overall, these results provide support for the industrial-scale production and application of site-specific, covalently immobilized ST2570.

Immobilization of Papain on Chitin and Chitosan and Recycling of Soluble Enzyme for Deflocculation of Saccharomyces cerevisiae from Bioethanol Distilleries

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Douglas Fernandes Silva

2015-01-01

Full Text Available Yeast flocculation (Saccharomyces cerevisiae is one of the most important problems in fuel ethanol production. Yeast flocculation causes operational difficulties and increase in the ethanol cost. Proteolytic enzymes can solve this problem since it does not depend on these changes. The recycling of soluble papain and the immobilization of this enzyme on chitin or chitosan were studied. Some cross-linking agents were evaluated in the action of proteolytic activity of papain. The glutaraldehyde (0.1–10% w·v−1, polyethyleneimine (0.5% v·v−1, and tripolyphosphate (1–10% w·v−1 inactivated the enzyme in this range, respectively. Glutaraldehyde inhibited all treatments of papain immobilization. The chitosan cross-linked with TPP in 5 h of reaction showed the yield of active immobilized enzyme of 15.7% and 6.07% in chitosan treated with 0.1% PEI. Although these immobilizations have been possible, these levels have not been enough to cause deflocculation of yeast cells. Free enzyme was efficient for yeast deflocculation in dosages of 3 to 4 g·L−1. Recycling of soluble papain by centrifugation was effective for 14 cycles with yeast suspension in time perfectly compatible to industrial conditions. The reuse of proteases applied after yeast suspension by additional yeast centrifugation could be an alternative to cost reduction of these enzymes.

Rapid micro-scale proteolysis of proteins for MALDI-MS peptide mapping using immobilized trypsin

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Gobom, Johan; Nordhoff, Eckhard; Ekman, Rolf; Roepstorff, Peter

1997-12-01

In this study we present a rapid method for tryptic digestion of proteins using micro-columns with enzyme immobilized on perfusion chromatography media. The performance of the method is exemplified with acyl-CoA-binding protein and reduced carbamidomethylated bovine serum albumin. The method proved to be significantly faster and yielded a better sequence coverage and an improved signal-to-noise ratio for the MALDI-MS peptide maps, compared to in-solution- and on-target digestion. Only a single sample transfer step is required, and therefore sample loss due to adsorption to surfaces is reduced, which is a critical issue when handling low picomole to femtomole amounts of proteins. An example is shown with on-column proteolytic digestion and subsequent elution of the digest into a reversed-phase micro-column. This is useful if the sample contains large amounts of salt or is too diluted for MALDI-MS analysis. Furthermore, by step-wise elution from the reversedphase column, a complex digest can be fractionated, which reduces signal suppression and facilitates data interpretation in the subsequent MS-analysis. The method also proved useful for consecutive digestions with enzymes of different cleavage specificity. This is exemplified with on-column tryptic digestion, followed by reversed-phase step-wise elution, and subsequent on-target V8 protease digestion.

Lipase from Aspergillus niger obtained from mangaba residue fermentation: biochemical characterization of free and immobilized enzymes on a sol-gel matrix

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Elis Augusta Leite dos Santos

2017-02-01

Full Text Available In this study, mangaba residue (seeds was used as a substrate for Aspergillus niger lipase production by solid-state fermentation. The partially purified enzyme was efficiently immobilized in a sol-gel matrix by covalent bonding with an immobilization yield of 91.2%. The immobilized biocatalyst and free lipase had an optimum pH of 2.0 and 5.0, respectively. However, greater stability was obtained at pH 4.0 and 7.0, respectively. The biocatalysts showed stability at the optimum temperature of 55°C, where the residual activity was above 87% after 240 min., of incubation. The lower deactivation constant (kd and higher half-life of the immobilized biocatalyst indicated greater thermal stability than those obtained with the free enzyme. The Michaelis Constant (Km (77 and 115 mM for free and immobilized lipase, respectively and maximum reaction rate (Vmax (1250 and 714 U mg-1 for free and immobilized lipase, respectively indicated that the immobilization process reduced enzyme-substrate affinity. Regarding the operational stability, the biocatalyst showed relative activity above 50% until seven cycles of reuse in olive oil hydrolysis. This novel biocatalyst obtained from a tropical fruit residue showed biochemical characteristics that support its application in future biocatalysis studies.

Immobilization of HRP Enzyme on Layered Double Hydroxides for Biosensor Application

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Zouhair M. Baccar

2011-01-01

Full Text Available We present a new biosensor for hydrogen peroxide (H2O2 detection. The biosensor was based on the immobilization of horseradish peroxidase (HRP enzyme on layered double hydroxides- (LDH- modified gold surface. The hydrotalcite LDH (Mg2Al was prepared by coprecipitation in constant pH and in ambient temperature. The immobilization of the peroxidase on layered hybrid materials was realized via electrostatic adsorption autoassembly process. The detection of hydrogen peroxide was successfully observed in PBS buffer with cyclic voltammetry and the chronoamperometry techniques. A limit detection of 9 μM of H2O2 was obtained with a good reproducibility. We investigate the sensitivity of our developed biosensor for H2O2 detection in raw milk.

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

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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.

Pepsin immobilized in dextran-modified fused-silica capillaries for on-line protein digestion and peptide mapping

Energy Technology Data Exchange (ETDEWEB)

Stigter, E.C.A. [Division of Biomedical Analysis, Department of Pharmaceutical Sciences, Faculty of Science, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht (Netherlands)], E-mail: e.c.a.stigter@uu.nl; Jong, G.J. de; Bennekom, W.P. van [Division of Biomedical Analysis, Department of Pharmaceutical Sciences, Faculty of Science, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht (Netherlands)

2008-07-07

On-line digestion of proteins under acidic conditions was studied using micro-reactors consisting of dextran-modified fused-silica capillaries with covalently immobilized pepsin. The proteins used in this study differed in molecular weight, isoelectric point and sample composition. The injected protein samples were completely digested in 3 min and the digest was analyzed with micro-high performance liquid chromatography (HPLC) and tandem mass spectrometry (MS/MS). The different proteins present in the samples could be identified with a Mascot database search on the basis of auto-MS/MS data. It proved also to be possible to digest and analyze protein mixtures with a sequence coverage of 55% and 97% for the haemoglobin {beta}- and {alpha}-chain, respectively, and 35-55% for the various casein variants. Protease auto-digestion, sample carry-over and loss of signal due to adsorption of the injected proteins were not observed. The backpressure of the reactor is low which makes coupling to systems such as Surface Plasmon Resonance biosensors, which do not tolerate too high pressure, possible. The reactor was stable for at least 40 days when used continuously.

Pepsin immobilized in dextran-modified fused-silica capillaries for on-line protein digestion and peptide mapping.

Science.gov (United States)

Stigter, E C A; de Jong, G J; van Bennekom, W P

2008-07-07

On-line digestion of proteins under acidic conditions was studied using micro-reactors consisting of dextran-modified fused-silica capillaries with covalently immobilized pepsin. The proteins used in this study differed in molecular weight, isoelectric point and sample composition. The injected protein samples were completely digested in 3 min and the digest was analyzed with micro-high performance liquid chromatography (HPLC) and tandem mass spectrometry (MS/MS). The different proteins present in the samples could be identified with a Mascot database search on the basis of auto-MS/MS data. It proved also to be possible to digest and analyze protein mixtures with a sequence coverage of 55% and 97% for the haemoglobin beta- and alpha-chain, respectively, and 35-55% for the various casein variants. Protease auto-digestion, sample carry-over and loss of signal due to adsorption of the injected proteins were not observed. The backpressure of the reactor is low which makes coupling to systems such as Surface Plasmon Resonance biosensors, which do not tolerate too high pressure, possible. The reactor was stable for at least 40 days when used continuously.

New stable isotope method to measure protein digestibility and response to pancreatic enzyme intake in cystic fibrosis.

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Engelen, M P K J; Com, G; Anderson, P J; Deutz, N E P

2014-12-01

Adequate protein intake and digestion are necessary to prevent muscle wasting in cystic fibrosis (CF). Accurate and easy-to-use methodology to quantify protein maldigestion is lacking in CF. To measure protein digestibility and the response to pancreatic enzyme intake in CF by using a new stable isotope methodology. In 19 CF and 8 healthy subjects, protein digestibility was quantified during continuous (sip) feeding for 6 h by adding (15)N-labeled spirulina protein and L-[ring-(2)H5]phenylalanine (PHE) to the nutrition and measuring plasma ratio [(15)N]PHE to [(2)H5]PHE. Pancreatic enzymes were ingested after 2 h in CF and the response in protein digestibility was assessed. To exclude difference in mucosal function, postabsorptive whole-body citrulline (CIT) production rate was measured by L-[5-(13)C-5,5-(2)H2]-CIT pulse and blood samples were taken to analyze tracer-tracee ratios. Protein digestibility was severely reduced in the CF group (47% of healthy subjects; P digestibility in CF until 90% of values obtained by healthy subjects. Maximal digestibility was reached at 100 min and maintained for 80 min. Stratification into CF children (n = 10) and adults showed comparable values for protein digestibility and similar kinetic responses to pancreatic enzyme intake. Whole-body citrulline production was elevated in CF indicating preserved mucosal function. Protein digestibility is severely compromised in patients with CF as measured by this novel and easy-to-use stable isotope approach. Pancreatic enzymes are able to normalize protein digestibility in CF, albeit with a severe delay. Registration ClinicalTrials.gov = NCT01494909. Copyright © 2013 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

Studies on the preparation of immobilized enzymes by radio-polymerization, 10. Preparation of. beta. -galactosidase and its utilization for the continuous determination of lactose. [Gamma radiation

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Amarakone, S P [Ceylon Inst. of Scientific and Industrial Research, Colombo (Sri Lanka); Hayashi, Toru; Kawashima, Koji

1983-03-01

..beta..-Galactosidase of E. coli origin was immobilized in the form of beads by the radiopolymerization of different combinations of monomers using a gamma irradiation technique. With the dialysed enzyme, recoveries of over 300 % could be obtained on suitable monomer combinations containing magnesium and sodium acrylates. The recovery of the enzyme also depended on the irradiation time. The immobilized enzyme had better pH and temperature stability and was less affected by the presence of metal ions in the medium, compared to the native enzyme. The optimum pH and temperatures of the immobilized enzyme were different from those of the native enzyme and were 7.0 to 7.5 and 50 deg C respectively. The immobilized enzyme was used in a column for the continuous determination of lactose with a standard type autoanalyser. Good linearity could be observed even up to 3% lactose in the sample.

High-pressure tolerance of earthworm fibrinolytic and digestive enzymes.

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Akazawa, Shin-Ichi; Tokuyama, Haruka; Sato, Shunsuke; Watanabe, Toshinori; Shida, Yosuke; Ogasawara, Wataru

2018-02-01

Earthworms contain several digestive and therapeutic enzymes that are beneficial to our health and useful for biomass utilization. Specifically, earthworms contain potent fibrinolytic enzymes called lumbrokinases, which are highly stable even at room temperature and remain active in dried earthworm powder. However, the high-temperature sterilization method leads to the inactivation of enzymes. Therefore, we investigated the effect of high-pressure treatment (HPT) (from 0.1 MPa to 500 MPa at 25°C and 50°C) on the enzymatic activity of lumbrokinase (LK), α-amylase (AMY), endoglucanase (EG), β-glucosidase (BGL), and lipase (LP) of the earthworm Eisenia fetida, Waki strain, and its sterilization ability in producing dietary supplement. LK showed thermo- and high-pressure tolerance. In addition, HPT may have resulted in pressure-induced stabilization and activation of LK. Although AMY activity was maintained up to 400 MPa at 25°C, the apparent activity decreased slightly at 50°C with HPT. EG showed almost the same pattern as AMY. However, it is possible that the effects of temperature and pressure compensated each other under 100 MPa at 50°C. BGL was shown to be a pressure- and temperature-sensitive enzyme, and LP showed a thermo- and high-pressure tolerance. The slight decrease in apparent activity occurred under 200 MPa at both temperatures. Furthermore, the low-temperature and pressure treatment completely sterilized the samples. These results provide a basis for the development of a novel earthworm dietary supplement with fibrinolytic and digestive activity and of high-pressure-tolerant enzymes to be used for biomass pretreatment. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Characteristics of Immobilized Urease on Grafted Alginate Bead Systems

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Enas N. Danial

2015-04-01

Full Text Available This study evaluated the biological importance of immobilized urease enzyme over the free urease. The support material used for urease immobilization was alginate. Generally, the immobilization of urease in alginate gel showed a marked increase in Km and Vmax. However, the immobilized urease showed higher thermal stability than that of free enzyme. The rate of thermal inactivation of the immobilized enzyme decreased due to entrapment in gel matrix. Also, the activity of the immobilized urease was more stable in retention than that of the free enzyme during the storage in solution, although the activity of the immobilized enzyme was lower in comparison with the free enzyme. A stable immobilized system and long storage life are convenient for applications that would not be feasible with a soluble enzyme system. These results highlighted the technical and biochemical benefits of immobilized urease over the free enzyme.

The Effects of Enzyme Complex on Performance, Intestinal Health and Nutrient Digestibility of Weaned Pigs

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J. Q. Yi

2013-08-01

Full Text Available Two experiments were conducted to evaluate the effect of supplementing a corn-soybean meal-based diet with an enzyme complex containing amylase, protease and xylanase on the performance, intestinal health, apparent ileal digestibility of amino acids and nutrient digestibility of weaned pigs. In Exp. 1, 108 piglets weaned at 28 d of age were fed one of three diets containing 0 (control, 100, or 150 ppm enzyme complex for 4 wks, based on a two-phase feeding program namely 1 to 7 d (phase 1 and 8 to 28 d (phase 2. At the end of the experiment, six pigs from the control group and the group supplemented with 150 ppm enzyme complex were chosen to collect digesta samples from intestine to measure viscosity and pH in the stomach, ileum, and cecum, as well as volatile fatty acid concentrations and composition of the microflora in the cecum and colon. There were linear increases (p<0.01 in weight gain, gain: feed ratio and digestibility of gross energy with the increasing dose rate of enzyme supplementation during the whole experiment. Supplementation with enzyme complex increased the digesta viscosity in the stomach (p<0.05 and significantly increased (p<0.01 the concentrations of acetic, propionic and butyric acid in the cecum and colon. Enzyme supplementation also significantly increased the population of Lactobacilli (p<0.01 in the cecum and decreased the population of E. coli (p<0.05 in the colon. In Exp. 2, six crossbred barrows (initial body weight: 18.26±1.21 kg, fitted with a simple T-cannula at the distal ileum, were assigned to three dietary treatments according to a replicated 3×3 Latin Square design. The experimental diets were the same as the diets used in phase 2 in Exp. 1. Apparent ileal digestibility of isoleucine (p<0.01, valine (p<0.05 and aspartic acid (p<0.05 linearly increased with the increasing dose rate of enzyme supplementation. In conclusion, supplementation of the diet with an enzyme complex containing amylase, protease and

Expression pattern of glycoside hydrolase genes in Lutzomyia longipalpis reveals key enzymes involved in larval digestion

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Caroline da Silva Moraes

2014-08-01

Full Text Available The sand fly Lutzomyia longipalpis is the most important vector of American Visceral Leishmaniasis. Adults are phytophagous (males and females or blood feeders (females only, and larvae feed on solid detritus. Digestion in sand fly larvae has scarcely been studied, but some glycosidase activities putatively involved in microorganism digestion were already described. Nevertheless, the molecular nature of these enzymes, as the corresponding genes and transcripts, were not explored yet. Catabolism of microbial carbohydrates in insects generally involves β-1,3-glucanases, chitinases and digestive lysozymes. In this work, the transcripts of digestive β-1,3-glucanase and chitinases were identified in the L. longipalpis larvae throughout analysis of sequences and expression patterns of glycoside hydrolases families 16, 18 and 22. The activity of one i-type lysozyme was also registered. Interestingly, this lysozyme seems to play a role in immunity, rather than digestion. This is the first attempt to identify the molecular nature of sand fly larval digestive enzymes.

Expression pattern of glycoside hydrolase genes in Lutzomyia longipalpis reveals key enzymes involved in larval digestion

Science.gov (United States)

Moraes, Caroline da Silva; Diaz-Albiter, Hector M.; Faria, Maiara do Valle; Sant'Anna, Maurício R. V.; Dillon, Rod J.; Genta, Fernando A.

2014-01-01

The sand fly Lutzomyia longipalpis is the most important vector of American Visceral Leishmaniasis. Adults are phytophagous (males and females) or blood feeders (females only), and larvae feed on solid detritus. Digestion in sand fly larvae has scarcely been studied, but some glycosidase activities putatively involved in microorganism digestion were already described. Nevertheless, the molecular nature of these enzymes, as the corresponding genes and transcripts, were not explored yet. Catabolism of microbial carbohydrates in insects generally involves β-1,3-glucanases, chitinases, and digestive lysozymes. In this work, the transcripts of digestive β-1,3-glucanase and chitinases were identified in the L. longipalpis larvae throughout analysis of sequences and expression patterns of glycoside hydrolases families 16, 18, and 22. The activity of one i-type lysozyme was also registered. Interestingly, this lysozyme seems to play a role in immunity, rather than digestion. This is the first attempt to identify the molecular nature of sand fly larval digestive enzymes. PMID:25140153

Bio-functionalization of electro-synthesized polypyrrole surface by heme enzyme using a mixture of Nafion and glutaraldehyde as synergetic immobilization matrix: Conformational characterization and electrocatalytic studies

International Nuclear Information System (INIS)

ElKaoutit, Mohammed; Naranjo-Rodriguez, Ignacio; Dominguez, Manuel; Hidalgo-Hidalgo-de-Cisneros, Jose Luis

2011-01-01

Use of a mixture of Nafion and glutaraldehyde as new immobilization matrix was described. The percentage of Nafion was optimized to prevent denaturation of horseradish peroxidase enzyme after its crosslinkage with glutaraldehyde on electro-synthesized polypyrrole surface. Topographic study by Atomic Force Microscopy (AFM) shows that the enzyme seems to have been introduced inside the ionic cluster of Nafion. The characterization of the resulting bio-interfaces by UV-vis and FT-IR shows that the intra-crosslinkage phenomena caused by the use of glutaraldehyde can be eliminated by the optimization of the concentration of Nafion additive. The secondary structure contents of native and immobilized enzyme were analyzed by a Gaussian curve fitting of the respective FT-IR spectra in the amide I region. Immobilized enzyme presented notable increasing percentages of globular and short helical structure compared with native enzyme. This indicates that immobilized enzyme was folded which is in accordance with AFM studies and supports the enzyme entrance inside ionic clutter of Nafion. Thanks to synergic effects of the polypyrrole conducting polymer and the perfluorosulfonic acid polymer Nafion, HRP enzyme was immobilized in its 'native' state, the resulting biosensor was able to sense peroxide without any chemical mediator and can be categorized as third generation.

Bio-functionalization of electro-synthesized polypyrrole surface by heme enzyme using a mixture of Nafion and glutaraldehyde as synergetic immobilization matrix: Conformational characterization and electrocatalytic studies

Energy Technology Data Exchange (ETDEWEB)

ElKaoutit, Mohammed, E-mail: elkaoutit@uca.es [Departamento de Quimica Analitica, Facultad de Ciencias, Universidad de Cadiz, 11510 Puerto Real, Cadiz (Spain); Naranjo-Rodriguez, Ignacio [Departamento de Quimica Analitica, Facultad de Ciencias, Universidad de Cadiz, 11510 Puerto Real, Cadiz (Spain); Dominguez, Manuel [Departamento de Fisica de la Materia Condensada, Facultad de Ciencias, Universidad de Cadiz, 11510 Puerto Real, Cadiz (Spain); Hidalgo-Hidalgo-de-Cisneros, Jose Luis [Departamento de Quimica Analitica, Facultad de Ciencias, Universidad de Cadiz, 11510 Puerto Real, Cadiz (Spain)

2011-10-01

Use of a mixture of Nafion and glutaraldehyde as new immobilization matrix was described. The percentage of Nafion was optimized to prevent denaturation of horseradish peroxidase enzyme after its crosslinkage with glutaraldehyde on electro-synthesized polypyrrole surface. Topographic study by Atomic Force Microscopy (AFM) shows that the enzyme seems to have been introduced inside the ionic cluster of Nafion. The characterization of the resulting bio-interfaces by UV-vis and FT-IR shows that the intra-crosslinkage phenomena caused by the use of glutaraldehyde can be eliminated by the optimization of the concentration of Nafion additive. The secondary structure contents of native and immobilized enzyme were analyzed by a Gaussian curve fitting of the respective FT-IR spectra in the amide I region. Immobilized enzyme presented notable increasing percentages of globular and short helical structure compared with native enzyme. This indicates that immobilized enzyme was folded which is in accordance with AFM studies and supports the enzyme entrance inside ionic clutter of Nafion. Thanks to synergic effects of the polypyrrole conducting polymer and the perfluorosulfonic acid polymer Nafion, HRP enzyme was immobilized in its 'native' state, the resulting biosensor was able to sense peroxide without any chemical mediator and can be categorized as third generation.

High Sensitivity Electrochemical Cholesterol Sensor Utilizing a Vertically Aligned Carbon Nanotube Electrode with Electropolymerized Enzyme Immobilization

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Ditsayut Phokharatkul

2009-10-01

Full Text Available In this report, a new cholesterol sensor is developed based on a vertically aligned CNT electrode with two-step electrochemical polymerized enzyme immobilization. Vertically aligned CNTs are selectively grown on a 1 mm2 window of gold coated SiO2/Si substrate by thermal chemical vapor deposition (CVD with gravity effect and water-assisted etching. CNTs are then simultaneously functionalized and enzyme immobilized by electrochemical polymerization of polyaniline and cholesterol enzymes. Subsequently, ineffective enzymes are removed and new enzymes are electrochemically recharged. Scanning electron microscopic characterization indicates polymer-enzyme nanoparticle coating on CNT surface. Cyclic voltammogram (CV measurements in cholesterol solution show the oxidation and reduction peaks centered around 450 and −220 mV, respectively. An approximately linear relationship between the cholesterol concentration and the response current could be observed in the concentration range of 50–300 mg/dl with a sensitivity of approximately 0.22 μA/mg·dl−1, which is considerably higher compared to previously reported CNT bioprobe. In addition, good specificity toward glucose, uric acid acetaminophen and ascorbic acid have been obtained. Moreover, sensors have satisfactory stability, repeatability and life time. Therefore, the electropolymerized CNT bioprobe is promising for cholesterol detection in normal cholesterol concentration in human blood.

Partial digestion with restriction enzymes of ultraviolet-irradiated human genomic DNA: a method for identifying restriction site polymorphisms

International Nuclear Information System (INIS)

Nobile, C.; Romeo, G.

1988-01-01

A method for partial digestion of total human DNA with restriction enzymes has been developed on the basis of a principle already utilized by P.A. Whittaker and E. Southern for the analysis of phage lambda recombinants. Total human DNA irradiated with uv light of 254 nm is partially digested by restriction enzymes that recognize sequences containing adjacent thymidines because of TT dimer formation. The products resulting from partial digestion of specific genomic regions are detected in Southern blots by genomic-unique DNA probes with high reproducibility. This procedure is rapid and simple to perform because the same conditions of uv irradiation are used for different enzymes and probes. It is shown that restriction site polymorphisms occurring in the genomic regions analyzed are recognized by the allelic partial digest patterns they determine

Enzymatic removal of phenol and p-chlorophenol in enzyme reactor: Horseradish peroxidase immobilized on magnetic beads

International Nuclear Information System (INIS)

Bayramoglu, Guelay; Arica, M. Yakup

2008-01-01

Horseradish peroxidase was immobilized on the magnetic poly(glycidylmethacrylate-co-methylmethacrylate) (poly(GMA-MMA)), via covalent bonding and used for the treatment of phenolic wastewater in continuous systems. For this purposes, horseradish peroxidase (HRP) was covalently immobilized onto magnetic poly(GMA-MMA) beds using glutaraldehyde (GA) as a coupling agent. The maximum HRP immobilization capacity of the magnetic poly(GMA-MMA)-GA beads was 3.35 mg g -1 . The immobilized HRP retained 79% of the activity of the free HRP used for immobilization. The immobilized HRP was used for the removal of phenol and p-chlorophenol via polymerization of dissolved phenols in the presence of hydrogen peroxide (H 2 O 2 ). The effect of pH and temperature on the phenol oxidation rate was investigated. The results were compared with the free HRP, which showed that the optimum pH value for the immobilized HRP is similar to that for the free HRP. The optimum pH value for free and immobilized HRP was observed at pH 7.0. The optimum temperature for phenols oxidation with immobilized HRP was between 25 and 35 deg. C and the immobilized HRP has more resistance to temperature inactivation than that of the free form. Finally, the immobilized HRP was operated in a magnetically stabilized fluidized bed reactor, and phenols were successfully removed in the enzyme reactor

Thermal Stabilization of Enzymes Immobilized within Carbon Paste Electrodes.

Science.gov (United States)

Wang, J; Liu, J; Cepra, G

1997-08-01

In this note we report on the remarkable thermal stabilization of enzymes immobilized in carbon paste electrodes. Amperometric biosensors are shown for the first time to withstand a prolonged high-temperature (>50 °C) stress. Nearly full activity of glucose oxidase is retained over periods of up to 4 months of thermal stress at 60-80 °C. Dramatic improvements in the thermostability are observed for polyphenol oxidase, lactate oxidase, alcohol oxidase, horseradish peroxidase, and amino acid oxidase. Such resistance to heat-induced denaturation is attributed to the conformational rigidity of these biocatalysts within the highly hydrophobic (mineral oil or silicone grease) pasting liquid. While no chemical stabilizer is needed for attaining such protective action, it appears that low humidity (i.e., low water content) is essential for minimizing the protein mobility. Besides their implications for electrochemical biosensors, such observations should lead to a new generation of thermoresistant enzyme reactors based on nonpolar semisolid supports.

Reversible thermal denaturation of immobilized rhodanese

International Nuclear Information System (INIS)

Horowitz, P.; Bowman, S.

1987-01-01

For the first time, the enzyme rhodanese had been refolded after thermal denaturation. This was previously not possible because of the strong tendency for the soluble enzyme to aggregate at temperatures above 37 degrees C. The present work used rhodanese that was covalently coupled to a solid support under conditions that were found to preserve enzyme activity. Rhodanese was immobilized using an N-hydroxymalonimidyl derivative of Sepharose containing a 6-carbon spacer. The number of immobilized competent active sites was measured by using [ 35 S]SO 3 (2-) to form an active site persulfide that is the obligatory catalytic intermediate. Soluble enzyme was irreversibly inactivated in 10 min at 52 degrees C. The immobilized enzyme regained at least 30% of its original activity even after boiling for 20 min. The immobilized enzyme had a Km and Vmax that were each approximately 3 times higher than the corresponding values for the native enzyme. After preincubation at high temperatures, progress curves for the immobilized enzyme showed induction periods of up to 5 min before attaining apparently linear steady states. The pH dependence of the activity was the same for both the soluble and the immobilized enzyme. These results indicate significant stabilization of rhodanese after immobilization, and instabilities caused by adventitious solution components are not the sole reasons for irreversibility of thermal denaturation seen with the soluble enzyme. The results are consistent with models for rhodanese that invoke protein association as a major cause of inactivation of the enzyme. Furthermore, the induction period in the progress curves is consistent with studies which show that rhodanese refolding proceeds through intermediate states

Digestive enzymes and gut morphometric parameters of threespine stickleback (Gasterosteus aculeatus): Influence of body size and temperature.

Science.gov (United States)

Hani, Younes Mohamed Ismail; Marchand, Adrien; Turies, Cyril; Kerambrun, Elodie; Palluel, Olivier; Bado-Nilles, Anne; Beaudouin, Rémy; Porcher, Jean-Marc; Geffard, Alain; Dedourge-Geffard, Odile

2018-01-01

Determining digestive enzyme activity is of potential interest to obtain and understand valuable information about fish digestive physiology, since digestion is an elementary process of fish metabolism. We described for the first time (i) three digestive enzymes: amylase, trypsin and intestinal alkaline phosphatase (IAP), and (ii) three gut morphometric parameters: relative gut length (RGL), relative gut mass (RGM) and Zihler's index (ZI) in threespine stickleback (Gasterosteus aculeatus), and we studied the effect of temperature and body size on these parameters. When mimicking seasonal variation in temperature, body size had no effect on digestive enzyme activity. The highest levels of amylase and trypsin activity were observed at 18°C, while the highest IAP activity was recorded at 20°C. When sticklebacks were exposed to three constant temperatures (16, 18 and 21°C), a temporal effect correlated to fish growth was observed with inverse evolution patterns between amylase activity and the activities of trypsin and IAP. Temperature (in both experiments) had no effect on morphometric parameters. However, a temporal variation was recorded for both RGM (in the second experiment) and ZI (in both experiments), and the later was correlated to fish body mass.

Digestive enzymes and gut morphometric parameters of threespine stickleback (Gasterosteus aculeatus): Influence of body size and temperature

Science.gov (United States)

Marchand, Adrien; Turies, Cyril; Kerambrun, Elodie; Palluel, Olivier; Bado-Nilles, Anne; Beaudouin, Rémy; Porcher, Jean-Marc; Geffard, Alain; Dedourge-Geffard, Odile

2018-01-01

Determining digestive enzyme activity is of potential interest to obtain and understand valuable information about fish digestive physiology, since digestion is an elementary process of fish metabolism. We described for the first time (i) three digestive enzymes: amylase, trypsin and intestinal alkaline phosphatase (IAP), and (ii) three gut morphometric parameters: relative gut length (RGL), relative gut mass (RGM) and Zihler’s index (ZI) in threespine stickleback (Gasterosteus aculeatus), and we studied the effect of temperature and body size on these parameters. When mimicking seasonal variation in temperature, body size had no effect on digestive enzyme activity. The highest levels of amylase and trypsin activity were observed at 18°C, while the highest IAP activity was recorded at 20°C. When sticklebacks were exposed to three constant temperatures (16, 18 and 21°C), a temporal effect correlated to fish growth was observed with inverse evolution patterns between amylase activity and the activities of trypsin and IAP. Temperature (in both experiments) had no effect on morphometric parameters. However, a temporal variation was recorded for both RGM (in the second experiment) and ZI (in both experiments), and the later was correlated to fish body mass. PMID:29614133

Significance of membrane bioreactor design on the biocatalytic performance of glucose oxidase and catalase: Free vs. immobilized enzyme systems

DEFF Research Database (Denmark)

Morthensen, Sofie Thage; Meyer, Anne S.; Jørgensen, Henning

2017-01-01

Membrane separation of xylose and glucose can be accomplished via oxidation of glucose to gluconic acid by enzymatic glucose oxidase catalysis. Oxygen for this reaction can be supplied via decomposition of hydrogen peroxide by enzymatic catalase catalysis. In order to maximize the biocatalytic...... productivity of glucose oxidase and catalase (gluconic acid yield per total amount of enzyme) the following system set-ups were compared: immobilization of glucose oxidase alone; co-immobilization of glucose oxidase and catalase; glucose oxidase and catalase free in the membrane bioreactor. Fouling......-induced enzyme immobilization in the porous support of an ultrafiltration membrane was used as strategy for entrapment of glucose oxidase and catalase. The biocatalytic productivity of the membrane reactor was found to be highly related to the oxygen availability, which in turn depended on the reactor...

Digestive enzymes in Rhinolophus euryale (Rhinolophidae, Chiroptera are active also during hibernation

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Maxinová Edita

2017-11-01

Full Text Available During the winter, bats use hibernation as a means of surviving the period of low prey offer. However, the Mediterranean horseshoe bat (Rhinolophus euryale arouses from torpor quite frequently. Based on the actual climatic conditions, it can profit from occasional foraging oportunities, when they occur. We analysed faeces collected on four nights during the period from November 2012 to February 2013 from the Domica-Baradla cave system (Slovakia and Hungary. In mid-November, the largest proportion of faecal contents were from Lepidoptera. Later on, the proportion of non-consumptive mass in the faeces increased and prey remnants disappeared. We analysed the activity of digestive enzymes (amylase, chitobiase, endochitinase and glukosaminidase in faeces. The activity of these enzymes was detected in fresh faeces throughout the whole winter. The faecal activity of the chitinases was relatively stable during the monitored period, whilst the activity of amylase was highest during late November and December. Some level of active digestive enzymes during the winter could be an adaptation to occasional winter foraging.

Immobilized enzymes to convert N-sulfo, N-acetyl heparosan to a critical intermediate in the production of bioengineered heparin.

Science.gov (United States)

Xiong, Jian; Bhaskar, Ujjwal; Li, Guoyun; Fu, Li; Li, Lingyun; Zhang, Fuming; Dordick, Jonathan S; Linhardt, Robert J

2013-09-10

Heparin is a critically important anticoagulant drug that is prepared from pig intestine. In 2007-2008, there was a crisis in the heparin market when the raw material was adulterated with the toxic polysaccharide, oversulfated chondroitin sulfate, which was associated with 100 deaths in the U.S. alone. As the result of this crisis, our laboratory and others have been actively pursuing alternative sources for this critical drug, including synthetic heparins and bioengineered heparin. In assessing the bioengineering processing costs it has become clear that the use of both enzyme-catalyzed cofactor recycling and enzyme immobilization will be needed for commercialization. In the current study, we examine the use of immobilization of C₅-epimerase and 2-O-sulfotransferase involved in the first enzymatic step in the bioengineered heparin process, as well as arylsulfotransferase-IV involved in cofactor recycling in all three enzymatic steps. We report the successful immobilization of all three enzymes and their use in converting N-sulfo, N-acetyl heparosan into N-sulfo, N-acetyl 2-O-sulfo heparin. Copyright © 2013 Elsevier B.V. All rights reserved.

Effect of immobilization stress on gene expression of catecholamine biosynthetic enzymes in heart auricles of socially isolated rats

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L. Gavrilovic

2009-12-01

Full Text Available Chronic stress is associated with the development of cardiovascular diseases. The sympathoneural system plays an important role in the regulation of cardiac function both in health and disease. In the present study, the changes in gene expression of the catecholamine biosynthetic enzymes tyrosine hydroxylase (TH, dopamine-β-hydroxylase (DBH and phenylethanolamine N-methyltransferase (PNMT and protein levels in the right and left heart auricles of naive control and long-term (12 weeks socially isolated rats were investigated by Taqman RT-PCR and Western blot analysis. The response of these animals to additional immobilization stress (2 h was also examined. Long-term social isolation produced a decrease in TH mRNA level in left auricles (about 70% compared to the corresponding control. Expression of the DBH gene was markedly decreased both in the right (about 62% and left (about 81% auricles compared to the corresponding control, group-maintained rats, whereas PNMT mRNA levels remained unchanged. Exposure of group-housed rats to acute immobilization for 2 h led to a significant increase of mRNA levels of TH (about 267%, DBH (about 37% and PNMT (about 60% only in the right auricles. Additional 2-h immobilization of individually housed rats did not affect gene expression of these enzymes in either the right or left auricle. Protein levels of TH, DBH and PNMT in left and right heart auricles were unchanged either in both individually housed and immobilized rats. The unchanged mRNA levels of the enzymes examined after short-term immobilization suggest that the catecholaminergic system of the heart auricles of animals previously exposed to chronic psychosocial stress was adapted to maintain appropriate cardiovascular homeostasis.

Inhibition of raw starch digestion by one glucoamylase preparation from black Aspergillus at high enzyme concentration

Energy Technology Data Exchange (ETDEWEB)

Saka, B C; Veda, S

1981-09-01

Raw starch digestion by glucoamylase I (Ab. G-I) preparation from black Aspergillus was inhibited significantly at relatively high concentration of the enzyme. The properties of this enzyme were studied together with those of another glucoamylase I (Nor. G-I), also from black Aspergillus. The two glucoamylases do not differ so much in their physico-chemical properties such as molecular weight, pH and thermal stability, pH and temperature optimum, substrate specificity, debranching activity, isoelectric point etc. The adsorption rate of both enzymes on raw starch increased by the increase of enzyme concentration. The raw starch digestion rate by adsorbed Ab. G-I, however, was decreased with the increase of concentration of enzyme whereas the same was increased in case of Nor. G-I. The inhibitory effect was weaker at 60 deg. Celcius or above. (Refs. 11).

Enzymatic Cellulose Hydrolysis: Enzyme Reusability and Visualization of beta-Glucosidase Immobilized in Calcium Alginate

DEFF Research Database (Denmark)

Tsai, Chien Tai; Meyer, Anne S.

2014-01-01

by confocal laser scanning microscopy (CLSM). The CLSM images, which we believe are the first to be published, corroborate that more BG aggregates were entrapped in the matrix when the enzymes were cross-linked by glutaraldehyde as opposed to when they are not cross-linked. The particles with the immobilized...

Preservation of Bacillus pumilus PU4-2 xylanases by immobilization technique into pollard and cation addition

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T Haryati

2010-03-01

Full Text Available Utilization of by-product from agriculture as alternative source of feedstuff has been widely practiced. However their usage is limited due to high fiber content and low nutrient digestibility. The use of specific hydrolizing enzymes, xylanases are gaining importance because of their wide application in various industrial sectors especially in bioconversion of hemicellulosic material. This experiment was done to evaluate the effect of cation addition and immobilization of enzyme into pollard on stability of B. pumilus xylanase. The enzyme extract was purified by precipitation with 75% ammonium sulphate. Four kinds of cation (Ca2+, Fe3+, Mg2+, Zn2+ were added to the purified enzyme, at concentration of 1m M and stored at 4 and 27˚C. For immobilization process, the optimum enzyme concentration that will be added to pollard has been evaluated by analysis of xylanase activity and their recovery. The specific activity of enzyme after precipitation increased 1.8 times, from 420.3 to 765.2 U/mg protein. All cations act as activator which relative activity become 130.6; 139.0; 103.8 and 163.5% respectively. Concentration of 0.5mM Ca2+ and Fe3+ were most able to keep xylanases activity stable at 4˚C. The optimum composition of enzymes and pollard was 1.5 ml for 5 gram of pollard with recovery of xylanases activity of 82.2%. In immobilized enzyme, the activity of enzyme without cation addition is higher than that with addition of Ca2+ and Fe3+. Activity of enzyme stored at 4˚C is more stable than that at 27˚C. Immobilized enzyme is more stable for storage, which lasted for 7 weeks at 27˚C and 12 weeks at 4˚C compared to liquid enzyme which lasted for only 7 days at 27˚C and 13 days at 4˚C.

RNA-Seq reveals the dynamic and diverse features of digestive enzymes during early development of Pacific white shrimp Litopenaeus vannamei.

Science.gov (United States)

Wei, Jiankai; Zhang, Xiaojun; Yu, Yang; Li, Fuhua; Xiang, Jianhai

2014-09-01

The Pacific white shrimp (Litopenaeus vannamei), with high commercial value, has a typical metamorphosis pattern by going through embryo, nauplius, zoea, mysis and postlarvae during early development. Its diets change continually in this period, and a high mortality of larvae also occurs in this period. Since there is a close relationship between diets and digestive enzymes, a comprehensive investigation about the types and expression patterns of all digestive enzyme genes during early development of L. vannamei is of considerable significance for shrimp diets and larvae culture. Using RNA-Seq data, the types and expression characteristics of the digestive enzyme genes were analyzed during five different development stages (embryo, nauplius, zoea, mysis and postlarvae) in L. vannamei. Among the obtained 66,815 unigenes, 296 were annotated as 16 different digestive enzymes including five types of carbohydrase, seven types of peptidase and four types of lipase. Such a diverse suite of enzymes illustrated the capacity of L. vannamei to exploit varied diets to fit their nutritional requirements. The analysis of their dynamic expression patterns during development also indicated the importance of transcriptional regulation to adapt to the diet transition. Our study revealed the diverse and dynamic features of digestive enzymes during early development of L. vannamei. These results would provide support to better understand the physiological changes during diet transition. Copyright © 2014 Elsevier Inc. All rights reserved.

Digestive enzyme ratios are good indicators of hatchling yolk reserve and digestive gland maturation in early life stages of cuttlefish Sepia officinalis L.: application of these new tools in ecology and aquaculture.

Science.gov (United States)

Safi, Georges; Martinez, A S; Le Pabic, C; Le Bihan, E; Robin, J P; Koueta, N

2018-01-01

In Sepia officinalis (Linnaeus, 1758), the digestive gland matures during the first month post-hatching, while a shift from intracellular acid to extracellular alkaline digestion occurs. The purpose of this study was to investigate the possibility of using enzymatic ratios for the description of digestive system maturation in early life stages of S. officinalis. Second, it is intended to apply these new tools as eco-physiological indicators for understanding the impact of cuttlefish eggs' life history from different spawning sites of the English Channel on digestive performance of juveniles. An experimental rearing was performed over 35 days after hatching (DAH) on juveniles from wild collected eggs in 2010 and 2011. Four digestive enzyme activities and their ratios [i.e., trypsin, cathepsin, acid (ACP), and alkaline (ALP) phosphatase, ALP/ACP, and trypsin/cathepsin] were studied along with histological features (e.g., internal yolk surface and digestive gland development). The two enzyme ratios were good indicators of digestive system maturation allowing the study of the digestive gland's development. They were highly correlated to juveniles' weight increase and histological features of the gland in early DAH. These ratios described more accurately the shift occurring between the intracellular acid and the extracellular alkaline modes of digestion in S. officinalis and were more specific than separated enzyme activities. Their application as eco-physiological tools revealed that enzyme ratios reflected yolk content and digestive gland development in new hatching juveniles. Finally, ALP/ACP ratio was shown to be a powerful tool to describe growth performance of S. officinalis which is useful for aquaculture optimization.

Characterization of modified zeolite as microbial immobilization media on POME anaerobic digestion

Science.gov (United States)

Cahyono, Rochim B.; Ismiyati, Sri; Ginting, Simparmin Br; Mellyanawaty, Melly; Budhijanto, Wiratni

2018-03-01

As the world’s biggest palm oil producer, Indonesia generates also huge amount of Palm Oil Mill Effluent (POME) wastewater and causes serious problem in environment. In conventional method, POME was converted into biogas using lagoon system which required extensive land area. Anaerobic Fluidized Bed Reactor (AFBR) proposes more effective biogas producing with smaller land area. In the proposed system, a immobilization media would be main factor for enhancing productivity. This research studied on characterization of Lampung natural zeolite as immobilization media in the AFBR system for POME treatment. Various activation method such as physical and chemical were attempted to create more suitable material which has larger surface area, pore size distribution as well as excellent surface structures. The physical method was applied by heating up the material till 400°C while HCl was used on the chemical activation. Based on the result, the chemical activation increased the surface area significantly into 71 m2/g compared to physical as well as original zeolite. The strong acid material was quite effective to enforce the impurities within zeolite pore structure compared to heating up the material. According to distribution data, the Lampung zeolite owned the pore size with the range of 3 – 5 μm which was mesopore material. The pore size was appropriate for immobilization media as it was smaller than size of biogas microbial. The XRD patterns verified that chemical activation could maintain the zeolite structure as the original. Obviously, the SEM photograph showed apparent structure and pore size on the modified zeolite using chemical method. The testing of modified zeolite on the batch system was done to evaluate the characterization process. The modified zeolite using chemical process resulted fast reduction of COD and stabilized the volatile fatty acid as the intermediate product of anaerobic digestion, especially in the beginning of the process. Therefore, the

Obtaining of Fibers and granules of carbon for the Immobilization of Enzymes

International Nuclear Information System (INIS)

Malagon M, Martha L; Rico R, Yolanda Rico R; Lopez de, Helda A; Caicedo M, Luis Alfonso

2002-01-01

Fibers and pellets of carbon were prepared from coal tar. The tar was filtrated and stabilized in a nitrogen atmosphere at 330 degrades Celsius. Extrusion and pellets prepared the fibers by injection on water. Lactase was immobilized by adsorption process. Pellets were better support than fibers, because produced lower pressure drop and upper enzyme retention. Pellets showed the following characteristics: density 2,407 g/cm3, porosity 81,69% and diameter 3 mm

Properties of immobilized papain by radiation polymerization

International Nuclear Information System (INIS)

Kumakura, Minoru; Kaetsu, Isao

1984-01-01

Papain was immobilized by the radiation polymerization of various monomers at low temperatures and the effects of the polymer matrix on the enzyme activity and thermal stability of the immobilized enzymes were studied. The activity of the immobilized enzymes prepared from monofunctional (acrylate and methacrylate) monomers was higher than that from bifunctional (bismethacrylate) monomers and that from polyoxyethylene dimethacrylate monomers increased with an increase in the number of oxyethylene units. The thermal stability of the immobilized enzymes prepared from hydrophilic monomers was higher than that from hydrophobic monomers and increased markedly with increasing monomer concentration. (author)

Halloysite Clay Nanotubes for Enzyme Immobilization.

Science.gov (United States)

Tully, Joshua; Yendluri, Raghuvara; Lvov, Yuri

2016-02-08

Halloysite clay is an aluminosilicate nanotube formed by rolling flat sheets of kaolinite clay. They have a 15 nm lumen, 50-70 nm external diameter, length of 0.5-1 μm, and different inside/outside chemistry. Due to these nanoscale properties, they are used for loading, storage, and controlled release of active chemical agents, including anticorrosions, biocides, and drugs. We studied the immobilization in halloysite of laccase, glucose oxidase, and lipase. Overall, negatively charged proteins taken above their isoelectric points were mostly loaded into the positively charged tube's lumen. Typical tube loading with proteins was 6-7 wt % from which one-third was released in 5-10 h and the other two-thirds remained, providing enhanced biocatalysis in nanoconfined conditions. Immobilized lipase showed enhanced stability at acidic pH, and the optimum pH shifted to more alkaline pH. Immobilized laccase was more stable with respect to time, and immobilized glucose oxidase showed retention of enzymatic activity up to 70 °C, whereas the native sample was inactive.

Bacteria and digestive enzymes in the alimentary tract of the giant ...

African Journals Online (AJOL)

A study was carried out to investigate the bacteria flora in the gut of the Giant African Land Snails (GALS), Archarchatina marginata and Achatina achatina. Microflora cultures from snail gut contents were prepared to isolate and identify microorganisms within the snail digestive tract. Enzyme assays were carried out on a few ...

Immobilization of cellulase using porous polymer matrix

International Nuclear Information System (INIS)

Kumakura, M.; Kaetsu, I.

1984-01-01

A new method is discussed for the immobilization of cellulase using porous polymer matrices, which were obtained by radiation polymerization of hydrophilic monomers. In this method, the immobilized enzyme matrix was prepared by enzyme absorbtion in the porous polymer matrix and drying treatment. The enzyme activity of the immobilized enzyme matrix varied with monomer concentration, cooling rate of the monomer solution, and hydrophilicity of the polymer matrix, takinn the change of the nature of the porous structure in the polymer matrix. The leakage of the enzymes from the polymer matrix was not observed in the repeated batch enzyme reactions

Polymer-assisted iron oxide magnetic nanoparticle immobilized keratinase

Energy Technology Data Exchange (ETDEWEB)

Konwarh, Rocktotpal; Karak, Niranjan [Advanced Polymer and Nanomaterial Laboratory, Department of Chemical Sciences, Tezpur University, Tezpur-784028, Assam (India); Rai, Sudhir Kumar; Mukherjee, Ashis Kumar [Department of Molecular Biology and Biotechnology, Tezpur University, Tezpur-784028, Assam (India)], E-mail: karakniranjan@yahoo.com

2009-06-03

Nanotechnology holds the prospect for avant-garde changes to improve the performance of materials in various sectors. The domain of enzyme biotechnology is no exception. Immobilization of industrially important enzymes onto nanomaterials, with improved performance, would pave the way to myriad application-based commercialization. Keratinase produced by Bacillus subtilis was immobilized onto poly(ethylene glycol)-supported Fe{sub 3}O{sub 4} superparamagnetic nanoparticles. The optimization process showed that the highest enzyme activity was noted when immobilized onto cyanamide-activated PEG-assisted MNP prepared under conditions of 25 deg. C and pH 7.2 of the reaction mixture before addition of H{sub 2}O{sub 2} (3% w/w), 2% (w/v) PEG{sub 6000} and 0.062:1 molar ratio of PEG to FeCl{sub 2}{center_dot}4H{sub 2}O. Further statistical optimization using response surface methodology yielded an R{sup 2} value that could explain more than 94% of the sample variations. Along with the magnetization studies, the immobilization of the enzyme onto the PEG-assisted MNP was characterized by UV, XRD, FTIR and TEM. The immobilization process had resulted in an almost fourfold increase in the enzyme activity over the free enzyme. Furthermore, the immobilized enzyme exhibited a significant thermostability, storage stability and recyclability. The leather-industry-oriented application of the immobilized enzyme was tested for the dehairing of goat-skin.

Polymer-assisted iron oxide magnetic nanoparticle immobilized keratinase

International Nuclear Information System (INIS)

Konwarh, Rocktotpal; Karak, Niranjan; Rai, Sudhir Kumar; Mukherjee, Ashis Kumar

2009-01-01

Nanotechnology holds the prospect for avant-garde changes to improve the performance of materials in various sectors. The domain of enzyme biotechnology is no exception. Immobilization of industrially important enzymes onto nanomaterials, with improved performance, would pave the way to myriad application-based commercialization. Keratinase produced by Bacillus subtilis was immobilized onto poly(ethylene glycol)-supported Fe 3 O 4 superparamagnetic nanoparticles. The optimization process showed that the highest enzyme activity was noted when immobilized onto cyanamide-activated PEG-assisted MNP prepared under conditions of 25 deg. C and pH 7.2 of the reaction mixture before addition of H 2 O 2 (3% w/w), 2% (w/v) PEG 6000 and 0.062:1 molar ratio of PEG to FeCl 2 ·4H 2 O. Further statistical optimization using response surface methodology yielded an R 2 value that could explain more than 94% of the sample variations. Along with the magnetization studies, the immobilization of the enzyme onto the PEG-assisted MNP was characterized by UV, XRD, FTIR and TEM. The immobilization process had resulted in an almost fourfold increase in the enzyme activity over the free enzyme. Furthermore, the immobilized enzyme exhibited a significant thermostability, storage stability and recyclability. The leather-industry-oriented application of the immobilized enzyme was tested for the dehairing of goat-skin.

Polymer-assisted iron oxide magnetic nanoparticle immobilized keratinase

Science.gov (United States)

Konwarh, Rocktotpal; Karak, Niranjan; Rai, Sudhir Kumar; Mukherjee, Ashis Kumar

2009-06-01

Nanotechnology holds the prospect for avant-garde changes to improve the performance of materials in various sectors. The domain of enzyme biotechnology is no exception. Immobilization of industrially important enzymes onto nanomaterials, with improved performance, would pave the way to myriad application-based commercialization. Keratinase produced by Bacillus subtilis was immobilized onto poly(ethylene glycol)-supported Fe3O4 superparamagnetic nanoparticles. The optimization process showed that the highest enzyme activity was noted when immobilized onto cyanamide-activated PEG-assisted MNP prepared under conditions of 25 °C and pH 7.2 of the reaction mixture before addition of H2O2 (3% w/w), 2% (w/v) PEG6000 and 0.062:1 molar ratio of PEG to FeCl2·4H2O. Further statistical optimization using response surface methodology yielded an R2 value that could explain more than 94% of the sample variations. Along with the magnetization studies, the immobilization of the enzyme onto the PEG-assisted MNP was characterized by UV, XRD, FTIR and TEM. The immobilization process had resulted in an almost fourfold increase in the enzyme activity over the free enzyme. Furthermore, the immobilized enzyme exhibited a significant thermostability, storage stability and recyclability. The leather-industry-oriented application of the immobilized enzyme was tested for the dehairing of goat-skin.

Expression of digestive enzymes and nutrient transporters in Eimeria-challenged broilers.

Science.gov (United States)

Su, S; Miska, K B; Fetterer, R H; Jenkins, M C; Wong, E A

2015-03-01

Avian coccidiosis is a disease caused by the intestinal protozoa Eimeria. The site of invasion and lesions in the intestine is species-specific, for example E. acervulina affects the duodenum, E. maxima the jejunum, and E. tenella the ceca. Lesions in the intestinal mucosa cause reduced feed efficiency and body weight gain. The growth reduction may be due to changes in expression of digestive enzymes and nutrient transporters in the intestine. The objective of this study was to compare the expression of digestive enzymes, nutrient transporters and an antimicrobial peptide in broilers challenged with either E. acervulina, E. maxima or E. tenella. The genes examined included digestive enzymes (APN and SI), peptide and amino acid transporters (PepT1, ASCT1, b(0,+)AT/rBAT, B(0)AT, CAT1, CAT2, EAAT3, LAT1, y(+)LAT1 and y(+)LAT2), sugar transporters (GLUT1, GLUT2, GLUT5 and SGLT1), zinc transporter (ZnT1) and an antimicrobial peptide (LEAP2). Duodenum, jejunum, ileum and ceca were collected 7 days post challenge. E. acervulina challenge resulted in downregulation of various nutrient transporters or LEAP2 in the duodenum and ceca, but not the jejunum or ileum. E. maxima challenge produced both downregulation and upregulation of nutrient transporters and LEAP2 in all three segments of the small intestine and ceca. E. tenella challenge resulted in the downregulation and upregulation of nutrient transporters and LEAP2 in the jejunum, ileum and ceca, but not the duodenum. At the respective target tissue, E. acervulina, E. maxima and E. tenella infection caused common downregulation of APN, b(0,+)AT, rBAT, EAAT3, SI, GLUT2, GLUT5, ZnT1 and LEAP2. The downregulation of nutrient transporters would result in a decrease in the efficiency of protein and polysaccharide digestion and uptake, which may partially explain the weight loss. The downregulation of nutrient transporters may also be a cellular response to reduced expression of the host defense protein LEAP2, which would

Induced and constitutive responses of digestive enzymes to plant toxins in an herbivorous mammal.

Science.gov (United States)

Kohl, Kevin D; Dearing, M Denise

2011-12-15

Many plants produce plant secondary compounds (PSCs) that bind and inhibit the digestive enzymes of herbivores, thus limiting digestibility for the herbivore. Herbivorous insects employ several physiological responses to overcome the anti-nutritive effects of PSCs. However, studies in vertebrates have not shown such responses, perhaps stemming from the fact that previously studied vertebrates were not herbivorous. The responses of the digestive system to dietary PSCs in populations of Bryant's woodrat (Neotoma bryanti) that vary in their ecological and evolutionary experience with the PSCs in creosote bush (Larrea tridentata) were compared. Individuals from naïve and experienced populations were fed diets with and without added creosote resin. Animals fed diets with creosote resin had higher activities of pancreatic amylase, as well as luminal amylase and chymotrypsin, regardless of prior experience with creosote. The experienced population showed constitutively higher activities of intestinal maltase and sucrase. Additionally, the naïve population produced an aminopeptidase-N enzyme that was less inhibited by creosote resin when feeding on the creosote resin diet, whereas the experienced population constitutively expressed this form of aminopeptidase-N. Thus, the digestive system of an herbivorous vertebrate responds significantly to dietary PSCs, which may be important for allowing herbivorous vertebrates to feed on PSC-rich diets.

Fibrolytic enzyme and ammonia application effects on the nutritive value, intake, and digestion kinetics of bermudagrass hay in beef cattle.

Science.gov (United States)

Romero, J J; Zarate, M A; Queiroz, O C M; Han, J H; Shin, J H; Staples, C R; Brown, W F; Adesogan, A T

2013-09-01

The objectives were to compare the effect of exogenous fibrolytic enzyme (Biocellulase A20) or anhydrous ammonia (4% DM) treatment on the nutritive value, voluntary intake, and digestion kinetics of bermudagrass (Cynodon dactylon cultivar Coastal) hay harvested after 2 maturities (5- and 13-wk regrowths). Six individually housed, ruminally cannulated Brangus steers (BW 325 ± 10 kg) were used in an experiment with a 6 × 6 Latin square design with a 3 (additives) × 2 (maturities) factorial arrangement of treatments. Each period consisted of 14 d of adaptation and 7, 4, 1, 1, and 4 d for measuring in vivo digestibility, in situ degradability, no measurements, rumen liquid fermentation and passage indices, and rate of solid passage, respectively. Steers were fed hay for ad libitum intake and supplemented with sugarcane molasses and distillers grain (supplement total of 2.88 kg DM/d). Enzyme did not affect the nutritional composition of hay but ammonia treatment decreased hay NDF, hemicellulose, and ADL concentrations and increased the CP concentration particularly for the mature lignified 13-wk hay. The enzyme increased NDF and hemicellulose digestibility of the 5-wk hay but decreased those of the 13-wk hay. Ammoniation decreased intake of hay but increased digestibility of DM, OM, NDF, hemicellulose, ADF, and cellulose and increased the ruminal in situ soluble and potentially digestible fractions and the rate of DM degradation of the 13-wk hay. Also, ammoniation increased the concentrations of ruminal NH3, total VFA, acetate, and butyrate but enzyme treatment did not. Neither enzyme addition nor ammoniation affected rate of liquid and solid passage. In conclusion, ammoniation decreased the concentration of most fiber fractions, decreased the intake of hays, and increased their CP concentration, in vivo digestibility, and in situ degradability at both maturities whereas enzyme application increased fiber digestibility of the 5-wk hay but decreased it in the case of

Thermal stability of the immobilized fructosyltransferase from Rhodotorula sp

Directory of Open Access Journals (Sweden)

E Aguiar-Oliveira

2011-09-01

Full Text Available The thermal stability of the extracellular fructosyltransferase (FTase from Rhodotorula sp., recovered from cultivation medium by ethanol precipitation and immobilized onto niobium ore, was studied by Arrhenius plot, half - life profile, half - inactivation temperature (T50 and thermodynamic parameters. The Arrhenius plot showed two different behaviors with different deactivation energies (Ead only after immobilization, the transition occurring in the temperature interval between 51 and 52ºC. T50 for the free enzyme was estimated to be around 62ºC and, after immobilization, 66ºC. After 15 minutes at 52ºC, it was also possible to observe enzymatic activation for both the free and immobilized forms, but greater activation was achieved at pH 4.5 with the immobilized enzyme. Between 47 - 51ºC the immobilized enzyme was more stable than the free enzyme, with pH 6.0 being the more stable condition for the immobilized enzyme. However, above 52ºC the free form was more stable.

Immobilized ligninolytic enzymes: An innovative and environmental responsive technology to tackle dye-based industrial pollutants - A review.

Science.gov (United States)

Bilal, Muhammad; Asgher, Muhammad; Parra-Saldivar, Roberto; Hu, Hongbo; Wang, Wei; Zhang, Xuehong; Iqbal, Hafiz M N

2017-01-15

In the twenty-first century, chemical and associated industries quest a transition prototype from traditional chemical-based concepts to a greener, sustainable and environmentally-friendlier catalytic alternative, both at the laboratory and industrial scale. In this context, bio-based catalysis offers numerous benefits along with potential biotechnological and environmental applications. The bio-based catalytic processes are energy efficient than conventional methodologies under moderate processing, generating no and negligible secondary waste pollution. Thanks to key scientific advances, now, solid-phase biocatalysts can be economically tailored on a large scale. Nevertheless, it is mandatory to recover and reprocess the enzyme for their commercial feasibility, and immobilization engineering can efficiently accomplish this challenge. The first part of the present review work briefly outlines the immobilization of lignin-modifying enzymes (LMEs) including lignin peroxidase (LiP), manganese peroxidase (MnP) and laccase of white-rot fungi (WRF). Whereas, in the second part, a particular emphasis has been given on the recent achievements of carrier-immobilized LMEs for the degradation, decolorization, or detoxification of industrial dyes and dye-based industrial wastewater effluents. Copyright © 2016 Elsevier B.V. All rights reserved.

Influence of probiotics on the growth and digestive enzyme activity of white Pacific shrimp ( Litopenaeus vannamei)

Science.gov (United States)

Gómez, R. Geovanny D.; Shen, M. A.

2008-05-01

The influence of Bacillus probiotics on the digestive enzyme activity and the growth of Litopenaeus vannamei were determined in this study. The shrimp was treated with five percentages (1.5, 3.0, 4.5, 6.0 and 7.5) of probiotics ( Bacillus spp.) supplemented to the feed and cultured for 45d. The growth measured as the weight gain at the end of culturing was significantly ( Pshrimps than that of the control (without receiving probiotics). Activities of protease and amylase, two digestive enzymes of the midgut gland and the intestine were significantly ( Pshrimp than in the control.

Kinetics of Anaerobic Digestion of Palm Oil Mill Effluent (POME) in Double-Stage Batch Bioreactor with Recirculation and Fluidization of Microbial Immobilization Media

Science.gov (United States)

Ramadhani, L. I.; Damayanti, S. I.; Sudibyo, H.; Budhijanto, W.

2018-03-01

Palm Oil Mill Effluent (POME) becomes big problem for palm oil industries, especially for Crude Palm Oil (CPO) industry since it produces 3 tons of POME for every ton of CPO production.The high amount of organic loading in POME makes it potential as a substrate in anaerobic digestion to generate biogas as renewable energy source. The most common but conventional method by using open lagoon is still preferred for most CPO industry in Indonesia to treat POME because of its simplicity and easiness. However, this method creates new major problem for the water bodies since it has no significant chemical oxygen demand (COD) removal and needs wide area. Besides, greenhouse gas (CH4) is also released during the process. An innovation was made in this study by designing vertical column process equipment to run an anaerobic digestion of POME. The vertical column was functioned as anaerobic fluidized bed reactor (AFBR). To enhance the digestion rate in AFBR, natural zeolite was used as the immobilization media and the inoculum was taken from digested biodiesel waste. This research aimed to determine the kinetic constants of double-stage anaerobic POME digestion for COD removal and biogas production. To get close to the real condition, the POME used in this experiment had 8,000 mg/L of sCOD (the real sCOD was ±16,000 mg/L). The experiment was conducted under room temperature with up-flow velocity between 1.75 and 2.3 cm/s for optimum fluidization of immobilization media.

Effects of sources of protein and enzyme supplementation on protein digestibility and chyme characteristics in broilers.

Science.gov (United States)

Yu, B; Lee, T T T; Chiou, P W S

2002-07-01

1. The purpose of this study was to evaluate the effects of protein source and enzyme supplementation on protein digestibility and chyme characteristics in broilers. 2. One hundred and twenty growing (13 d old) and 60 finishing (34 d old) Arbor Acre strain commercial male broilers were selected and placed into individual metabolic cages. 3. The experiment was a 5 x 2 factorial arrangement with 5 different sources of protein: casein, fish meal, soybean meal (SBM), soy protein concentrate (SPC), maize gluten meal (MGM) and two levels of protease (bromelain), 0 and 65 CDU/kg diets. 4. The diets were iso-nitrogenous and semi-purified, with Cr2O3 as an indicator for determination of ileal digestibility and chyme characteristics. 5. Apparent ileal protein digestibility (AIPD) in both growing and finishing chickens was highest on the casein diet, followed by fish meal, SBM, SPC and MGM. 6. Enzyme inclusion did not improve protein digestibility, but significantly decreased the digesta pH value in the gizzard and increased pH in the ileum in the 3-week-old broilers. 7. The digesta pH values in the gizzard and duodenum were significantly lower in the SBM and fish meal groups compared with the other protein groups. The molecular weight distribution pattern of the soluble protein in the chyme of the gastrointestinal (GI) segments showed a similar trend, regardless of the enzyme inclusion or the stage of growth. 8. The molecular weight profile of soluble protein changed dynamically in the casein fed broilers from the gizzard to ileum and the low molecular weight proteins, < 7 kDa, reached maximum levels at the ileum. The molecular weight profile of the soluble protein in the SBM and SPC changed between the jejunum and the ileum and in the intermediate molecular soluble protein weight (7 to 10 kDa) was significantly decreased. This indicated that the hydrolysis process began from the middle to the posterior end of the small intestine. 9. Similar profiles were also shown with

Effect of enzyme location on activity and stability of trypsin and urease immobilized on porous membranes by using layer-by-layer self-assembly of polyelectrolyte

OpenAIRE

Guedidi, Sadika; Yürekli, Yılmaz; Deratani, André; Déjardin, Philippe; Innocent, Christophe; Altınkaya, Sacide; Roudesli, Sadok; Yemenicioğlu, Ahmet

2010-01-01

The layer-by-layer (LbL) self-assembly of polyelectrolyte is one of the simplest ways to immobilize enzyme on membrane. In this paper, the immobilization of trypsin (TRY) and urease (URE) on polyacrylonitrile based membranes using the LbL assembly technique was presented. The studied systems consisted in bilayered assemblies with the enzyme layer as the outer layer and trilayered assemblies with the enzyme layer as the inner sandwiched layer. The membrane pore size was chosen so that the smal...

Digestibility, productive performance, and egg quality of laying hens as affected by dried cassava pulp replacement with corn and enzyme supplementation.

Science.gov (United States)

Khempaka, Sutisa; Maliwan, Prapot; Okrathok, Supattra; Molee, Wittawat

2018-02-24

Two experiments were conducted to investigate the potential use of dried cassava pulp (DCP) supplemented with enzymes as an alternative feed ingredient in laying hen diets. In experiment 1, 45 laying hens (Isa Brown) aged 45 weeks were placed in individual cages to measure nutrient digestibility for 10 days. Nine dietary treatments were control and DCP as a replacement for corn at 20, 25, 30, and 35% supplemented with mixed enzymes (cellulase, glucanase, and xylanase) at 0.10 and 0.15%. Results showed that the use of DCP at 20-35% added with mixed enzymes had no negative effects on dry matter digestibility, while organic matter digestibility and nitrogen retention decreased with increased DCP up to 30-35% in diets. Both enzyme levels (0.10 and 0.15%) showed similar results on nutrient digestibility and retention. In experiment 2, a total of 336 laying hens aged 32 weeks were randomly allocated to seven dietary treatments (control and DCP-substituted diets at 20, 25, and 30%) supplemented with mixed enzymes (0.10 and 0.15%). Diets incorporated with 20-30% of DCP and supplemented with mixed enzymes at both levels had no significant effects on egg production, egg weight, feed intake, egg mass, feed conversion ratio, protein efficiency ratio, or egg quality, except for egg yolk color being decreased with an increase of DCP in diets (P digestibility, productive performance, or egg quality.

Development of digestive enzymes in larvae of Mayancichlid Cichlasoma urophthalmus

OpenAIRE

López Ramírez, G.; Cuenca Soria, C.A.; Álvarez González, C.A.; Tovar Ramírez, D.; Ortiz Galindo, José Luis; Perales García, N.; Márquez Couturier, G.; Arias Rodríguez, L.; Indy, J.R.; Contreras Sánchez, W.M.; Gisbert, E.; Moyano, F.J.

2011-01-01

The development of digestive enzymes during the early ontogeny of the Mayan cichlid (Cichlasoma urophthalmus) was studied using biochemical and electrophoretic techniques. From yolk absorption (6 days after hatching: dah), larvae were fed Artemia nauplii until 15 dah, afterward they were fed with commercial microparticulated trout food (45% protein and 16% lipids) from 16 to 60 dah. Several samples were collected including yolk-sac larvae (considered as day 1 after hatching) and specimens up ...

A Urea Biosensor from Stacked Sol-Gel Films with Immobilized Nile Blue Chromoionophore and Urease Enzyme

OpenAIRE

Alqasaimeh, Muawia Salameh; Heng, Lee Yook; Ahmad, Musa

2007-01-01

An optical urea biosensor was fabricated by stacking several layers of sol-gel films. The stacking of the sol-gel films allowed the immobilization of a Nile Blue chromoionophore (ETH 5294) and urease enzyme separately without the need of any chemical attachment procedure. The absorbance response of the biosensor was monitored at 550 nm, i.e. the deprotonation of the chromoionophore. This multi-layer sol-gel film format enabled higher enzyme loading in the biosensor to be achieved. The urea op...

OLED-based biosensing platform with ZnO nanoparticles for enzyme immobilization

Science.gov (United States)

Cai, Yuankun; Shinar, Ruth; Shinar, Joseph

2009-08-01

Organic light-emitting diode (OLED)-based sensing platforms are attractive for photoluminescence (PL)-based monitoring of a variety of analytes. Among the promising OLED attributes for sensing applications is the thin and flexible size and design of the OLED pixel array that is used for PL excitation. To generate a compact, fielddeployable sensor, other major sensor components, such as the sensing probe and the photodetector, in addition to the thin excitation source, should be compact. To this end, the OLED-based sensing platform was tested with composite thin biosensing films, where oxidase enzymes were immobilized on ZnO nanoparticles, rather than dissolved in solution, to generate a more compact device. The analytes tested, glucose, cholesterol, and lactate, were monitored by following their oxidation reactions in the presence of oxygen and their respective oxidase enzymes. During such reactions, oxygen is consumed and its residual concentration, which is determined by the initial concentration of the above-mentioned analytes, is monitored. The sensors utilized the oxygen-sensitive dye Pt octaethylporphyrin, embedded in polystyrene. The enzymes were sandwiched between two thin ZnO layers, an approach that was found to improve the stability of the sensing probes.

Immobilization of Isolated Lipase From Moldy Copra (Aspergillus Oryzae)

OpenAIRE

Dali, Seniwati; Patong, A. B. D. Rauf; Jalaluddin, M. Noor; Pirman; Hamzah, Baharuddin

2011-01-01

Enzyme immobilization is a recovery technique that has been studied in several years, using support as a media to help enzyme dissolutions to the reaction substrate. Immobilization method used in this study was adsorption method, using specific lipase from Aspergillus oryzae. Lipase was partially purified from the culture supernatant of Aspergillus oryzae. Enzyme was immobilized by adsorbed on silica gel. Studies on free and immobilized lipase systems for determination of optimum pH, optimum ...

Properties and Gamma Radiation Stability of Immobilized Alpha Amylase on Synthetic and Natural Polymer Blends

International Nuclear Information System (INIS)

Ismaill, S.A.; Mobasher, E.F.; Shousha, M.A.

2009-01-01

αAmylase was immobilized onto two different copolymers. One of them was chitosan/alginate copolymer. The other copolymer was N- isopropyl acrylamide and alginate. αAmylase was immobilized by entrapment method. The optimum temperature and thermal inactivation of the free enzyme and the immobilized one were investigated. The activity of the immobilized enzyme was stable at higher temperature. Immobilized enzyme was stable under different ph. The immobilized enzymes showed a slight decrease in the relative activity after being used 12 times. Storage of the free and immobilized enzymes for 2 months showed that the free αamylase lost most of its catalytic activity after storage at this period. The storage of the immobilized enzyme in dry state was much better than that in the wet state. Storage at room temperature showed much less stability of the immobilized enzyme than in 4 degree C. Exposure the free and immobilized enzymes to gamma- radiation at doses (0-50 kGy) showed complete loss in activity of free enzyme at 5 kGy, while the immobilized enzyme showed high resistance to gamma- radiation. The kinetic studies of free and immobilized enzymes showed that the immobilization process increased Km and decreased V m ax values of the enzyme

Properties and Gamma Radiation Stability of Immobilized Alpha Amylase on Synthetic and Natural Polymer Blends

International Nuclear Information System (INIS)

Ismaill, S.A.; Mobasher, E.F.; Shousha, M.A.

2008-01-01

αAmylase was immobilized onto two different copolymers. One of them was chitosan/alginate copolymer. The other copolymer was N- isopropyl acrylamide and alginate. αAmylase was immobilized by entrapment method. The optimum temperature and thermal inactivation of the free enzyme and the immobilized one were investigated. The activity of the immobilized enzyme was stable at higher temperature. Immobilized enzyme was stable under different ph. The immobilized enzymes showed a slight decrease in the relative activity after being used 12 times. Storage of the free and immobilized enzymes for 2 months showed that the free αamylase lost most of its catalytic activity after storage at this period. The storage of the immobilized enzyme in dry state was much better than that in the wet state. Storage at room temperature showed much less stability of the immobilized enzyme than in 4 degree C. Exposure the free and immobilized enzymes to gamma- radiation at doses (0-50 kGy) showed complete loss in activity of free enzyme at 5 kGy, while the immobilized enzyme showed high resistance to gamma- radiation. The kinetic studies of free and immobilized enzymes showed that the immobilization process increased Km and decreased V m ax values of the enzyme

Development of high-performance functional materials for enzyme immobilization by the use of ionizing radiation

International Nuclear Information System (INIS)

SALIM, R.D.M.

2013-01-01

Isomerization of glucose to fructose was carried out using Glucose isomerase (GI) that immobilized by entrapment into Poly (acrylic acid) P (AA) and Poly (acrylic acid-co- 2-Acrylamido 2- methyl Propane sulfonic acid) P (AA-co-AMPS) polymer networks, the enzyme carriers were prepared by radiation induced co-polymerization in presence of (Methylene- bis acrylamide) (MBAA) as a crosslinking agent. Effects of immobilization conditions such as irradiation dose, methylene bis acrylamide concentration, comonomer composition, and amount of GI were investigated. The influence of reaction conditions on the activity of immobilized GI were studied, the optimum ph value of reaction solution is 7.5 and reaction temperature is 65 degree C. The immobilized GI into P (AA-co-AMPS) and P (AA) polymer networks retained 81% and 69%,respectively, of its initial activity after recycled for 15 times while it retained 87% and 71% ,respectively ,of its initial activity after stored at 4 degree C for 48 days , The Km values of free and immobilized GI onto P(AA-co-AMPS) and onto P(AA) matrices were found to be 34, 29.2 , 14.5 mg/ml respectively while the Vmax Values calculated to be 3.87 ,1.6,0.79 mg/ml.min, respectively, Therefore , the bio conversion of glucose to fructose can be successfully performed by GI entrapped into P (AA-co-AMPS) hydrogel .

Porous chitosan beads of superior mechanical properties for the covalent immobilization of enzymes.

Science.gov (United States)

Wahba, Marwa I

2017-12-01

Porous chitosan beads of superior mechanical properties were produced via a two stepped treatment process. First, the chitosan ionotropic gelation solution was supplemented with Na 2 CO 3 , which acted as a porogen. Afterwards, the beads were chemically cross-linked with glutaraldehyde. This treatment also caused the produced porous chitosan beads to acquire higher observed activities of immobilized β-d-galactosidase (β-gal). The observed activities of the β-gal immobilized onto the 0.2M and the 0.35M Na 2 CO 3 treated beads were 1.63 and 1.91 fold respectively, higher than the activity offered by the control beads. Nevertheless, both the control beads and the 0.2M Na 2 CO 3 beads caused the optimum pH range of β-gal to shift from 4.6-5.1 to ∼2.7-5. The enzyme's optimum temperature shifted from 55 to 60°C after its immobilization onto the control chitosan beads whereas the β-gal immobilized onto the 0.2M Na 2 CO 3 chitosan beads exhibited a temperature optimum of 55-60°C. The reusability study revealed the superiority of the 0.2M Na 2 CO 3 treated beads which retained 59.1% of their initial activity during the 13th enzymatic cycle. On the other hand, the control chitosan beads were fragmented and lost their activity after only four enzymatic cycles. Copyright © 2017 Elsevier B.V. All rights reserved.

Advantages of the Pre-Immobilization of Enzymes on Porous Supports for Their Entrapment in Sol-Gels

Czech Academy of Sciences Publication Activity Database

Betancor, L.; López-Gallego, F.; Hidalgo, A.; Fuentes, M.; Podrazký, Ondřej; Kuncová, Gabriela; Guisán, J.M.; Fernández-Lafuente, R.

2005-01-01

Roč. 6, č. 2 (2005), s. 1027-1030 ISSN 1525-7797 Grant - others:CICYT(ES) BIO2000/0747/C05/02; CICYT(ES) BIO2001/2259 Institutional research plan: CEZ:AV0Z40720504 Keywords : immobilization * enzymes * sol-gel Subject RIV: CE - Biochemistry Impact factor: 3.618, year: 2005

Molecular view of the interaction between iota-carrageenan and a phospholipid film and its role in enzyme immobilization.

Science.gov (United States)

Nobre, Thatyane M; de Sousa e Silva, Heurison; Furriel, Rosa P M; Leone, Francisco A; Miranda, Paulo B; Zaniquelli, Maria Elisabete D

2009-05-28

Proteins incorporated into phospholipid Langmuir-Blodgett (LB) films are a good model system for biomembranes and enzyme immobilization studies. The specific fluidity of biomembranes, an important requisite for enzymatic activity, is naturally controlled by varying phospholipid compositions. In a model system, instead, LB film fluidity may be varied by covering the top layer with different substances able to interact simultaneously with the phospholipid and the protein to be immobilized. In this study, we immobilized a carbohydrate rich Neurospora crassa alkaline phosphatase (NCAP) in monolayers of the sodium salt of dihexadecylphosphoric acid (DHP), a synthetic phospholipid that provides very condensed Langmuir films. The binding of NCAP to DHP Langmuir-Blodgett (LB) films was mediated by the anionic polysaccharide iota-carrageenan (iota-car). Combining results from surface isotherms and the quartz crystal microbalance technique, we concluded that the polysaccharide was essential to promote the interaction between DHP and NCAP and also to increase the fluidity of the film. An estimate of DHP:iota-car ratio within the film also revealed that the polysaccharide binds to DHP LB film in an extended conformation. Furthermore, the investigation of the polysaccharide conformation at molecular level, using sum-frequency vibrational spectroscopy (SFG), indicated a preferential conformation of the carrageenan molecules with the sulfate groups oriented toward the phospholipid monolayer, and both the hydroxyl and ether groups interacting preferentially with the protein. These results demonstrate how interfacial electric fields can reorient and induce conformational changes in macromolecules, which may significantly affect intermolecular interactions at interfaces. This detailed knowledge of the interaction mechanism between the enzyme and the LB film is relevant to design strategies for enzyme immobilization when orientation and fluidity properties of the film provided by the

The influence of carbon nanotubes on enzyme activity and structure: investigation of different immobilization procedures through enzyme kinetics and circular dichroism studies

International Nuclear Information System (INIS)

Cang-Rong, Jason Teng; Pastorin, Giorgia

2009-01-01

In the last decade, many environmental organizations have devoted their efforts to identifying renewable biosystems, which could provide sustainable fuels and thus enhance energy security. Amidst the myriad of possibilities, some biofuels make use of different types of waste biomasses, and enzymes are often employed to hydrolyze these biomasses and produce sugars that will be subsequently converted into ethanol. In this project, we aimed to bridge nanotechnology and biofuel production: here we report on the activity and structure of the enzyme amyloglucosidase (AMG), physically adsorbed or covalently immobilized onto single-walled carbon nanotubes (SWNTs) and multi-walled carbon nanotubes (MWNTs). In fact, carbon nanotubes (CNTs) present several properties that render them ideal support systems, without the diffusion limitations displayed by porous material and with the advantage of being further functionalizable at their surface. Chemical ligation was achieved both on oxidized nanotubes (via carbodiimide chemistry), as well as on amino-functionalized nanotubes (via periodate-oxidized AMG). Results showed that AMG retained a certain percentage of its specific activity for all enzyme-carbon nanotubes complexes prepared, with the physically adsorbed samples displaying better catalytic efficiency than the covalently immobilized samples. Analysis of the enzyme's structure through circular dichroism (CD) spectroscopy revealed significant structural changes in all samples, the degree of change being consistent with the activity profiles. This study proves that AMG interacts differently with carbon nanotubes depending on the method employed. Due to the higher activity reported by the enzyme physically adsorbed onto CNTs, these samples demonstrated a vast potential for further development. At the same time, the possibility of inducing magnetic properties into CNTs offers the opportunity to easily separate them from the original solution. Hence, substances to which they

Rheostatic control of tryptic digestion in a microscale fluidic system

International Nuclear Information System (INIS)

Percy, Andrew J.; Schriemer, David C.

2010-01-01

Integrated fluidic systems that unite bottom-up and top-down proteomic approaches have the potential to deliver complete protein characterization. To circumvent fraction collection, as is conducted in current blended approaches, a technique to regulate digestion efficiency in a flow-through system is required. The present study examined the concept of regulating tryptic digestion in an immobilized enzyme reactor (IMER), incorporating mixed solvent systems for digestion acceleration. Using ovalbumin, cytochrome c, and myoglobin as protein standards, we demonstrate that tryptic digestion can be efficiently regulated between complete digestion and no digestion extremes by oscillating between 45 and 0% acetonitrile in the fluid stream. Solvent composition was tuned using programmable solvent waveforms in a closed system consisting of the IMER, a sample delivery stream, a dual gradient pumping system and a mass spectrometer. Operation in this rheostatic digestion mode provides access to novel peptide mass maps (due to substrate unfolding hysteresis) as well as the intact protein, in a reproducible and stable fashion. Although cycle times were on the order of 90 s for testing purposes, we show that regulated digestion is sufficiently rapid to be limited by solvent switching efficiency and kinetics of substrate unfolding/folding. Thus, regulated digestion should be useful in blending bottom-up and top-down proteomics in a single closed fluidic system.

Mesoporous silica nanoparticles supported copper(II) and nickel(II) Schiff base complexes: Synthesis, characterization, antibacterial activity and enzyme immobilization

Science.gov (United States)

Tahmasbi, Leila; Sedaghat, Tahereh; Motamedi, Hossein; Kooti, Mohammad

2018-02-01

Mesoporous silica nanoparticles (MSNs) were prepared by sol-gel method and functionalized with 3-aminopropyltriethoxysilane. Schiff base grafted mesoporous silica nanoparticle was synthesized by the condensation of 2-hydroxy-3-methoxybenzaldehyde and amine-functionalized MSNs. The latter material was then treated with Cu(II) and Ni(II) salts separately to obtain copper and nickel complexes anchored mesoporous composites. The newly prepared hybrid organic-inorganic nanocomposites have been characterized by several techniques such as FT-IR, LA-XRD, FE-SEM, TEM, EDS, BET and TGA. The results showed all samples have MCM-41 type ordered mesoporous structure and functionalization occurs mainly inside the mesopore channel. The presence of all elements in synthesized nanocomposites and the coordination of Schiff base via imine nitrogen and phenolate oxygen were confirmed. MSNs and all functionalized MSNs have uniform spherical nanoparticles with a mean diameter less than 100 nm. The as-synthesized mesoporous nanocomposites were investigated for antibacterial activity against Gram-positive (B. subtilis and S. aureus) and Gram-negative (E. coli and P. aeruginosa) bacteria, as carrier for gentamicin and also for immobilization of DNase, coagulase and amylase enzymes. MSN-SB-Ni indicated bacteriocidal effect against S.aureus and all compounds were found to be good carrier for gentamicin. Results of enzyme immobilization for DNase and coagulase and α-amylase revealed that supported metal complexes efficiently immobilized enzymes.

Production of cellulase from immobilized Trichoderma reesei

International Nuclear Information System (INIS)

Kasai, Noboru; Tamada, Masao; Kumakura, Minoru

1989-05-01

This report completed the results that obtained on the study of the enzyme activity in the culture of immobilized Trichoderma reesei cells in flask scale (100ml) and bench scale (30l). In the flask scale culture, the batch and repeated batch culture were carried out, and in the bench scale culture, the batch, repeated batch and continuous culture were done by using a culture equipment that is an unit process of the bench scale test plant for saccharification of cellulosic wastes. The enzyme activity of the immobilized cells was higher than that of the intact cells in the flask scale culture and it was confirmed that the enzyme activity was not decreased on the repeated batch culture of six times even. In the bench scale culture, it was found that a optimum culture condition of the immobilized cells was not different from that of the free cells and the immobilized cells gave the enzyme solution with a high enzyme activity in the culture condition of 450rpm stirring speed and air supply of 0.1v/v/m above. The technique of the repeated batch and continuous culture for long times in bench scale without contamination was established. The enzyme activity of the immobilized cells in continuous culture became to be 85 % to that in batch culture and it was found that the enzyme solution with high enzyme activity was continuously obtained in the continuous culture for long times. (author)

Lipase immobilization and production of fatty acid methyl esters from canola oil using immobilized lipase

International Nuclear Information System (INIS)

Yuecel, Yasin; Demir, Cevdet; Dizge, Nadir; Keskinler, Buelent

2011-01-01

Lipase enzyme from Aspergillus oryzae (EC 3.1.1.3) was immobilized onto a micro porous polymeric matrix which contains aldehyde functional groups and methyl esters of long chain fatty acids (biodiesel) were synthesized by transesterification of crude canola oil using immobilized lipase. Micro porous polymeric matrix was synthesized from styrene-divinylbenzene (STY-DVB) copolymers by using high internal phase emulsion technique and two different lipases, Lipozyme TL-100L ® and Novozym 388 ® , were used for immobilization by both physical adsorption and covalent attachment. Biodiesel production was carried out with semi-continuous operation. Methanol was added into the reactor by three successive additions of 1:4 M equivalent of methanol to avoid enzyme inhibition. The transesterification reaction conditions were as follows: oil/alcohol molar ratio 1:4; temperature 40 o C and total reaction time 6 h. Lipozyme TL-100L ® lipase provided the highest yield of fatty acid methyl esters as 92%. Operational stability was determined with immobilized lipase and it indicated that a small enzyme deactivation occurred after used repeatedly for 10 consecutive batches with each of 24 h. Since the process is yet effective and enzyme does not leak out from the polymer, the method can be proposed for industrial applications. -- Research highlights: → Lipozyme TL-100L and Novozym 388 were immobilized onto micro porous polymeric matrix by both physical adsorption and covalent linking. → Immobilized enzymes were used for synthesis of fatty acid methyl esters by transesterification of canola oil and methanol using semi-continuous operation system. → According to chromatographic analysis, Lipase Lipozyme TL-100L resulted in the highest yield of methyl ester as 92%.

Functional and transcriptomic analysis of leaf litter digestion by millipedes: Role of microorganisms and digestive enzymes. An introduction of a starting project

Czech Academy of Sciences Publication Activity Database

Tajovský, Karel; Šustr, Vladimír; Chroňáková, Alica; Chrudimský, Tomáš; Macková, Jana; Koubová, Anna; Šimek, Miloslav

-, Suppl. 5 (2017), s. 61 ISSN 1513-9700. [International Congress of Myriapodology /17./. 23.07.2017-26.07.2017, Krabi] Institutional support: RVO:60077344 Keywords : millipedes * digestive enzymes * gut microbial communities * cellulose degradation * methane release Subject RIV: EG - Zoology OBOR OECD: Zoology

Prolidase is a critical enzyme for complete gliadin digestion in Tenebrio molitor larvae.

Science.gov (United States)

Tereshchenkova, Valeriia F; Goptar, Irina A; Zhuzhikov, Dmitry P; Belozersky, Mikhail A; Dunaevsky, Yakov E; Oppert, Brenda; Filippova, Irina Yu; Elpidina, Elena N

2017-08-01

Prolidase is a proline-specific metallopeptidase that cleaves imidodipeptides with C-terminal Pro residue. Prolidase was purified and characterized from the Tenebrio molitor larval midgut. The enzyme was localized in the soluble fraction of posterior midgut tissues, corresponding to a predicted cytoplasmic localization of prolidase according to the structure of the mRNA transcript. Expression of genes encoding prolidase and the major digestive proline-specific peptidase (PSP)-dipeptidyl peptidase 4-were similar. The pH optimum of T. molitor prolidase was 7.5, and the enzyme was inhibited by Z-Pro, indicating that it belongs to type I prolidases. In mammals, prolidase is particularly important in the catabolism of a proline-rich protein-collagen. We propose that T. molitor larval prolidase is a critical enzyme for the final stages of digestion of dietary proline-rich gliadins, providing hydrolysis of imidodipeptides in the cytoplasm of midgut epithelial cells. We propose that the products of hydrolysis are absorbed from the luminal contents by peptide transporters, which we have annotated in the T. molitor larval gut transcriptome. The origin of prolidase substrates in the insect midgut is discussed in the context of overall success of grain feeding insects. © 2017 Wiley Periodicals, Inc.

Digestive Enzyme Supplementation for Autism Spectrum Disorders: A Double-Blind Randomized Controlled Trial

Science.gov (United States)

Munasinghe, Sujeeva A.; Oliff, Carolyn; Finn, Judith; Wray, John A.

2010-01-01

To examine the effects of a digestive enzyme supplement in improving expressive language, behaviour and other symptoms in children with Autism Spectrum Disorder. Randomized, double-blind placebo-controlled trial using crossover design over 6 months for 43 children, aged 3-8 years. Outcome measurement tools included monthly Global Behaviour Rating…

Evaluation of fungal laccase immobilized on natural nanostructured bacterial cellulose

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Lin eChen

2015-11-01

Full Text Available The aim of this work was to assess the possibility of using native bacterial nanocellulose (BC as a carrier for laccase immobilization. BC was synthesized by Gluconacetobacter xylinus, which was statically cultivated in a mannitol-based medium and was freeze-dried to form BC sponge after purification. For the first time, fungal laccase from Trametes versicolor was immobilized on the native nanofibril network-structured BC sponge through physical adsorption and cross-linking with glutaraldehyde. The properties including morphologic and structural features of the BC as well as the immobilized enzyme were thoroughly investigated. It was found that enzyme immobilized by cross-linking exhibited broader pH operation range of high catalytic activity as well as higher running stability compared to free and adsorbed enzyme. Using ABTS as substrate, the optimum pH value was 3.5 for the adsorption-immobilized laccase and 4.0 for the crosslinking-immobilized laccase. The immobilized enzyme retained 69% of the original activity after being recycled 7 times. Novel applications of the BC-immobilized enzyme tentatively include active packaging, construction of biosensors, and establishment of bioreactors.

Relationship between digestive enzymes and food habit of Lutzomyia longipalpis (Diptera: Psychodidae) larvae: Characterization of carbohydrases and digestion of microorganisms.

Science.gov (United States)

Moraes, C S; Lucena, S A; Moreira, B H S; Brazil, R P; Gontijo, N F; Genta, F A

2012-08-01

The sandfly Lutzomyia longipalpis (Lutz and Neiva, 1912) is the main vector of American Visceral Leishmaniasis. In spite of its medical importance and several studies concerning adult digestive physiology, biochemistry and molecular biology, very few studies have been carried out to elucidate the digestion in sandfly larvae. Even the breeding sites and food sources of these animals in the field are largely uncharacterized. In this paper, we describe and characterize several carbohydrases from the gut of L. longipalpis larvae, and show that they are probably not acquired from food. The enzyme profile of this insect is consistent with the digestion of fungal and bacterial cells, which were proved to be ingested by larvae under laboratory conditions. In this respect, sandfly larvae might have a detritivore habit in nature, being able to exploit microorganisms usually encountered in the detritus as a food source. Copyright © 2012 Elsevier Ltd. All rights reserved.

The effect of water temperature on food transit time and digestive enzymes activity in Caspian kutum (Rutilus kutum larvae

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Nahid Ghysvandi

2014-07-01

Full Text Available The present study investigates the effects of water temperature on digestive enzymes activity and food transit time in Caspian kutum (Rutilus kutum larvae. Caspian kutum larvae (532 ± 0.05 and 543 ± 0.02 mg were divided into two groups with three replicates and reared at different water temperature i.e. 25.6 ± 0.4°C (T1 and 18.4 ± 0.1°C (T2. At the end of the experiment, sampling of intestine was performed at 0, 1, 3, 5, 8, 16, 24 and 30 h after feeding from each treatment. In T2, food was observed until 24 h after feeding and the intestine was empty 29 h after feeding, while in T1 19 h after feeding the intestine was empty. Digestive enzymes activities were higher in T2 treatment. The peaks of trypsin and alkaline phosphatase enzymes activity were found 8 h after feeding in T1, while occurred 16 h after feeding in T2. The highest chymotrypsin and alpha-amylase enzymes activity were observed 5 and 8h after feeding in T1 and T2, respectively. These results confirmed remarkable effects of temperature on food transit time and digestive enzymes activity of Caspian kutum.

EFFECTS OF EXOGENOUS ENZYMES ON NUTRIENTS DIGESTIBILITY AND GROWTH PERFORMANCE IN SHEEP AND GOATS

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Abdel-Fattah Z.M. Salem

2011-07-01

Full Text Available Six crossbred sheep (32.00±0.603 kg BW and 6 Baladi goats (18.00±0.703 kg BW were used in 2×2 factorial design to evaluate the effect of exogenous enzymes of ZADO® (i.e., ENZ and on digestibility and growth performance. Animals were fed on wheat straw ad libitum and restricted amount of commercial concentrate with (+ENZ or without (-ENZ 10 g/animal/day of ZADO to cover 120% of their maintenance requirements. Nutrients digestibilities were increased (P

Cascade catalysis in membranes with enzyme immobilization for multienzymatic conversion of CO2 to methanol

DEFF Research Database (Denmark)

Luo, Jianquan; Meyer, Anne S.; Mateiu, Ramona Valentina

2015-01-01

.e. by directing membrane fouling formation), without any addition of organic solvent. Such coimmobilization and sequential immobilization systems were examined for the production of methanol from CO2 with formate dehydrogenase (FDH), formaldehyde dehydrogenase (FaldDH) and alcohol dehydrogenase (ADH). Enzyme...... for multi-enzymatic cascade systems, but also reveals the reaction bottleneck and provides possible solutions for the bioconversion of CO2 to methanol....

Utilization of newly developed immobilized enzyme reactors for preparation and study of immunoglobulin G fragments

Czech Academy of Sciences Publication Activity Database

Korecká, L.; Bílková, Z.; Holčapek, M.; Královský, J.; Beneš, Milan J.; Lenfeld, Jiří; Minc, N.; Cecal, R.; Viovy, J.-L.; Przybylski, M.

2004-01-01

Roč. 808, č. 1 (2004), s. 15-24 ISSN 1570-0232. [International Symposium on Polymer Design for BioSeparation and Nanobiotechnology /8./. Compiegne, 27.11.2003-29.11.2003] Grant - others:GA ČR(CZ) GA203/02/0023 Program:GA Institutional research plan: CEZ:AV0Z4050913 Keywords : immobilized enzyme reactors * immunoglobulin G Subject RIV: CE - Biochemistry Impact factor: 2.176, year: 2004

Evaluation of the Optimal Reaction Conditions for the Methanolysis and Ethanolysis of Castor Oil Catalyzed by Immobilized Enzymes

DEFF Research Database (Denmark)

Andrade, Thalles Allan; Al-Kabalawi, Ibrahim; Errico, Massimiliano

This study aims to compare the efficiency of the transesterification of castor oil with methanol and ethanol as part of the biodiesel production, using immobilized enzyme Lipozyme IM as catalyst. Different reaction conditions were evaluated and optimized, including the reaction temperature, alcohol...

Immobilization of Chloroperoxidase on Aminopropyl-Glass

Science.gov (United States)

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

Enhanced activity and stability of L-arabinose isomerase by immobilization on aminopropyl glass.

Science.gov (United States)

Zhang, Ye-Wang; Jeya, Marimuthu; Lee, Jung-Kul

2011-03-01

Immobilization of Bacillus licheniformis L: -arabinose isomerase (BLAI) on aminopropyl glass modified with glutaraldehyde (4 mg protein g support⁻¹) was found to enhance the enzyme activity. The immobilization yield of BLAI was proportional to the quantity of amino groups on the surface of support. Reducing particle size increased the adsorption capacity (q(m)) and affinity (k(a)). The pH and temperature for immobilization were optimized to be pH 7.1 and 33 °C using response surface methodology (RSM). The immobilized enzyme was characterized and compared to the free enzyme. There is no change in optimal pH and temperature before and after immobilization. However, the immobilized BLAI enzyme achieved 145% of the activity of the free enzyme. Correspondingly, the catalytic efficiency (k(cat)/K(m)) was improved 1.47-fold after immobilization compared to the free enzyme. The thermal stability was improved 138-fold (t₁/₂) increased from 2 to 275 h) at 50 °C following immobilization.

Symbiotic Chlorella variabilis incubated under constant dark conditions for 24 hours loses the ability to avoid digestion by host lysosomal enzymes in digestive vacuoles of host ciliate Paramecium bursaria.

Science.gov (United States)

Kodama, Yuuki; Fujishima, Masahiro

2014-12-01

Endosymbiosis between symbiotic Chlorella and alga-free Paramecium bursaria cells can be induced by mixing them. To establish the endosymbiosis, algae must acquire temporary resistance to the host lysosomal enzymes in the digestive vacuoles (DVs). When symbiotic algae isolated from the alga-bearing paramecia are kept under a constant dark conditions for 24 h before mixing with the alga-free paramecia, almost all algae are digested in the host DVs. To examine the cause of algal acquisition to the host lysosomal enzymes, the isolated algae were kept under a constant light conditions with or without a photosynthesis inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea for 24 h, and were mixed with alga-free paramecia. Unexpectedly, most of the algae were not digested in the DVs irrespective of the presence of the inhibitor. Addition of 1 mM maltose, a main photosynthetic product of the symbiotic algae or of a supernatant of the isolated algae kept for 24 h under a constant light conditions, did not rescue the algal digestion in the DVs. These observations reveal that unknown factors induced by light are a prerequisite for algal resistance to the host lysosomal enzymes. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Catalytical Properties of Free and Immobilized Aspergillus niger Tannase

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Abril Flores-Maltos

2011-01-01

Full Text Available A fungal tannase was produced, recovered, and immobilized by entrapment in calcium alginate beads. Catalytical properties of the immobilized enzyme were compared with those of the free one. Tannase was produced intracellularly by the xerophilic fungus Aspergillus niger GH1 in a submerged fermentation system. Enzyme was recovered by cell disruption and the crude extract was partially purified. The catalytical properties of free and immobilized tannase were evaluated using tannic acid and methyl gallate as substrates. KM and Vmax values for free enzyme were very similar for both substrates. But, after immobilization, KM and Vmax values increased drastically using tannic acid as substrate. These results indicated that immobilized tannase is a better biocatalyst than free enzyme for applications on liquid systems with high tannin content, such as bioremediation of tannery or olive-mill wastewater.

Catalytical Properties of Free and Immobilized Aspergillus niger Tannase.

Science.gov (United States)

Flores-Maltos, Abril; Rodríguez-Durán, Luis V; Renovato, Jacqueline; Contreras, Juan C; Rodríguez, Raúl; Aguilar, Cristóbal N

2011-01-01

A fungal tannase was produced, recovered, and immobilized by entrapment in calcium alginate beads. Catalytical properties of the immobilized enzyme were compared with those of the free one. Tannase was produced intracellularly by the xerophilic fungus Aspergillus niger GH1 in a submerged fermentation system. Enzyme was recovered by cell disruption and the crude extract was partially purified. The catalytical properties of free and immobilized tannase were evaluated using tannic acid and methyl gallate as substrates. K(M) and V(max) values for free enzyme were very similar for both substrates. But, after immobilization, K(M) and V(max) values increased drastically using tannic acid as substrate. These results indicated that immobilized tannase is a better biocatalyst than free enzyme for applications on liquid systems with high tannin content, such as bioremediation of tannery or olive-mill wastewater.

Stabilization of dimeric β-glucosidase from Aspergillus niger via glutaraldehyde immobilization under different conditions.

Science.gov (United States)

Vazquez-Ortega, Perla Guadalupe; Alcaraz-Fructuoso, Maria Teresa; Rojas-Contreras, Juan A; López-Miranda, Javier; Fernandez-Lafuente, Roberto

2018-03-01

The dimeric enzyme β-glucosidase from Aspergillus niger has been immobilized on different amino-agarose beads at pH 5 and 7, exploiting the versatility of glutaraldehyde. The stability of the free enzyme depended on enzyme concentration. Immobilization via ion exchange improved enzyme stability/activity, depending on the immobilization pH. However, the enzyme was desorbed in 75 mM NaCl at pH 7 and some stability/enzyme concentration dependence still existed. of these biocatalysts with glutaraldehyde increased enzyme stability (e.g. at pH 5, after incubation under conditions where the enzyme just ionically exchanged was fully inactivated, the activity of the glutaraldehyde treated enzyme remained unaltered). Immobilization on glutaraldehyde pre-activated supports yielded a higher increase in enzyme activity, but the stabilization was lower. While when measuring the enzyme activity at pH 4 there were no changes after immobilization, all immobilized enzymes were more active than the free enzyme at pH 6 and 7 (2-3 times). The Ki/Km ratio did not significantly decrease in any immobilized biocatalysts, and in some cases it worsened in a significant way (by a 9 fold factor using preactivated supports). The new biocatalysts are significantly more stable and avoid enzyme subunit desorption, being the immobilization pH a key point in their design. Copyright © 2017 Elsevier Inc. All rights reserved.

Digestibility of the cottonseed meal with or without addition of protease and phytase enzymes in swine diet - doi: 10.4025/actascianimsci.v34i3.12360

Directory of Open Access Journals (Sweden)

Maria do Carmo Mouhaupt Marques Ludke

2012-05-01

Full Text Available This study evaluated the digestibility of cottonseed meal with or without addition of enzymes (phytase and protease for growing pigs. It was used 18 barrows, housed in metabolism cages, distributed in a completely randomized design, standardizing body weight (bw with average of 25.8 ± 3.6 kg, with three treatments and six repetitions. The treatments consisted of a reference diet based on corn and soybean meal, the second treatment with replacement of 30% of the reference diet by cottonseed meal without enzymes, and the third with 30% of the reference diet replaced by cottonseed meal with added enzymes. Was determined the digestible protein, digestible energy, digestibility of dry matter, energy and protein. It was also registered the balance of nitrogen and phosphorus. The use of cottonseed meal with the addition of enzymes in diets for growing pigs has no effect on the digestibility of dry matter, gross energy and crude protein, but improved the absorption of phosphorus, consequently reducing its excretion in the feces. There was no improvement in nitrogen balance in the diets containing cottonseed meal with enzymes.

Characteristics of immobilized aminoacylase from Aspergillus oryzae on macroporous copolymers.

Science.gov (United States)

He, B L; Jiang, P; Qiu, Y B

1990-01-01

Aminoacylase from Aspergillus oryzae was adsorbed on functionallized macroporous copolymers where the enzyme showed excellent catalyzing activity and operation stability. Various factors which effect the activity of the immobilized aminoacylase such as temperature, pH and ionic strength were investigated. The continuous operation of the enzyme immobilized on macroporous copolymers was compared with that of the enzyme immobilized on DEAE-Sephadex.

Effects of treating sorghum wet distillers grains with solubles with fibrolytic enzymes on nutrient digestibility and performance in finishing beef steers

Science.gov (United States)

Two experiments were conducted to determine the effects of treating sorghum WDG with solubles (SWDG) with an enzyme, or enzyme-buffer combination on diet digestibility and feedlot performance. Experimental treatments are; 1) untreated SWDG (control), 2) addition of an enzyme complex to SWDG (enzyme...

PREPARATION AND CHARACTERIZATION OF BIOCATALYSTS BASED ON IMMOBILIZED GLYCOSIDASES

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O. L. Meshcheriakova

2014-01-01

Full Text Available Summary. Enzymes subclass glycosidases cleaving poly- and oligosaccharides to simple sugars, are of great practical importance for a variety of industries. Such enzymes include α-L-fucosidase and β-fructofuranosidase. α-L-fucosidase splits fucoidan kelp to fucose and fucooligosaccharides. Fucose has prebiotic, immunotropic action, and a wide spectrum of biological activity in vertebrates, fucooligosaccharides - antioxidant and prebiotic properties. In this regard, and fucose polymers may be demanded in the food, feed and pharmaceutical industry. β-fructofuranosidase sucrose hydrolysis with the formation of invert syrup high quality and biological value that is of interest to the sugar industry. In order to intensify the processes of hydrolysis of fucoidan and sucrose due to the higher stability and reusability of enzyme preparations carried immobilization α-L-fucosidase on chitosan and β-fructofuranosidase of ion exchange brand FIBAN A-6 adsorption method. Activity of the immobilized α-L-fucosidase and β-fructofuranosidase were 80 and 70% of the activity of the free enzyme, respectively. Found that immobilized β-fructofuranosidase exhibits maximal activity at pH 4,0-4,1, the immobilized α-L-fucosidase - at pH 7,0. The optimal pH of immobilized enzymes similar to those for the free enzyme. Optimal temperature hydrolysis substrates immobilized α-L-fucosidase and β-fructofuranosidase was 50 and 70 ° C respectively, 10 ° C and 20 ° C higher compared to free enzymes. Studies have shown sufficient stability of immobilized glycosidases, so at 4-fold using their enzymatic activity decreased by 1.5 times; Biocatalysts obtained in storage in the refrigerator for 4-6 months retained 80% of the catalytic activity of enzymes.

Immobilization of Isolated Lipase From Moldy Copra (Aspergillus Oryzae

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Seniwati Dali

2011-01-01

Full Text Available Enzyme immobilization is a recovery technique that has been studied in several years, using support as a media to help enzyme dissolutions to the reaction substrate. Immobilization method used in this study was adsorption method, using specific lipase from Aspergillus oryzae. Lipase was partially purified from the culture supernatant of Aspergillus oryzae. Enzyme was immobilized by adsorbed on silica gel. Studies on free and immobilized lipase systems for determination of optimum pH, optimum temperature, thermal stability and reusability were carried out. The results showed that free lipase had optimum pH 8,2 and optimum temperature 35 °C while the immobilized lipase had optimum 8,2 and optimum temperature 45 °C. The thermal stability of the immobilized lipase, relative to that of the free lipase, was markedly increased. The immobilized lipase can be reused for at least six times.

Two-stage in vitro digestibility assay, a tool for formulating non-starch polysaccharide degrading enzyme combinations for commonly used feed ingredients of poultry rations

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Y. Ramana Reddy

2013-05-01

Full Text Available Aim: An attempt was made to assess the effect of pure enzyme combinations with the objective of formulating customized enzyme mixtures based on sugar release when subjected to two-stage in vitro digestion assay. Materials and Methods: A two-stage in vitro digestibility assay was carried out for commonly used feed ingredients for poultry viz., maize, soy bean meal, sunflower cake, and de-oiled rice bran supplemented with three concentrations of xylanase (5000; 7500 and 10000 IU/kg, cellulase (50; 100 and 400 IU/kg and â-D-glucanase (100; 200 and 400 IU/kg were used to formulate various NSP enzymes combinations. In total 27 NSP enzyme combinations (3x3x3 were formulated and the sugar released due to NSP digestion was quantified by phenol sulphuric acid method. Results: The total sugar release was significantly (P<0.05 higher with supplementation of various enzymes combinations for maize, sunflower cake and de-oiled rice bran where as no significant (P<0.05 interaction of various NSP enzymes combinations was observed for soy bean meal. The NSP digestibility was highest in combination (xylanase-5000, cellulase-50 and â-D-glucanase-400 IU/kg, (xylanase-10000, cellulase-50 and â-D-glucanase-200 IU/kg and (xylanase-7500, cellulase- 100 and â-D-glucanase-100 IU/kg for maize, sunflower cake and de-oiled rice bran respectively. In case of sunflower cake, significant (P<0.01 three way interaction was observed among the xylanase, cellulose, and â-D-glucanase enzymes and the two-way interactions between the enzymes were also significant (P<0.01. Conclusion: It is concluded that 'n' number of non-starch Polysaccharide enzymes combinations can be screened for their efficiency to digest non-starch Polysaccharides present in various feed ingredients commonly used in poultry rations by employing two-stage in vitro digestibility assay as a tool. [Vet World 2013; 6(8.000: 525-529

Expression of digestive enzymes and nutrient transporters in Eimeria acervulina-challenged layers and broilers

Science.gov (United States)

Avian coccidiosis is a disease caused by the intestinal protozoa Eimeria. Eimeria-infected chickens develop lesions in the intestinal mucosa, which result in reduced feed efficiency and body weight gain. This growth reduction may be due to changes in expression of digestive enzymes and nutrient tran...

Effect of acidic treatment on carbon nano tubes for immobilization of cellulase enzyme

International Nuclear Information System (INIS)

Al-Khatib, M.F.R.; Mohd Zahangir Alam; Rasha Mohammed

2009-01-01

Full text: The effect of acidic treatment on MWCNTs functionalization was studied by mixing different ratios (1:1, 1:2, and 1:3 v/v %) of nitric acid and sulphuric acid, respectively. The effect of these treatments on the structure of MWCNTs was characterized by Fourier transform infrared spectroscopy (FTIR) and Filed emission scanning electron microscopy (FESEM). Results showed that the optimum ratio 1:3 (v/v %) is best suitable in imparting carboxylic acid and hydroxyl groups which are required for immobilization of cellulase enzyme on functionalized CNTs. (author)

A general overview of support materials for enzyme immobilization: Characteristics, properties, practical utility

DEFF Research Database (Denmark)

Zdarta, Jakub; Meyer, Anne S.; Jesionowski, Teofil

2018-01-01

on the properties of the produced catalytic system. A large variety of inorganic and organic as well as hybrid and composite materials may be used as stable and efficient supports for biocatalysts. This review provides a general overview of the characteristics and properties of the materials applied for enzyme...... immobilization. For the purposes of this literature study, support materials are divided into two main groups, called Classic and New materials. The review will be useful in selection of appropriate support materials with tailored properties for the production of highly effective biocatalytic systems for use...

In vitro digestion of purified β-casein variants A(1), A(2), B, and I: effects on antioxidant and angiotensin-converting enzyme inhibitory capacity.

Science.gov (United States)

Petrat-Melin, B; Andersen, P; Rasmussen, J T; Poulsen, N A; Larsen, L B; Young, J F

2015-01-01

Genetic polymorphisms of bovine milk proteins affect the protein profile of the milk and, hence, certain technological properties, such as casein (CN) number and cheese yield. However, reports show that such polymorphisms may also affect the health-related properties of milk. Therefore, to gain insight into their digestion pattern and bioactive potential, β-CN was purified from bovine milk originating from cows homozygous for the variants A(1), A(2), B, and I by a combination of cold storage, ultracentrifugation, and acid precipitation. The purity of the isolated β-CN was determined by HPLC, variants were verified by mass spectrometry, and molar extinction coefficients at λ=280nm were determined. β-Casein from each of the variants was subjected to in vitro digestion using pepsin and pancreatic enzymes. Antioxidant and angiotensin-converting enzyme (ACE) inhibitory capacities of the hydrolysates were assessed at 3 stages of digestion and related to that of the undigested samples. Neither molar extinction coefficients nor overall digestibility varied significantly between these 4 variants; however, clear differences in digestion pattern were indicated by gel electrophoresis. In particular, after 60min of pepsin followed by 5min of pancreatic enzyme digestion, one ≈4kDa peptide with the N-terminal sequence (106)H-K-E-M-P-F-P-K- was absent from β-CN variant B. This is likely a result of the (122)Ser to (122)Arg substitution in variant B introducing a novel trypsin cleavage site, leading to the changed digestion pattern. All investigated β-CN variants exhibited a significant increase in antioxidant capacity upon digestion, as measured by the Trolox-equivalent antioxidant capacity assay. After 60min of pepsin + 120min of pancreatic enzyme digestion, the accumulated increase in antioxidant capacity was ≈1.7-fold for the 4 β-CN variants. The ACE inhibitory capacity was also significantly increased by digestion, with the B variant reaching the highest inhibitory

Adsorptive control of water in esterification with immobilized enzymes: II. fixed-bed reactor behavior.

Science.gov (United States)

Mensah, P; Gainer, J L; Carta, G

1998-11-20

Experimental and theoretical studies are conducted to understand the dynamic behavior of a continuous-flow fixed-bed reactor in which an esterification is catalyzed by an immobilized enzyme in an organic solvent medium. The experimental system consists of a commercial immobilized lipase preparation known as Lipozyme as the biocatalyst, with propionic acid and isoamyl alcohol (dissolved in hexane) as the reaction substrates. A complex dynamic behavior is observed experimentally as a result of the simultaneous occurrence of reaction and adsorption phenomena. Both propionic acid and water are adsorbed by the biocatalyst resulting in lower reaction rates. In addition, an excessive accumulation of water in the reactor leads to a rapid irreversible inactivation of the enzyme. A model based on previously-obtained adsorption isotherms and kinetic expressions, as well as on adsorption rate measurements obtained in this work, is used to predict the concentration and thermodynamic activity of water along the reactor length. The model successfully predicts the dynamic behavior of the reactor and shows that a maximum thermodynamic activity of water occurs at a point at some distance from the reactor entrance. A cation exchange resin in sodium form, packed in the reactor as a selective water adsorbent together with the catalyst particles, is shown to be an effective means for preventing an excessive accumulation of water formed in the reaction. Its use results in longer cycle times and greater productivity. As predicted by the model, the experimental results show that the water adsorbed on the catalyst and on the ion exchange resin can be removed with isoamyl alcohol with no apparent loss in enzyme activity. Copyright 1998 John Wiley & Sons, Inc.

Influence of acetylcholinesterase immobilization on the photoluminescence properties of mesoporous silicon surface

Energy Technology Data Exchange (ETDEWEB)

Saleem, Muhammad [Department of Chemistry, Kongju National University, Gongju, Chungnam 314-701 (Korea, Republic of); Rafiq, Muhammad; Seo, Sung-Yum [Department of Biology, Kongju National University, Gongju, Chungnam 314-701 (Korea, Republic of); Lee, Ki Hwan, E-mail: khlee@kongju.ac.kr [Department of Chemistry, Kongju National University, Gongju, Chungnam 314-701 (Korea, Republic of)

2014-07-01

Acetylcholinesterase immobilized p-type porous silicon surface was prepared by covalent attachment. The immobilization procedure was based on support surface chemical oxidation, silanization, surface activation with cyanuric chloride and finally covalent attachment of free enzyme on the cyanuric chloride activated porous silicon surface. Different pore diameter of porous silicon samples were prepared by electrochemical etching in HF based electrolyte solution and appropriate sample was selected suitable for enzyme immobilization with maximum trapping ability. The surface modification was studied through field emission scanning electron microscope, EDS, FT-IR analysis, and photoluminescence measurement by utilizing the fluctuation in the photoluminescence of virgin and enzyme immobilized porous silicon surface. Porous silicon showed strong photoluminescence with maximum emission at 643 nm and immobilization of acetylcholinesterase on porous silicon surface cause considerable increment on the photoluminescence of porous silicon material while acetylcholinesterase free counterpart did not exhibit any fluorescence in the range of 635–670 nm. The activities of the free and immobilized enzymes were evaluated by spectrophotometric method by using neostigmine methylsulfate as standard enzyme inhibitor. The immobilized enzyme exhibited considerable response toward neostigmine methylsulfate in a dose dependent manner comparable with that of its free counterpart alongside enhanced stability, easy separation from the reaction media and significant saving of enzyme. It was believed that immobilized enzyme can be exploited in organic and biomolecule synthesis possessing technical and economical prestige over free enzyme and prominence of easy separation from the reaction mixture.

Influence of acetylcholinesterase immobilization on the photoluminescence properties of mesoporous silicon surface

International Nuclear Information System (INIS)

Saleem, Muhammad; Rafiq, Muhammad; Seo, Sung-Yum; Lee, Ki Hwan

2014-01-01

Acetylcholinesterase immobilized p-type porous silicon surface was prepared by covalent attachment. The immobilization procedure was based on support surface chemical oxidation, silanization, surface activation with cyanuric chloride and finally covalent attachment of free enzyme on the cyanuric chloride activated porous silicon surface. Different pore diameter of porous silicon samples were prepared by electrochemical etching in HF based electrolyte solution and appropriate sample was selected suitable for enzyme immobilization with maximum trapping ability. The surface modification was studied through field emission scanning electron microscope, EDS, FT-IR analysis, and photoluminescence measurement by utilizing the fluctuation in the photoluminescence of virgin and enzyme immobilized porous silicon surface. Porous silicon showed strong photoluminescence with maximum emission at 643 nm and immobilization of acetylcholinesterase on porous silicon surface cause considerable increment on the photoluminescence of porous silicon material while acetylcholinesterase free counterpart did not exhibit any fluorescence in the range of 635–670 nm. The activities of the free and immobilized enzymes were evaluated by spectrophotometric method by using neostigmine methylsulfate as standard enzyme inhibitor. The immobilized enzyme exhibited considerable response toward neostigmine methylsulfate in a dose dependent manner comparable with that of its free counterpart alongside enhanced stability, easy separation from the reaction media and significant saving of enzyme. It was believed that immobilized enzyme can be exploited in organic and biomolecule synthesis possessing technical and economical prestige over free enzyme and prominence of easy separation from the reaction mixture.

The Choice of Enzyme for Human Pancreas Digestion is a Critical Factor for Increasing the Success of Islet Isolation.

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Qi, Meirigeng; Valiente, Luis; McFadden, Brian; Omori, Keiko; Bilbao, Shiela; Juan, Jemily; Rawson, Jeffrey; Scott, Stephen; Ferreri, Kevin; Mullen, Yoko; El-Shahawy, Mohamed; Dafoe, Donald; Kandeel, Fouad; Al-Abdullah, Ismail H

2015-05-01

We evaluated three commercially available enzymes for pancreatic digestion by comparing key parameters during the islet isolation process, as well as islet quality post-isolation. Retrospectively compared and analyzed islet isolations from pancreata using three different enzyme groups: Liberase HI (n=63), Collagenase NB1/Neutral Protease (NP) (n=43), and Liberase Mammalian Tissue Free Collagenase/Thermolysin (MTF C/T) (n=115). A standardized islet isolation and purification method was used. Islet quality assessment was carried out using islet count, viability, in vitro glucose-stimulated insulin secretion (GSIS), glucose-stimulated oxygen consumption rate (ΔOCR), and in vivo transplantation model in mice. Donor characteristics were not significantly different among the three enzyme groups used in terms of age, sex, hospital stay duration, cause of death, body mass index (BMI), hemoglobin A1c (HbA1c), cold ischemia time (CIT), and pancreas weight. Digestion efficacy (percentage of digested tissue by weight) was significantly higher in the Liberase MTF C/T group (73.5 ± 1.5 %) when compared to the Liberase HI group (63.6 ± 2.3 %) (psuccess rate of transplantation in diabetic NOD Scid mice (65%), which was significantly higher than the Liberase HI (42%, p=0.001) and the Collagenase NB1/NP enzymes (41%, psuccess rate of transplantation in diabetic NOD Scid mice compared to Liberase HI and Collagenase NB1/NP enzymes.

Enhanced starch hydrolysis using α-amylase immobilized on cellulose ultrafiltration affinity membrane.

Science.gov (United States)

Konovalova, Viktoriia; Guzikevich, Kateryna; Burban, Anatoliy; Kujawski, Wojciech; Jarzynka, Karolina; Kujawa, Joanna

2016-11-05

In order to prepare ultrafiltration membranes possessing biocatalytic properties, α-amylase has been immobilized on cellulose membranes. Enzyme immobilization was based on a covalent bonding between chitosan and a surface of cellulose membrane, followed by an attachment of Cibacron Blue F3G-A dye as affinity ligand. Various factors affecting the immobilization process, such as enzyme concentration, pH of modifying solution, zeta-potential of membrane surface, and stability of immobilized enzyme were studied. The applicability of immobilized α-amylase has been investigated in ultrafiltration processes. The immobilization of α-amylase on membrane surface allows to increase the value of mass transfer coefficient and to decrease the concentration polarization effect during ultrafiltration of starch solutions. The enzyme layer on the membrane surface prevents a rapid increase of starch concentration due to the amylase hydrolysis of starch in the boundary layer. The presented affinity immobilization technique allows also for the regeneration of membranes from inactivated enzyme. Copyright © 2016 Elsevier Ltd. All rights reserved.

Antioxidant properties of digestive enzyme-treated fibre-rich fractions from wheat, finger millet, pearl millet and sorghum: A comparative evaluation

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Aisha Siddiq A.

2015-12-01

Full Text Available Whole grains are rich in antioxidant components (AC, most of which are bound to fibre fraction and released during digestion. The study investigated the effect of digestive enzymes on the antioxidant properties of fibre-rich fractions from wheat (Triticum aestivum, finger millet (Eleusine coracana, pearl millet (Pennisetum typhoides and sorghum (Sorghum bicolor. Coarse (CF and fine fractions (FF of milled flour were separated using a standard sieve and analysed for nutritional composition, AC extractable in different solvents and antioxidant activity (AA in untreated and enzyme-treated fractions. The CF had a higher range of insoluble dietary fibre (17.26–20.93% than FF (10.65–17.29%. The highest amount of polyphenols and flavonoids was extractable in different solvents from finger millet and pearl millet, respectively. FF of pearl millet showed higher total AA in all solvents. Enzyme-treated samples had a much higher content of AC as well as higher total AA. Free radical scavenging assay revealed that enzyme-treated millet flours had higher activity in comparison to wheat. Between fractions, wheat exhibited variable results. Among millets, CF of finger millet and FF of pearl millet and sorghum had higher AA. In conclusion, digestive enzyme treatment released more AC from grains, and exhibited a higher AA.

pH-dependent immobilization of urease on glutathione-capped gold nanoparticles.

Science.gov (United States)

Garg, Seema; De, Arnab; Mozumdar, Subho

2015-05-01

Urease is a nickel-dependent metalloenzyme that catalyzes the hydrolysis of urea to form ammonia and carbon dioxide. Although the enzyme serves a significant role in several detoxification and analytical processes, its usability is restricted due to high cost, availability in small amounts, instability, and a limited possibility of economic recovery from a reaction mixture. Hence, there is a need to develop an efficient, simple, and reliable immobilization strategy for the enzyme. In this study, the carboxyl terminated surface of glutathione-capped gold nanoparticles have been utilized as a solid support for the covalent attachment of urease. The immobilization has been carried out at different pH conditions so as to elucidate its effect on the immobilization efficiency and enzyme bioactivity. The binding of the enzyme has been quantitatively and qualitatively analyzed through techniques like ultraviolet-visible spectroscopy, intrinsic steady state fluorescence, and circular dichorism. The bioactivity of the immobilized enzyme was investigated with respect to the native enzyme under different thermal conditions. Recyclability and shelf life studies of the immobilized enzyme have also been carried out. Results reveal that the immobilization is most effective at pH of 7.4 followed by that in an acidic medium and is least in alkaline environment. The immobilized enzyme also exhibits enhance activity in comparison to the native form at physiological temperature. The immobilized urease (on gold glutathione nanoconjugates surface) can be effectively employed for biosensor fabrication, immunoassays and as an in vivo diagnostic tool in the future. © 2014 Wiley Periodicals, Inc.

Examination of digestive enzyme distribution in gut tract and functions of intestinal caecum, in megascolecid earthworms (Oligochaeta: Megascolecidae) in Japan.

Science.gov (United States)

Nozaki, Mana; Ito, Katsutoshi; Miura, Chiemi; Miura, Takeshi

2013-09-01

Earthworms ingest various materials in addition to food items, such as soil particles. Most earthworms of the family Megascolecidae, a dominant family in Japan, have intestinal caeca connected directly to the intestinal tract. The function of the caeca has not been demonstrated, although it is thought to be associated with digestion. We investigated the activity of the digestive enzymes amylase, phosphatase, cellulase, and protease in different regions of the gut, including the intestinal caeca, in three species of megascolecid earthworms, Pheretima heteropoda, Pheretima hilgendorfi, and Pheretima sieboldi. Activities of several enzymes were high in the intestinal caeca; in particular, protease activity was higher in the caeca than that in the anterior gut, foregut, midgut, and hindgut in all three species. Moreover, the ratio of enzyme activities in the intestinal caeca to whole-gut tended to be higher in manicate intestinal caeca than in simple intestinal caeca. These results suggest that the digestive system of earthworms relies on the intestinal caeca.

Consequences of lower food intake on the digestive enzymes activities, the energy reserves and the reproductive outcome in Gammarus fossarum.

Directory of Open Access Journals (Sweden)

Laetitia Charron

Full Text Available Digestive enzyme activity is often used as a sensitive response to environmental pollution. However, only little is known about the negative effects of stress on digestive capacities and their consequences on energy reserves and reproduction, although these parameters are important for the maintenance of populations. To highlight if changes in biochemical responses (digestive enzymes and reserves led to impairments at an individual level (fertility, Gammarus fossarum were submitted to a lower food intake throughout a complete female reproductive cycle (i.e. from ovogenesis to offspring production. For both males and females, amylase activity was inhibited by the diet stress, whereas trypsin activity was not influenced. These results underline similar sensitivity of males and females concerning their digestive capacity. Energy reserves decreased with food starvation in females, and remained stable in males. The number of embryos per female decreased with food starvation. Lower digestive activity in males and females therefore appears as an early response. These results underline the ecological relevance of digestive markers, as they make it possible to anticipate upcoming consequences on reproduction in females, a key biological variable for population dynamics.

Anaerobic treatment of palm oil mill effluent in batch reactor with digested biodiesel waste as starter and natural zeolite for microbial immobilization

Science.gov (United States)

Setyowati, Paulina Adina Hari; Halim, Lenny; Mellyanawaty, Melly; Sudibyo, Hanifrahmawan; Budhijanto, Wiratni

2017-05-01

Palm oil mill effluent (POME) is the wastewater discharged from sludge separation, sterilization, and clarification process of palm oil industries. Each ton of palm oil produces about half ton of high organic load wastewater. Up to now, POME treatment is done in lagoon, leaving major problems in land requirement and greenhouse gasses release. The increasing of palm oil production provokes the urgency of appropriate technology application in treating POME to prevent the greenhouse gasses emission while exploit POME as renewable energy source. The purposes of this study were firstly to test the effectiveness of using the digested biodiesel waste as the inoculum and secondly to evaluate the effectiveness of natural zeolite addition in minimizing the inhibitory effect in digesting POME. It was expected that the oil-degrading bacteria in the inoculum would shorten the adaptation period in digesting POME. Furthermore, the consortium formation of anaerobic bacteria accelerated by natural zeolite powder addition would increase the microbial resistance to the inhibitors contained in the POME. The batch digesters, containing 0 (control); 17; 38; and 63 g natural zeolite/g sCOD substrate were observed for 43 days. The result showed that zeolite addition did not give significant effect on sCOD reduction (97.3-98.6% of initial sCOD). Moreover, addition of immobilization media up to 17 g natural zeolite/g stimulated the acidification and biogas production up to 10% higher than control. The purity of methane produced with various amount of immobilization media did not differ for each variation, i.e. 50-54% v/v methane. The increasing amount of natural zeolite up to 63 g/g sCOD did not significantly enhance biogas product rate nor methane content.

Production of Biodiesel Using Immobilized Lipase and the Characterization of Different Co-Immobilizing Agents and Immobilization Methods

Directory of Open Access Journals (Sweden)

Kang Zhao

2016-08-01

Full Text Available Lipase from Candida sp. 99–125 is widely employed to catalyzed transesterification and can be used for biodiesel production. In this study, the lipase was immobilized by combined adsorption and entrapment to catalyze biodiesel production from waste cooking oil (WCO via transesterification, and investigating co-immobilizing agents as additives according to the enzyme activity. The addition of the mixed co-immobilizing agents has positive effects on the activities of the immobilized lipase. Three different immobilizing methods were compared by the conversion ratio of biodiesel and structured by Atom Force Microscopy (AFM and Scanning Electron Microscopy (SEM, respectively. It was found that entrapment followed by adsorption was the best method. The effect of the co-immobilizing agent amount, lipase dosage, water content, and reuse ability of the immobilized lipase was investigated. By comparison with previous research, this immobilized lipase showed good reuse ability: the conversion ratio excesses 70% after 10 subsequent reactions, in particular, was better than Novozym435 and TLIM on waste cooking oil for one unit of lipase.

Physical immobilization of 60 kDa chaperonin linked lipase from pseudomonas aeruginosa BN-1

International Nuclear Information System (INIS)

Syed, M.N.; Mehmood, S.; Bashir, A.; Ashraf, F.

2012-01-01

Abstract: The 60 kDa chaperone linked lipase from Pseudomonas aeruginosa was subjected to physical adsorption on silica 60 and acrylic beads. It was found that higher enzyme loading was achieved on silica gel than acrylic bead. The half life of immobilized enzyme was greater compared to the free enzyme. The adsorption of the enzyme onto a solid phase also resulted in increased thermo and solvent stability. It was observed that soluble enzyme showed maximum stability at 70 degree C while immobilized enzyme showed stability up to 80 degree C for 45 minutes. The stability of immobilized enzyme increased up to 48 hours from 24 hours against different organic solvent at 1.0 M concentration. It was noted that enzyme immobilized on acrylic beads have greater reusability compared to silica immobilized enzyme. (author)

Covalent immobilization of β-glucosidase on magnetic particles for lignocellulose hydrolysis.

Science.gov (United States)

Alftrén, Johan; Hobley, Timothy John

2013-04-01

β-Glucosidase hydrolyzes cellobiose to glucose and is an important enzyme in the consortium used for hydrolysis of cellulosic and lignocellulosic feedstocks. In the present work, β-glucosidase was covalently immobilized on non-porous magnetic particles to enable re-use of the enzyme. It was found that particles activated with cyanuric chloride and polyglutaraldehyde gave the highest bead-related immobilized enzyme activity when tested with p-nitrophenyl-β-D-glucopyranoside (104.7 and 82.2 U/g particles, respectively). Furthermore, the purified β-glucosidase preparation from Megazyme gave higher bead-related enzyme activities compared to Novozym 188 (79.0 and 9.8 U/g particles, respectively). A significant improvement in thermal stability was observed for immobilized enzyme compared to free enzyme; after 5 h (at 65 °C), 36 % of activity remained for the former, while there was no activity in the latter. The performance and recyclability of immobilized β-glucosidase on more complex substrate (pretreated spruce) was also studied. It was shown that adding immobilized β-glucosidase (16 U/g dry matter) to free cellulases (8 FPU/g dry matter) increased the hydrolysis yield of pretreated spruce from ca. 44 % to ca. 65 %. In addition, it was possible to re-use the immobilized β-glucosidase in the spruce and retain activity for at least four cycles. The immobilized enzyme thus shows promise for lignocellulose hydrolysis.

Potential Applications of Immobilized β-Galactosidase in Food Processing Industries

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Parmjit S. Panesar

2010-01-01

Full Text Available The enzyme β-galactosidase can be obtained from a wide variety of sources such as microorganisms, plants, and animals. The use of β-galactosidase for the hydrolysis of lactose in milk and whey is one of the promising enzymatic applications in food and dairy processing industries. The enzyme can be used in either soluble or immobilized forms but the soluble enzyme can be used only for batch processes and the immobilized form has the advantage of being used in batch wise as well as in continuous operation. Immobilization has been found to be convenient method to make enzyme thermostable and to prevent the loss of enzyme activity. This review has been focused on the different types of techniques used for the immobilization of β-galactosidase and its potential applications in food industry.

Properties of Immobilized Candida antarctica Lipase B on Highly Macroporous Copolymer

International Nuclear Information System (INIS)

Handayani, N.; Achmad, S.; Wahyuningrum, D.

2011-01-01

In spite of their excellent catalytic properties, enzymes should be improved before their implementation both in industrial and laboratorium scales. Immobilization of enzyme is one of the ways to improve their properties. Candida antarctica lipase B (Cal-B) has been reported in numerous publications to be a particularly useful enzyme catalizing in many type of reaction including regio- and enantio- synthesis. For this case, cross-linking of immobilized Cal-B with 1,2,7,8 diepoxy octane is one of methods that proved significantly more stable from denaturation by heat, organic solvents, and proteolysis than lyophilized powder or soluble enzymes. More over, the aim of this procedure is to improve the activity and reusability of lipase. Enzyme kinetics test was carried out by transesterification reaction between 4-nitrophenyl acetate (pNPA) and methanol by varying substrate concentrations, and the result is immobilized enzymes follows the Michaelis-Menten models and their activity is match with previous experiment. Based on the V max values, the immobilized enzymes showed higher activity than the free enzyme. Cross-linking of immobilized lipase indicate that cross-linking by lower concentration of cross-linker, FIC (immobilized lipase that was incubated for 24 h) gave the highest activity and cross-linking by higher concentration of cross-linker, PIC (immobilized lipase that was incubated for 2 h) gives the highest activity. However, pore size and saturation level influenced their activity. (author)

Characterization of cellulose acetate micropore membrane immobilized acylase I.

Science.gov (United States)

Guo, Yong-Sheng; Wang, Jie; Song, Xi-Jin

2004-12-01

This paper describes an innovative method for the immobilization of acylase I, which was entrapped into the CA-CTA micropore membrane. The most suitable casting solutions proportion for immobilizing the enzyme was obtained through orthogonal experiment. Properties of the enzyme membrane were investigated and compared with those of free enzyme and blank membrane. The thermal stability and pH stability of the enzyme inside the membrane were changed by immobilization. The optimum pH was found to be 6.0, which changes 1.0 unit compared with that of free acylase I. The optimum temperature was found to be about 90 degrees C, which is higher than that of free acylase I (60 degrees C). Experimental results showed that immobilization had effects on the kinetic parameters of acylase I.

Andrographolide powder treatment as antifeedant decreased digestive enzyme activity from Plutella xylostella (L.) larvae midgut

Science.gov (United States)

Madihah, Malini, Desak Made; Roviani, Hana; Rani, Nessa Vidya; Hermawan, Wawan

2018-02-01

Andrographolide, an active compound of Andrographis paniculata, has shown antifeedant activity against Plutella xylostella larvae by disrupting the midgut histological structures. This study aims to determine the activity of andrographolide in crystallized powder form against several digestive enzymes from the midgut of 4th instar P. xylostella larvae. The concentrations used were 0 (control), 1000, 1600, 2500, 4000 and 6500 ppm with four replications each. No-choice antifeedant test with leaf disc method is used in a bioassay for 24 hours. The midgut was dissected from 2nd until 6th segment of 4th instar larvae and was homogenized in iced-buffer solution. Furthermore, larvae's midgut samples were centrifuged at 10,000 rpm, 4°C for 20 min and the supernatant is used as enzyme source. The results showed that andrographolide significantly reduces the amylase, invertase, protease and trypsin activity, as well as total protein concentration compared with control (p<0.05) in a dose-dependent manner. This study provides information about the mode of action of andrographolide in inhibiting feed activity by the reduced digestive enzyme activity of 4th instar P. xylostella larvae.

Preparation of reusable bioreactors using reversible immobilization of enzyme on monolithic porous polymer support with attached gold nanoparticles.

Science.gov (United States)

Lv, Yongqin; Lin, Zhixing; Tan, Tianwei; Svec, Frantisek

2014-01-01

Porcine lipase has been reversibly immobilized on a monolithic polymer support containing thiol functionalities prepared within confines of a fused silica capillary and functionalized with gold nanoparticles. Use of gold nanoparticles enabled rejuvenation of the activity of the deactivated reactor simply by stripping the inactive enzyme from the nanoparticles using 2-mercaptoethanol and subsequent immobilization of fresh lipase. This flow through enzymatic reactor was then used to catalyze the hydrolysis of glyceryl tributyrate (tributyrin). The highest activity was found within a temperature range of 37-40°C. The reaction kinetics is characterized by Michaelis-Menten constant, Km  = 10.9 mmol/L, and maximum reaction rate, Vmax  = 5.0 mmol/L min. The maximum reaction rate for the immobilized enzyme is 1,000 times faster compared to lipase in solution. The fast reaction rate enabled to achieve 86.7% conversion of tributyrin in mere 2.5 min and an almost complete conversion in 10 min. The reactor lost only less than 10% of its activity even after continuous pumping through it a solution of substrate equaling 1,760 reactor volumes. Finally, potential application of this enzymatic reactor was demonstrated with the transesterification of triacylglycerides from kitchen oil to fatty acid methyl esters thus demonstrating the ability of the reactor to produce biodiesel. © 2013 Wiley Periodicals, Inc.

Immobilization of α-amylase onto poly(glycidyl methacrylate) grafted electrospun fibers by ATRP

International Nuclear Information System (INIS)

Oktay, Burcu; Demir, Serap; Kayaman-Apohan, Nilhan

2015-01-01

In this study, novel α-amylase immobilized poly(vinyl alcohol) (PVA) nanofibers were prepared. The PVA nanofiber surfaces were functionalized with 2-bromoisobutyryl bromide (BiBBr) and followed by surface initiated atom transfer radical polymerization (SI-ATRP) of glycidyl methacrylate (GMA). The morphology of the poly(glycidyl methacrylate) (PGMA) grafted PVA nanofibers was characterized by scanning electron microscopy (SEM). Also PGMA brushes were confirmed by X-ray photo electron microscopy (XPS). α-Amylase was immobilized in a one step process onto the PGMA grafted PVA nanofiber. The characteristic properties of the immobilized and free enzymes were examined. The thermal stability of the enzyme was improved and showed maximum activity at 37 °C by immobilization. pH values of the maximum activity of the free and immobilized enzymes were also found at 6.0 and 6.5, respectively. Free enzyme lost its activity completely within 15 days. The immobilized enzyme lost only 23.8% of its activity within 30 days. - Highlights: • Electrospun photocrosslinkable PVA nanofiber was prepared. • PGMA brushes were conducted on PVA nanofiber via SI-ATRP. • The immobilized enzyme showed maximum activity at pH 6.0 and at 37 °C. • Functionalized nanofibers may be used as promising supports for enzyme immobilization

Determination of some properties of free and immobilized urease from aspergillus fumigatus and its application in urea assay

International Nuclear Information System (INIS)

Tetiker, A.T.; Ertan, F.

2016-01-01

Urease enzyme was extracted from Apergillus fumigatus and immobilized in calcium alginate beads. The immobilization efficiency was calculated as 82.5 %. Optimum pH and temperature for free and immobilized enzymes were found to be 7.0 and 40 degree C, respectively. The immobilized urease had a better Km value but, catalytic efficiencies (kcat/Km) were very similar. Immobilized enzyme maintained 44% of its initial activity after 5 repeated use of enzyme. It was found that storage stability of immobilized enzyme was better than that of the free enzyme. Immobilized urease enzyme was used for the determination of urea amounts in animal feed. (author)

Immobilization of urease on grafted starch by radiation method

International Nuclear Information System (INIS)

Nguyenanh Dung; Nguyendinh Huyen

1995-01-01

The acrylamide was grafted by radiation onto starch which is a kind of polymeric biomaterial. The urease was immobilized on the grafted starch. Some experiments to observe the quantitative relationships between the percent graft and the activity of immobilized enzyme were determined. The enzyme activity was maintained by more than seven batch enzyme reactions. (author)

Studies on the preparation of immobilized enzymes by radiopolymerization, (9)

International Nuclear Information System (INIS)

Kawashima, Koji; Fujino, Satomi; Hayashi, Toru; Kim, Sung-K.

1982-01-01

Glucose Oxidase (GOD, EC 1, 1, 3, 4) was immobilized in the form of the beads by the radiation polymerization method under low temperature and the enzymatic characteristics were investigated. 1) Polyethyleneglycol dimethacrylate and acrylamide were favorable compounds for the immobilization of GOD. 2) Neither optimum pH nor pH stability was changed after immobilization treatment. 3) Optimum reaction temperature was shifted by 5 0 C to the higher side and heat stability was improved. 4) Immobilized GOD showed activity up to 60U per gram of dried polymer. 5) The small beads had retained high activities (10 - 80%) 6) The immobilized GOD was not leached out from the polymer matrix. (author)

Soybean hull and enzyme inclusion effects on diet digestibility and growth performance in beef steers consuming corn-based diets.

Science.gov (United States)

Russell, J R; Sexten, W J; Kerley, M S

2016-06-01

A beef feedlot study was conducted to determine the effects of increasing soybean hull (SH) inclusion and enzyme addition on diet digestibility and animal performance. The hypothesis was SH inclusion and enzyme addition would increase fiber digestibility with no negative effect on animal performance. Eight treatments (TRT) were arranged in a 4 × 2 factorial using four diets and two enzyme (ENZ) inclusion rates. The diets were composed primarily of whole shell corn (WSC) with 0%, 7%, 14%, or 28% SH replacing corn. The ENZ was a commercial proprietary mix of , and (Cattlemace, R&D Life Sciences, Menomonie, WI) included in the diets at 0% (S0, S7, S14, S28) or 0.045% DM basis (S0e, S7e, S14e, S28e). Eighty steers (287 ± 31 kg, SD) were stratified by weight and blocked into pens with 1 heavy and 1 light pen per TRT (2 pen/TRT, 5 steers/pen). Steers were fed for 70 d with titanium dioxide included in the diets for the final 15 d. Fecal samples were collected on d 70 to determine diet digestibility. Diets were balanced for AA and RDP requirement based on available ME. Individual DMI was measured using a GrowSafe system. Diet, ENZ, and diet × ENZ effects were analyzed using the MIXED procedure of SAS. Initial BW was applied as a covariate for final BW (FBW), and DMI was included as a covariate for all digestibility measures. The diet × ENZ interaction had no effect on FBW, ADG, DMI, or any digestibility measure ( ≥ 0.11). Steers fed ENZ tended to have greater FBW ( = 0.09) and had numerically greater ADG than steers not fed ENZ. Diet influenced DMI ( digestibility ( ≥ 0.2). Diet had an effect on NDF and ADF digestibility ( ≤ 0.04) which decreased as SH inclusion increased. The addition of ENZ tended to decrease NDF digestibility ( = 0.08) but had no effect on ADF digestibility ( = 0.8). Fiber digestibility in WSC diets did not improve with SH inclusion or ENZ addition but steers fed diets with 14% to 28% of WSC replaced by SH and the addition of 0.045% ENZ

Immobilization of cellulase by radiation polymerization

International Nuclear Information System (INIS)

Kumakura, M.; Kaetsu, I.

1983-01-01

Immobilization of cellulase by radiation polymerization at low temperatures was studied. The enzymatic activity of immobilized cellulase pellets varied with the monomer, enzyme concentration, and the thickness of immobilized cellulase pellets. The optimum monomer concentration in the immobilization of cellulase was 30-50% at the pellet thickness of 1.0 mm, in which the enzymatic activity was 50%. The enzymatic activity of immobilized cellulase pellets was examined using various substrates such as cellobiose, carboxymethylcellulose, and paper pretreated by radiation. It was found that irradiated paper can be hydrolyzed by immobilized cellulase pellets. (author)

Production and immobilization of alpha amylase using biotechnology techniques for use in biological and medical applications

International Nuclear Information System (INIS)

Mobasher, E.E.F.

2009-01-01

The immobilized enzymes on polymeric supports are prepared for purpose of repeated use and the possibilities of continuous reaction system. One of the most important properties is the stability of proteins when they are used in some medical and industrial applications. The immobilization of the enzymes improves this property as well as many other properties.In this study, alpha amylase was purified and immobilized onto two different polymers. α- amylase was used in this study for its biological and industrial applications. It is used in paper textile, pharmaceutical applications, food, and detergent industries. α- amylase was found in plants, animals, and microorganisms. Purification of α-amylase from microorganisms is the main source of α-amylase because it was excreted from many bacteria and fungi. In this study, α-amylase was purified from Aspergillus niger. Fractional precipitation of the α- amylase produced by A. niger with 80% ammonium sulphate saturation. The crude enzyme was applied on column chromatography packed with Sephadex G 100 for purification. The active eluents containing partially purified enzyme were collected for further investigation. The specific activity of α-amylase was (34.9 U/mg) which was corresponding to 2.09 fold purification for the tested organism. The purified α-amylase was immobilized by entrapment method into two types of polymers. One of them was natural consist of chitosan and alginate. The other polymer was synthetic consist of N- isopropyl acrylamide and alginate. The temperature optimum and thermal inactivation showed a severe loss in the activity of the free enzymes, while the temperature profile of the immobilized enzymes was much broader at higher temperatures demonstrating the effectiveness of the polymer protecting the enzymes. Also, the immobilized enzymes (natural polymer and synthetic polymer) showed higher thermal stability. Optimum ph and stability showed that immobilization of enzymes resulted in more

Immobilization of alpha-amylase from Bacillus circulans GRS 313 on coconut fiber.

Science.gov (United States)

Dey, Gargi; Nagpal, Varima; Banerjee, Rintu

2002-01-01

A simple and inexpensive method for immobilizing alpha-amylase from Bacillus circulans GRS 313 on coconut fiber was developed. The immobilization conditions for highest efficiency were optimized with respect to immobilization pH of 5.5, 30 degrees C, contact time of 4 h, and enzyme to support a ratio of 1:1 containing 0.12 mg/mL of protein. The catalytic properties of the immobilized enzyme were compared with that of the free enzyme. The activity of amylase adsorbed on coconut fiber was 38.7 U/g of fiber at its optimum pH of 5.7 and 48 degrees C, compared with the maximum activity of 40.2 U/mL of free enzyme at the optimum pH of 4.9 and 48 degrees C. The reutilization capacity of the immobilized enzyme was up to three cycles.

Preparation and activity of bubbling-immobilized cellobiase within chitosan-alginate composite.

Science.gov (United States)

Wang, Fang; Su, Rong-Xin; Qi, Wei; Zhang, Ming-Jia; He, Zhi-Min

2010-01-01

Cellobiase can hydrolyze cellobiose into glucose; it plays a key role in the process of cellulose hydrolysis by reducing the product inhibition. To reuse the enzyme and improve the economic value of cellulosic ethanol, cellobiase was immobilized using sodium alginate and chitosan as carriers by the bubbling method. The immobilization conditions were optimized as follows: enzyme loading of 100 U cellobiase/g carrier, 30 min immobilization, 3.5 wt% sodium alginate, 0.25 wt% chitosan, and 2 wt% calcium chloride. Compared to free enzyme, the immobilized cellobiase had a decreased apparent K(m) and the maximum activity at a lower pH, indicating its higher acidic and thermal stability. The immobilized cellobiase was further tested in the hydrolysis of cellobiose and various cellulosic substrates (microcrystalline cellulose, filter paper, and ammonia-pretreated corn cobs). Together with cellulases, the immobilized cellobiase converted the cellulosic substrates into glucose with the rate and extent similar to the free enzyme.

Assessment of digestive enzymes activity during the fry development of the endangered Caspian brown trout Salmo caspius.

Science.gov (United States)

Zamani, A; Hajimoradloo, A; Madani, R; Farhangi, M

2009-09-01

The study of digestive enzymes activity at Salmo caspius fry showed that enzymes were available at the moment of mouth opening on the first day post hatching (dph) and the activity of enzymes showed no significant difference from the hatching day 28 dph. An increased activity was seen between 32 and 43 dph and this activity was significantly higher than the activity during the first 28 days. In the primary stages after yolk sac resorption (43-58 dph), enzymes activity showed an increased profile, however none of them showed a significant difference between 43 and 58 dph.

Covalent immobilization of invertase on PAMAM-dendrimer modified superparamagnetic iron oxide nanoparticles

International Nuclear Information System (INIS)

Uzun, K.; Cevik, E.; Senel, M.; Soezeri, H.; Baykal, A.; Abasiyanik, M. F.; Toprak, M. S.

2010-01-01

In this study, polyamidoamine (PAMAM) dendrimer was synthesized on the surface of superparamagnetite nanoparticles to enhance invertase immobilization. The amount of immobilized enzyme on the surface-hyperbranched magnetite nanoparticle was up to 2.5 times (i.e., 250%) as much as that of magnetite nanoparticle modified with only amino silane. Maximum reaction rate (V max ) and Michaelis-Menten constant (K m ) were determined for the free and immobilized enzymes. Various characteristics of immobilized invertase such as; the temperature activity, thermal stability, operational stability, and storage stability were evaluated and results revealed that stability of the enzyme is improved upon immobilization.

Extraction and identification of α-amylase inhibitor peptides from Nephelium lappacheum and Nephelium mutabile seed protein using gastro-digestive enzymes.

Science.gov (United States)

Evaristus, Natashya Anak; Wan Abdullah, Wan Nadiah; Gan, Chee-Yuen

2018-04-01

The potential of N. lappacheum and N. mutabile seed as a source of α-amylase inhibitor peptides was explored based on the local traditional practice of using the seed. Different gastro-digestive enzymes (i.e. pepsin or chymotrypsin) or a sequential digestion were used to extract the peptides. The effects of digestion time and enzyme to substrate (E:S) ratio on the α-amylase inhibitory activity were investigated. Results showed that chymotrypsin was effective in producing the inhibitor peptides from rambutan seed protein at E:S ratio 1:20 for 1 h, whereas pepsin was more effective for pulasan seed protein under the same condition. A total of 20 and 31 novel inhibitor peptides were identified, respectively. These peptides could bind with the subsites of α-amylase (i.e. Trp58, Trp59, Tyr62, Asp96, Arg195, Asp197, Glu233, His299, Asp300, and His305) and formed a sliding barrier that preventing the formation of enzyme/substrate intermediate leading to lower α-amylase activity. Copyright © 2018 Elsevier Inc. All rights reserved.

Modeling and simulation of enzymatic gluconic acid production using immobilized enzyme and CSTR-PFTR circulation reaction system.

Science.gov (United States)

Li, Can; Lin, Jianqun; Gao, Ling; Lin, Huibin; Lin, Jianqiang

2018-04-01

Production of gluconic acid by using immobilized enzyme and continuous stirred tank reactor-plug flow tubular reactor (CSTR-PFTR) circulation reaction system. A production system is constructed for gluconic acid production, which consists of a continuous stirred tank reactor (CSTR) for pH control and liquid storage and a plug flow tubular reactor (PFTR) filled with immobilized glucose oxidase (GOD) for gluconic acid production. Mathematical model is developed for this production system and simulation is made for the enzymatic reaction process. The pH inhibition effect on GOD is modeled by using a bell-type curve. Gluconic acid can be efficiently produced by using the reaction system and the mathematical model developed for this system can simulate and predict the process well.

Electrografting of carboxyphenyl thin layer onto gold for DNA and enzyme immobilization

International Nuclear Information System (INIS)

Nowicka, Anna M.; Fau, Michal; Kowalczyk, Agata; Strawski, Marcin; Stojek, Zbigniew

2014-01-01

The convenient functionalization of metal surfaces by carboxyphenyl groups in aprotic media is not possible for two reasons. First, carboxy derivatives of diazonium salts are very unstable and, second, the electroreduction product is soluble in the solvent. So, the optimization of the conditions of the electrografting of the metal surfaces by applying aqueous solutions is much needed. Compared to earlier cyclic voltammetry approaches we have shown that the chronoamperometric deposition is more convenient. The constant potential equal to the voltammetric peak potential and the molar ratio 1:1 for the substrates: 4-aminobenzoic acid and NaNO 2 as the diazotization agent, in 0.5 M HCl, appeared to be very satisfying conditions for the deposition of a thin layer of deposit of perpendicularly oriented carboxyphenyl groups at the Au surface and for maximal elimination of the influence of the side-reactions products. Under the determined conditions the immobilization of DNA strands was optimal and the deposited laccase layer was tightly packed and very efficient toward the electroreduction of oxygen. Electrochemical impedance spectroscopy, electrochemical quartz crystal microbalance, cyclic voltammetry, chronocoulometry, atomic force microscopy, contact angle measurements and UV–Vis spectroscopy of the solution were used to characterize the electrografted carboxyphenyl layers and subsequent oligonucleotide and enzyme immobilization process

Preparation and characterization of two types of covalently immobilized amyloglucosidase

Directory of Open Access Journals (Sweden)

ZORAN VUJCIC

2005-05-01

Full Text Available Amyloglucosidase from A. niger was covalently immobilized onto poly (GMA-co-EGDMA by the glutaraldehyde and periodate method. The immobilization of amyloglucosidase after periodate oxidation gave a preparate with the highest specific activity reported so far on similar polymers. The obtained immobilized preparates show the same pH optimum, but a higher temperature optimum compared with the soluble enzyme. The kinetic parameters for the hydrolysis of soluble starch by free and both immobilized enzymes were determined.

Immobilization of Candida antarctica Lipase B by Adsorption to Green Coconut Fiber

Science.gov (United States)

Brígida, Ana I. S.; Pinheiro, Álvaro D. T.; Ferreira, Andrea L. O.; Gonçalves, Luciana R. B.

An agroindustrial residue, green coconut fiber, was evaluated as support for immobilization of Candida antarctica type B (CALB) lipase by physical adsorption. The influence of several parameters, such as contact time, amount of enzyme offered to immobilization, and pH of lipase solution was analyzed to select a suitable immobilization protocol. Kinetic constants of soluble and immobilized lipases were assayed. Thermal and operational stability of the immobilized enzyme, obtained after 2 h of contact between coconut fiber and enzyme solution, containing 40 U/ml in 25 mM sodium phosphate buffer pH 7, were determined. CALB immobilization by adsorption on coconut fiber promoted an increase in thermal stability at 50 and 60 °C, as half-lives (t 1/2) of the immobilized enzyme were, respectively, 2- and 92-fold higher than the ones for soluble enzyme. Furthermore, operational stabilities of methyl butyrate hydrolysis and butyl butyrate synthesis were evaluated. After the third cycle of methyl butyrate hydrolysis, it retained less than 50% of the initial activity, while Novozyme 435 retained more than 70% after the tenth cycle. However, in the synthesis of butyl butyrate, CALB immobilized on coconut fiber showed a good operational stability when compared to Novozyme 435, retaining 80% of its initial activity after the sixth cycle of reaction.

Improving off-line accelerated tryptic digestion. Towards fast-lane proteolysis of complex biological samples.

Science.gov (United States)

Vukovic, Jadranka; Loftheim, Håvard; Winther, Bjørn; Reubsaet, J Léon E

2008-06-27

Off-line digestion of proteins using immobilized trypsin beads is studied with respect to the format of the digestion reactor, the digestion conditions, the comparison with in-solution digestion and its use in complex biological samples. The use of the filter vial as the most appropriate digestion reactor enables simple, efficient and easy-to-handle off-line digestion of the proteins on trypsin beads. It was shown that complex proteins like bovine serum albumin (BSA) need much longer time (89 min) and elevated temperature (37 degrees C) to be digested to an acceptable level compared to smaller proteins like cytochrome c (5 min, room temperature). Comparing the BSA digestion using immobilized trypsin beads with conventional in-solution digestion (overnight at 37 degrees C), it was shown that comparable results were obtained with respect to sequence coverage (>90%) and amount of missed cleavages (in both cases around 20 peptides with 1 or 2 missed cleavages were detected). However, the digestion using immobilized trypsin beads was considerable less time consuming. Good reproducibility and signal intensities were obtained for the digestion products of BSA in a complex urine sample. In addition to this, peptide products of proteins typically present in urine were identified.

Influences of apolipoprotein E on soluble and heparin-immobilized hepatic lipase

International Nuclear Information System (INIS)

Landis, B.A.; Rotolo, F.S.; Meyers, W.C.; Clark, A.B.; Quarfordt, S.H.

1987-01-01

The effect of human apolipoprotein E (apoE), either alone or in combination with apoC, on the lipolysis of a radiolabeled triglyceride emulsion was studied with hepatic lipase in solution and immobilized on heparin-Sepharose. The soluble hepatic lipase was inhibited, whereas the heparin-immobilized lipase was stimulated by apoE. This stimulation was attenuated by combining apoE with either apoC-II or C-III. The heparin-immobilized lipase demonstrated much less lipolysis of the zwitterionic phosphatidylcholine-stabilized triglyceride emulsion than did the soluble enzyme. This difference was less when the emulsion was stabilized by a nonionic detergent. apoE inhibited lipase activity when assayed under conditions (0.4 M NaCl) of bound enzyme and unbound substrate. Increasing the emulsion apoE content beyond optimum inhibited lipolysis by the immobilized enzyme. Kinetic analysis of phosphatidylcholine-stabilized triglyceride emulsions revealed a significant decrease in immobilized enzyme K/sub m/ and an increase in V/sub max/ when the emulsion was supplemented with apoE. Distributing the immobilized lipase in clustered aggregates produced more lipolysis than when the same enzyme content was uniformly bound

Cellulase immobilization on superparamagnetic nanoparticles for reuse in cellulosic biomass conversion

Directory of Open Access Journals (Sweden)

Fernando Segato

2016-07-01

Full Text Available Current cellulosic biomass hydrolysis is based on the one-time use of cellulases. Cellulases immobilized on magnetic nanocarriers offer the advantages of magnetic separation and repeated use for continuous hydrolysis. Most immobilization methods focus on only one type of cellulase. Here, we report co-immobilization of two types of cellulases, β-glucosidase A (BglA and cellobiohydrolase D (CelD, on sub-20 nm superparamagnetic nanoparticles. The nanoparticles demonstrated 100% immobilization efficiency for both BglA and CelD. The total enzyme activities of immobilized BglA and CelD were up to 67.1% and 41.5% of that of the free cellulases, respectively. The immobilized BglA and CelD each retained about 85% and 43% of the initial immobilized enzyme activities after being recycled 3 and 10 times, respectively. The effects of pH and temperature on the immobilized cellulases were also investigated. Co-immobilization of BglA and CelD on MNPs is a promising strategy to promote synergistic action of cellulases while lowering enzyme consumption.

Immobilization and characterization of inulinase from Ulocladium

Indian Academy of Sciences (India)

Ulocladium atrum inulinase was immobilized on different composite membranes composed of chitosan/nonwoven fabrics. Km values of free and immobilized U. atrum inulinase on different composite membranes were calculated. The enzyme had optimum pH at 5.6 for free and immobilized U. atrum inulinase on polyester ...

Catalytical Properties of Free and Immobilized Aspergillus niger Tannase

OpenAIRE

Abril Flores-Maltos; Luis V. Rodríguez-Durán; Jacqueline Renovato; Juan C. Contreras; Raúl Rodríguez; Cristóbal N. Aguilar

2011-01-01

A fungal tannase was produced, recovered, and immobilized by entrapment in calcium alginate beads. Catalytical properties of the immobilized enzyme were compared with those of the free one. Tannase was produced intracellularly by the xerophilic fungus Aspergillus niger GH1 in a submerged fermentation system. Enzyme was recovered by cell disruption and the crude extract was partially purified. The catalytical properties of free and immobilized tannase were evaluated using tannic acid and methy...

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

Science.gov (United States)

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

2018-07-01

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

Effects of Enzyme Complex Supplementation to a Paddy-based Diet on Performance and Nutrient Digestibility of Meat-type Ducks

Directory of Open Access Journals (Sweden)

P. Kang

2013-02-01

Full Text Available Paddy rice is rarely used as a feed because of its high fiber content. In this study, two experiments were conducted to study the effects of supplementing an enzyme complex consisting of xylanase, beta-glucanase and cellulase, to paddy-based diets on the performance and nutrient digestibility in meat-type ducks. In the both experiments, meat-type ducks (Cherry Valley were randomly assigned to four treatments. Treatment 1 was a basal diet of corn-soybean; treatment 2 was a basal diet of corn-paddy-soybean; treatment 3, had enzyme complex added to the corn-paddy-soybean basal diet at levels of 0.5 g/kg diet; and treatment 4, had enzyme complex added to the corn-paddy-soybean diet at levels of 1.0 g/kg diet. The results showed that the enzyme complex increased the ADG, and decreased the ADFI and F/G significantly (p0.05. The outcome of this research indicates that the application of enzyme complex made up of xylanase, beta-glucanase, and cellulase, in the corn-paddy-soybean diet, can improve performance and nutrition digestibility in meat-type ducks.

Immobilization and kinetics of catalase on calcium carbonate nanoparticles attached epoxy support.

Science.gov (United States)

Preety; Hooda, Vinita

2014-01-01

A novel hybrid epoxy/nano CaCO3 composite matrix for catalase immobilization was prepared by polymerizing epoxy resin in the presence of CaCO3 nanoparticles. The hybrid support was characterized using scanning electron microscopy and Fourier transform infrared spectroscopy. Catalase was successfully immobilized onto epoxy/nano CaCO3 support with a conjugation yield of 0.67 ± 0.01 mg/cm(2) and 92.63 ± 0.80 % retention of activity. Optimum pH and optimum temperature of free and immobilized catalases were found to be 7.0 and 35 °C. The value of Km for H2O2 was higher for immobilized enzyme (31.42 mM) than native enzyme (27.73 mM). A decrease in Vmax value from 1,500 to 421.10 μmol (min mg protein)(-1) was observed after immobilization. Thermal and storage stabilities of catalase improved immensely after immobilization. Immobilized enzyme retained three times than the activity of free enzyme when kept at 75 °C for 1 h and the half-life of enzyme increased five times when stored in phosphate buffer (0.01 M, pH 7.0) at 5 °C. The enzyme could be reused 30 times without any significant loss of its initial activity. Desorption of catalase from the hybrid support was minimum at pH 7.0.

Characterizations of substrate and enzyme specificity of glucoamylase assays of mucosal starch digestion with determinations of group and single biopsy reference values

Science.gov (United States)

Carbohydrate digesting enzyme activities are measured in duodenal biopsies to detect deficiencies of lactase and sucrase activities, however glucoamylase (GA) assays for starch digestion are not included. Because food starch represents half of energy intake in the human diet, assays for starch diges...

Sol-gel immobilization of serine proteases for application in organic solvents

NARCIS (Netherlands)

van Unen, D.J.; Engbersen, Johannes F.J.; Reinhoudt, David

2001-01-01

The serine proteases α-chymotrypsin, trypsin, and subtilisin Carlsberg were immobilized in a sol-gel matrix and the effects on the enzyme activity in organic media are evaluated. The percentage of immobilized enzyme is 90% in the case of α-chymotrypsin and the resulting specific enzyme activity in

Effects of supplemental enzymes on apparent nutrient digestibility in rainbow trout (Oncorhynchus mykiss) fed plant-based diets

DEFF Research Database (Denmark)

Dalsgaard, Anne Johanne Tang; Hjermitslev, Niels Harthøj; Ekmann, Kim Schøn

2010-01-01

in fish feed due to growing demands for and high price variations in fish meal, but high inclusion levels in diets for carnivorous fish are hampered by a great variety of anti-nutritional factors (ANFs), which reduce nutrient utilisation. Exogenous dietary enzymes may potentially help to alleviate...... on the effects of enzymes in fish feed apart from phytase. Phytase works by hydrolyzing phytic acid, and numerous studies have documented that phytase supplementation increases phosphorus availability in fish fed diets with high inclusion levels of plant proteins. Plant derived proteins are increasingly used...... these effects, and the objective of the present study was to evaluate the effects of supplementing protease and pectinase to a diet containing approximately 30% soybean meal, rapeseed meal or sunflower meal on nutrient digestibility in juvenile rainbow trout (Oncorhynchus mykiss). Digestibility trials were...

Immobilization of Aspergillus awamori β-glucosidase on commercial gelatin: An inexpensive and efficient process.

Science.gov (United States)

Nishida, Verônica S; de Oliveira, Roselene F; Brugnari, Tatiane; Correa, Rúbia Carvalho G; Peralta, Rosely A; Castoldi, Rafael; de Souza, Cristina G M; Bracht, Adelar; Peralta, Rosane M

2018-05-01

In this work, a β-glucosidase of Aspergillus awamori with a molecular weight of 180 kDa was produced in solid-state cultures using a mixture of pineapple crown leaves and wheat bran. Maximum production of the enzyme (820 ± 30 U/g substrate) was obtained after 8 days of culture at 28 °C and initial moisture of 80%. The crude enzyme was efficiently immobilized on glutaraldehyde cross-linked commercial gelatin. Immobilization changed the kinetics of the enzyme, whose behavior could no longer be described by a saturation function of the Michaelis-Menten type. Comparative evaluation of the free and immobilized enzyme showed that the immobilized enzyme was more thermostable and less inhibited by glucose than the free form. In consequence of these properties, the immobilized enzyme was able to hydrolyze cellobiose more extensively. In association with Trichoderma reesei cellulase, the free and immobilized β-glucosidase increased the liberation of glucose from cellulose 3- and 5-fold, respectively. Immobilization of the A. awamori β-glucosidase on glutaraldehyde cross-linked commercial gelatin is an efficient and cheap method allowing the reuse of the enzyme by at least 10 times. Copyright © 2018 Elsevier B.V. All rights reserved.

Improvement of thermal-stability of enzyme immobilized onto mesoporous zirconia

Directory of Open Access Journals (Sweden)

Yuichi Masuda

2014-03-01

Thereafter, FDH immobilized on MPZ showed higher catalytic activity than that on MPS. Enhancement of catalytic activity was obtained by improving the substrate affinity derived from interparticle voids of MPZ. In addition, the FDH immobilized on MPZ had a very great higher thermal stability. Further investigation using transmittance Infrared spectroscopy indicated that the high-order structure of the FDH immobilized on MPZ did not get altered after the heat-treatment.

Three-dimensional immobilization of beta-galactosidase on a silicon surface.

Science.gov (United States)

Betancor, Lorena; Luckarift, Heather R; Seo, Jae H; Brand, Oliver; Spain, Jim C

2008-02-01

Many alternative strategies to immobilize and stabilize enzymes have been investigated in recent years for applications in biosensors. The entrapment of enzymes within silica-based nanospheres formed through silicification reactions provides high loading capacities for enzyme immobilization, resulting in high volumetric activity and enhanced mechanical stability. Here we report a strategy for chemically associating silica nanospheres containing entrapped enzyme to a silicon support. beta-galactosidase from E. coli was used as a model enzyme due to its versatility as a biosensor for lactose. The immobilization strategy resulted in a three-dimensional network of silica attached directly at the silicon surface, providing a significant increase in surface area and a corresponding 3.5-fold increase in enzyme loading compared to enzyme attached directly at the surface. The maximum activity recovered for a silicon square sample of 0.5 x 0.5 cm was 0.045 IU using the direct attachment of the enzyme through glutaraldehyde and 0.16 IU when using silica nanospheres. The immobilized beta-galactosidase prepared by silica deposition was stable and retained more than 80% of its initial activity after 10 days at 24 degrees C. The ability to generate three-dimensional structures with enhanced loading capacity for biosensing molecules offers the potential to substantially amplify biosensor sensitivity. (c) 2007 Wiley Periodicals, Inc.

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

Science.gov (United States)

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.

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

Energy Technology Data Exchange (ETDEWEB)

Moreno-Cortez, Iván E. [Centro de Investigación en Química Aplicada (CIQA), Blvd. Enrique Reyna # 140, San José de los Cerritos, Saltillo, Coahuila 25100, México (Mexico); Universidad Autónoma de Nuevo León (UANL), Fac. de Ingeniería Mecánica y Eléctrica (FIME), Av. Universidad S/N, Cd. Universitaria, San Nicolás de los Garza, Nuevo León C.P. 66450, México (Mexico); Universidad Autónoma de Nuevo León (UANL), Centro de Innovación, Investigación y Desarrollo en Ingeniería y Tecnología (CIIDIT), Apodaca, Nuevo León, México (Mexico); Romero-García, Jorge, E-mail: jromero@ciqa.mx [Centro de Investigación en Química Aplicada (CIQA), Blvd. Enrique Reyna # 140, San José de los Cerritos, Saltillo, Coahuila 25100, México (Mexico); and others

2015-07-01

In this paper, papain enzyme (E.C. 3.4.22.2, 1.6 U/mg) was successfully immobilized in poly(vinyl alcohol) (PVA) nanofibers prepared by electrospinning. The morphology of the electrospun nanofibers was characterized by scanning electron microscopy (SEM) and the diameter distribution was in the range of 80 to 170 nm. The presence of the enzyme within the PVA nanofibers was confirmed by infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS) and energy dispersive X-ray spectroscopy (EDXS) analyses. The maximum catalytic activity was reached when the enzyme loading was 13%. The immobilization of papain in the nanofiber membrane was achieved by chemical crosslinking with a glutaraldehyde vapor treatment (GAvt). The catalytic activity of the immobilized papain was 88% with respect to the free enzyme. The crosslinking time by GAvt to immobilize the enzyme onto the nanofiber mat was 24 h, and the enzyme retained its catalytic activity after six cycles. The crosslinked samples maintained 40% of their initial activity after being stored for 14 days. PVA electrospun nanofibers are excellent matrices for the immobilization of enzymes due to their high surface area and their nanoporous structure. - Highlights: • Successfully attempt to immobilize the papain enzyme in electrospun nanofibers • The morphology of nanofibers did not change at moderate enzyme concentrations. • The retained activity of the immobilized enzyme was 88% relative to the free enzyme. • The immobilized enzyme retains 40% of the initial activity after 14 days of storage. • Potential application of this work in the fabrication of biosensors specialized in the detection of metal ions.

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

International Nuclear Information System (INIS)

Moreno-Cortez, Iván E.; Romero-García, Jorge

2015-01-01

In this paper, papain enzyme (E.C. 3.4.22.2, 1.6 U/mg) was successfully immobilized in poly(vinyl alcohol) (PVA) nanofibers prepared by electrospinning. The morphology of the electrospun nanofibers was characterized by scanning electron microscopy (SEM) and the diameter distribution was in the range of 80 to 170 nm. The presence of the enzyme within the PVA nanofibers was confirmed by infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS) and energy dispersive X-ray spectroscopy (EDXS) analyses. The maximum catalytic activity was reached when the enzyme loading was 13%. The immobilization of papain in the nanofiber membrane was achieved by chemical crosslinking with a glutaraldehyde vapor treatment (GAvt). The catalytic activity of the immobilized papain was 88% with respect to the free enzyme. The crosslinking time by GAvt to immobilize the enzyme onto the nanofiber mat was 24 h, and the enzyme retained its catalytic activity after six cycles. The crosslinked samples maintained 40% of their initial activity after being stored for 14 days. PVA electrospun nanofibers are excellent matrices for the immobilization of enzymes due to their high surface area and their nanoporous structure. - Highlights: • Successfully attempt to immobilize the papain enzyme in electrospun nanofibers • The morphology of nanofibers did not change at moderate enzyme concentrations. • The retained activity of the immobilized enzyme was 88% relative to the free enzyme. • The immobilized enzyme retains 40% of the initial activity after 14 days of storage. • Potential application of this work in the fabrication of biosensors specialized in the detection of metal ions

Industrial Applications of Enzymes: Recent Advances, Techniques, and Outlooks

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Jordan Chapman

2018-06-01

Full Text Available Enzymes as industrial biocatalysts offer numerous advantages over traditional chemical processes with respect to sustainability and process efficiency. Enzyme catalysis has been scaled up for commercial processes in the pharmaceutical, food and beverage industries, although further enhancements in stability and biocatalyst functionality are required for optimal biocatalytic processes in the energy sector for biofuel production and in natural gas conversion. The technical barriers associated with the implementation of immobilized enzymes suggest that a multidisciplinary approach is necessary for the development of immobilized biocatalysts applicable in such industrial-scale processes. Specifically, the overlap of technical expertise in enzyme immobilization, protein and process engineering will define the next generation of immobilized biocatalysts and the successful scale-up of their induced processes. This review discusses how biocatalysis has been successfully deployed, how enzyme immobilization can improve industrial processes, as well as focuses on the analysis tools critical for the multi-scale implementation of enzyme immobilization for increased product yield at maximum market profitability and minimum logistical burden on the environment and user.

Enhanced thermostability of silica-immobilized lipase from Bacillus coagulans BTS-3 and synthesis of ethyl propionate.

Science.gov (United States)

Kumar, Satyendra; Pahujani, Shweta; Ola, R P; Kanwar, S S; Gupta, Reena

2006-06-01

A lipase from the thermophilic isolate Bacillus coagulans BTS-3 was produced and purified. The enzyme was purified 40-fold to homogeneity by ammonium sulfate precipitation and DEAE-Sepharose column chromatography. Its molecular weight was 31 kDa on SDS-PAGE. The purified lipase was immobilized on silica and its binding efficiency was found to be 60%. The enzyme took 60 min to bind maximally onto the support. The pH and temperature optima of immobilized lipase were same as those of the free enzyme, i.e. 8.5 and 55 degrees C, respectively. The immobilized enzyme had shown marked thermostability on the elevated temperatures of 55, 60, 65 and 70 degrees C. The immobilized enzyme was reused for eigth cycles as it retained almost 80% of its activity. The catalytic activity of immobilized enzyme was enhanced in n-hexane and ethanol. The immobilized enzyme when used for esterification of ethanol and propionic acid showed 96% conversion in n-hexane in 12 h at 55 degrees C.

Effective L-Tyrosine Hydroxylation by Native and Immobilized Tyrosinase.

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Małgorzata Cieńska

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

Effective L-Tyrosine Hydroxylation by Native and Immobilized Tyrosinase.

Science.gov (United States)

Cieńska, Małgorzata; Labus, Karolina; Lewańczuk, Marcin; Koźlecki, Tomasz; Liesiene, Jolanta; Bryjak, Jolanta

2016-01-01

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

Effective L-Tyrosine Hydroxylation by Native and Immobilized Tyrosinase

Science.gov (United States)

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

2016-01-01

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

Catalytic Properties and Immobilization Studies of Catalase from Malva sylvestris L.

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G. Arabaci

2013-01-01

Full Text Available Catalase was partially purified from Malva sylvestris L. and immobilized onto chitosan. Then, its catalytic properties were investigated. (NH42SO4 precipitation and dialysis were performed in the extracted enzyme. Further purification was performed with sephadex G-200 column. Kinetic studies of the purified enzyme activity were measured and characterized. The inhibitory effects of KCN, NaN3, CuSO4, and EDTA on M. sylvestris L. catalase activity were observed except NaCl. Furthermore, M. sylvestris L. catalase was immobilized covalently with glutaraldehyde onto chitosan particles. The pH and temperature optima as well as the changes in the kinetics (Km, Vmax of the immobilized and free M. sylvestris L. catalase were determined. The Km value for immobilized catalase (23.4 mM was higher than that of free enzyme (17.6 mM. Optimum temperature was observed higher than that of the free enzyme. The optimum pH was the same for both free and immobilized catalases (pH 7.50. Immobilized catalase showed higher storage and thermal stabilities than free catalases. Free catalase lost all its activity within 60 days whereas immobilized catalase lost 45% of its activity during the same incubation period at 4°C. The remaining immobilized catalase activity was about 70% after 8 cycles of batch operations.

Digestive peptidase evolution in holometabolous insects led to a divergent group of enzymes in Lepidoptera

KAUST Repository

Dias, Renata O.; Via, Allegra; Brandã o, Marcelo M.; Tramontano, Anna; Silva-Filho, Marcio C.

2015-01-01

© 2015 Elsevier Ltd. Trypsins and chymotrypsins are well-studied serine peptidases that cleave peptide bonds at the carboxyl side of basic and hydrophobic l-amino acids, respectively. These enzymes are largely responsible for the digestion of proteins. Three primary processes regulate the activity of these peptidases: secretion, precursor (zymogen) activation and substrate-binding site recognition. Here, we present a detailed phylogenetic analysis of trypsins and chymotrypsins in three orders of holometabolous insects and reveal divergent characteristics of Lepidoptera enzymes in comparison with those of Coleoptera and Diptera. In particular, trypsin subsite S1 was more hydrophilic in Lepidoptera than in Coleoptera and Diptera, whereas subsites S2-S4 were more hydrophobic, suggesting different substrate preferences. Furthermore, Lepidoptera displayed a lineage-specific trypsin group belonging only to the Noctuidae family. Evidence for facilitated trypsin auto-activation events were also observed in all the insect orders studied, with the characteristic zymogen activation motif complementary to the trypsin active site. In contrast, insect chymotrypsins did not seem to have a peculiar evolutionary history with respect to their mammal counterparts. Overall, our findings suggest that the need for fast digestion allowed holometabolous insects to evolve divergent groups of peptidases with high auto-activation rates, and highlight that the evolution of trypsins led to a most diverse group of enzymes in Lepidoptera.

Digestive peptidase evolution in holometabolous insects led to a divergent group of enzymes in Lepidoptera

KAUST Repository

Dias, Renata O.

2015-03-01

© 2015 Elsevier Ltd. Trypsins and chymotrypsins are well-studied serine peptidases that cleave peptide bonds at the carboxyl side of basic and hydrophobic l-amino acids, respectively. These enzymes are largely responsible for the digestion of proteins. Three primary processes regulate the activity of these peptidases: secretion, precursor (zymogen) activation and substrate-binding site recognition. Here, we present a detailed phylogenetic analysis of trypsins and chymotrypsins in three orders of holometabolous insects and reveal divergent characteristics of Lepidoptera enzymes in comparison with those of Coleoptera and Diptera. In particular, trypsin subsite S1 was more hydrophilic in Lepidoptera than in Coleoptera and Diptera, whereas subsites S2-S4 were more hydrophobic, suggesting different substrate preferences. Furthermore, Lepidoptera displayed a lineage-specific trypsin group belonging only to the Noctuidae family. Evidence for facilitated trypsin auto-activation events were also observed in all the insect orders studied, with the characteristic zymogen activation motif complementary to the trypsin active site. In contrast, insect chymotrypsins did not seem to have a peculiar evolutionary history with respect to their mammal counterparts. Overall, our findings suggest that the need for fast digestion allowed holometabolous insects to evolve divergent groups of peptidases with high auto-activation rates, and highlight that the evolution of trypsins led to a most diverse group of enzymes in Lepidoptera.

Preparation of immobilized growing cells and enzymatic hydrolysis of sawdust

International Nuclear Information System (INIS)

Kumakura, M.; Kaetsu, I.

1984-01-01

Trichoderma reesei cells were immobilized by radiation polymerization using porous materials such as non-woven material and sawdust, and the enzymatic hydrolysis of sawdust with the enzyme solution from the immobilized growing cells was studied. The filter paper activity, which shows the magnitude of cellulase production in the immobilized cells, was comparable with that in the intact cells. The filter paper activity was affected by addition concentration of monomer and porous materials. The cells in the immobilized cells grew to be adhered on the surface of the fibrous polymers. Sawdust, which was pretreated by irradiation technique, was effectively hydrolyzed with the enzyme solution resulting from the culture of the immobilized cells, in which the glucose yield increased increasing the culture time of the immobilized cells. (author)

Immobilization of Mucor miehei Lipase onto Macroporous Aminated Polyethersulfone Membrane for Enzymatic Reactions

NARCIS (Netherlands)

Handayani, Nurrahmi; Loos, Katja; Wahyuningrum, Deana; Buchari, [No Value; Zulfikar, Muhammad Ali

2012-01-01

Immobilization of enzymes is one of the most promising methods in enzyme performance enhancement, including stability, recovery, and reusability. However, investigation of suitable solid support in enzyme immobilization is still a scientific challenge. Polyethersulfone (PES) and aminated PES

Covalent immobilization of lipases on monodisperse magnetic microspheres modified with PAMAM-dendrimer

Energy Technology Data Exchange (ETDEWEB)

Zhu, Weiwei [Lanzhou University, State Key Laboratory of Applied Organic Chemistry, Key Laboratory of Nonferrous Metal Chemistry and Resources Utilization of Gansu Province, College of Chemistry and Chemical Engineering, Institute of Biochemical Engineering and Environmental Technology (China); Zhang, Yimei [Suzhou Research Academy of North China Electric Power University (China); Hou, Chen; Pan, Duo; He, Jianjun; Zhu, Hao, E-mail: zhuhao07@lzu.edu.cn [Lanzhou University, State Key Laboratory of Applied Organic Chemistry, Key Laboratory of Nonferrous Metal Chemistry and Resources Utilization of Gansu Province, College of Chemistry and Chemical Engineering, Institute of Biochemical Engineering and Environmental Technology (China)

2016-02-15

This paper reported an immobilization of Candida rugosa lipase (CRL) onto PAMAM-dendrimer-grafted magnetic nanoparticles synthesized by a modified solvothermal reduction method. The dendritic magnetic nanoparticles were amply characterized by several instrumental measurements, and the CRL was covalently anchored on the three generation supports with glutaraldehyde as coupling reagent. The amount of immobilized enzyme was up to 150 mg/g support and the factors related with the enzyme activity were investigated. The immobilization of lipase improved their performance in wider ranges of pH and temperature. The immobilized lipase exhibited excellent thermal stability and reusability in comparison with free enzyme and can be reused 10 cycles with the enzymatic activity remained above 90 %. The properties of lipase improved obviously after being immobilized on the dendritic supports. The inactive immobilized lipase could be regenerated with glutaraldehyde and Cu{sup 2+}, respectively. This synthetic strategy was facile and eco-friendly for applications in lipase immobilization.

Characterization of immobilized post-carbohydrate meal salivary α ...

African Journals Online (AJOL)

The effect of experimental parameters like pH, temperature and substrate concentration on the activity of the immobilized post-carbohydrate meal salivary ... of immobilized post-carbohydrate meal salivary α-amylase in this study show that immobilization had no significant effect on the enzyme and compared to kinetic ...

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

Directory of Open Access Journals (Sweden)

Hui Ren

2016-06-01

Full Text Available 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.

Digestive enzyme activity and trophic behavior in two predator aquatic insects (Plecoptera, Perlidae): a comparative study.

Science.gov (United States)

López-Rodríguez, M J; Trenzado, C E; Tierno de Figueroa, J M; Sanz, A

2012-05-01

Plecoptera (Perlidae) are among the major macroinvertebrate predators in stream ecosystems and one of the insect families with lower tolerance to environmental alterations, being usually employed as bioindicators of high water ecological quality. The differences in the trophic roles of the coexisting species have been exclusively studied from their gut contents, while no data are available on the comparative digestive capacity. In the present paper, we make a comparative study of the activity of several digestive enzymes, namely proteases (at different pH), amylase, lipase, trypsin and chymotrypsin, in two species of stoneflies, Perla bipunctata and Dinocras cephalotes, which cohabit in the same stream. The study of digestive enzyme activity together with the analysis of gut contents can contribute to a better understanding of the ecology of these aquatic insects and their role in freshwater food webs. Thus, our results show that the two studied predator species inhabiting in the same stream present specializations on their feeding behaviors, facilitating their coexistence, and also differences in their capacity of use the resources. One of the main findings of this study is that D. cephalotes is able to assimilate a wider trophic resource spectrum and this could be one of the reasons why this species has a wider global distribution in all its geographical range. Copyright © 2012 Elsevier Inc. All rights reserved.

Enzymatic transesterification of soybean oil with ethanol using lipases immobilized on highly crystalline PVA microspheres

International Nuclear Information System (INIS)

Bergamasco, Juliana; Araujo, Marcelo V. de; Vasconcellos, Adriano de; Luizon Filho, Roberto A.; Hatanaka, Rafael R.; Giotto, Marcus V.; Aranda, Donato A.G.; Nery, José G.

2013-01-01

Polyvinyl alcohol (PVA) microspheres with different degree of crystallinity were used as solid supports for Rhizomucor miehei lipase immobilization, and the enzyme-PVA complexes were used as biocatalysts for the transesterification of soybean oil to fatty acid ethyl esters (FAEE). The amounts of immobilized enzyme on the polymeric supports were similar for both the amorphous microspheres (PVA4) and the high crystalline microspheres (PVA25). However, the enzymatic activity of the immobilized enzymes was depended on the crystallinity degree of the PVA microspheres: enzymes immobilized on the PVA4 microspheres have shown low enzymatic activity (6.13 U mg −1 ), in comparison with enzymes immobilized on the high crystalline PVA25 microspheres (149.15 U mg −1 ). A synergistic effect was observed for the enzyme-PVA25 complex during the transesterification reaction of soybean oil to FAEE: transesterification reactions with free enzyme with the equivalent amount of enzyme that were immobilized onto the PVA25 microspheres (5.4 U) have yielded only 20% of FAEE, reactions with the pure highly crystalline microsphere PVA25 have not yielded FAEE, however reactions with the enzyme-PVA25 complexes have yielded 66.3% of FAEE. This synergistic effect of an immobilized enzyme on a polymeric support has not been observed before for transesterification reaction of triacylglycerides into FAEE. Based on ATR-FTIR, 23 Na- and 13 C-NMR-MAS spectroscopic data and the interaction of the polymeric network intermolecular hydrogen bonds with the lipases residual amino acids a possible explanation for this synergistic effect is provided. Highlights: • Rhizomucor miehei lipase was immobilized on PVA microspheres (PVA4, PVA12, PVA25). • Polymer-enzyme complex was characterized by XDR, SEM, ATR-FTIR, 13 C-CPMAS-NMR, 23 Na-MAS-NMR. • Polymer-enzymes (PVA12 and PVA25) enzymes yielded considerable amount of ethyl esters. • Synergistic effect was observed for the polymer-enzyme complexes

Study on the immobilization of alpha-amylase by radiation-induced polymerization at low-temperature, (2)

International Nuclear Information System (INIS)

Yoshida, Masaru; Kumakura, Minoru; Kaetsu, Isao

1975-07-01

The immobilization α-amylase in low concentration (50-250μg) by radiation induced polymerization at low temperature, with HEMA has been studied. The immobilization was performed in the temperature range of -196 0 C to +40 0 C. Activity of the immobilized enzyme decreases at temperatures above 0 0 C. The optimum temperatures for immobilization of α-amylase are -78 0 C - -24 0 C, where only the polymerization by irradiation is effective. HEMA is a suitable monomer as the immobilization carrier, because of its high polymerization rate of 100% in the temperature range. The suitable concentration of HEMA is less than 30%, and above this concentration the activity of enzyme decreases considerably. The optimum irradiation dose for immobilization is 1 x 10 6 R, and the activity of enzyme decreases at 5 x 10 6 R. The polymerization composition is porous gel structure, so the enzymatic reaction can be carried out merely by introducing a substrate to the composition. The activity attained in the immobilized enzyme is 75-80% that of the native α-amylase. The immobilized enzyme is more heat-resistant than the native one. (auth.)

Thrombin immobilization to methacrylic acid grafted poly(3-hydroxybutyrate) and its in vitro application.

Science.gov (United States)

Akkaya, Alper; Pazarlioglu, Nurdan

2013-01-01

Poly(3-hydroxybutyrate) is nontoxic and biodegradable, with good biocompatibility and potential support for long-term implants. For this reason, it is a good support for enzyme immobilization. Enzyme immobilization could not be done directly because poly(3-hydroxybutyrate) has no functional groups. Therefore, modification should be done for enzyme immobilization. In this study, methacrylic acid was graft polymerized to poly(3-hydroxybutyrate) and thrombin was immobilized to polymethacrylic acid grafted poly(3-hydroxybutyrate). In fact, graft polymerization of methacrylic acid to poly(3-hydroxybutyrate) and thrombin immobilization was a model study. Biomolecule immobilized poly(3-hydroxybutyrate) could be used as an implant. Thrombin was selected as a biomolecule for this model study and it was immobilized to methacrylic acid grafted poly(3-hydroxybutyrate). Then the developed product was used to stop bleeding.

Preparation and characterization of immobilized lipase on magnetic hydrophobic microspheres

DEFF Research Database (Denmark)

Guo, Zheng; Bai, Shu; Sun, Yan

2003-01-01

H for the immobilized CCL were determined. Activity amelioration of the immobilized CCL for the hydrolysis of olive oil was observed, indicating an interfacial activation of the enzyme after immobilization. Moreover, the immobilized CCL showed enhanced thermal stability and good durability in the repeated use after...

Bioaccessibility and inhibitory effects on digestive enzymes of carnosic acid in sage and rosemary.

Science.gov (United States)

Ercan, Pınar; El, Sedef Nehir

2018-04-28

In this study, the aim was to determine the bioaccessibilities of carnosic acid in sage and rosemary and in vitro inhibitory effects of these samples on lipid and starch digestive enzymes by evaluating the lipase, α-amylase and α-glucosidase enzyme inhibition activities. The content of carnosic acid in rosemary (18.72 ± 0.33 mg/g) was found to be higher than that content of that in sage (3.76 ± 0.13 mg/g) (p sage and rosemary, respectively. The tested sage and rosemary showed inhibitory activity against α-glucosidase (Concentration of inhibitor required to produce a 50% inhibition of the initial rate of reaction - IC 50 88.49 ± 2.35, 76.80 ± 1.68 μg/mL, respectively), α-amylase (IC 50 107.65 ± 12.64, 95.65 ± 2.73 μg/mL, respectively) and lipase (IC 50 6.20 ± 0.63, 4.31 ± 0.62 μg/mL, respectively). Furthermore, to the best of our knowledge, this is the first work that carnosic acid standard equivalent inhibition capacities (CAEIC 50 ) for these food samples were determined and these values were in agreement with the IC 50 values. These results show that sage and rosemary are potent inhibitors of lipase, α-amylase and α-glucosidase digestive enzymes. Copyright © 2018 Elsevier B.V. All rights reserved.

Covalent Immobilization of β-Glucosidase on Magnetic Particles for Lignocellulose Hydrolysis

DEFF Research Database (Denmark)

Alftrén, Johan; Hobley, Timothy John

2013-01-01

β-Glucosidase hydrolyzes cellobiose to glucose and is an important enzyme in the consortium used for hydrolysis of cellulosic and lignocellulosic feedstocks. In the present work, β-glucosidase was covalently immobilized on non-porous magnetic particles to enable re-use of the enzyme. It was found...... that particles activated with cyanuric chloride and polyglutaraldehyde gave the highest bead-related immobilized enzyme activity when tested with p-nitrophenyl-β-D-glucopyranoside (104.7 and 82.2 U/g particles, respectively). Furthermore, the purified β-glucosidase preparation from Megazyme gave higher bead......-related enzyme activities compared to Novozym 188 (79.0 and 9.8 U/g particles, respectively). A significant improvement in thermal stability was observed for immobilized enzyme compared to free enzyme; after 5 h (at 65 °C), 36 % of activity remained for the former, while there was no activity in the latter...

Immobilization of xanthine oxidase on a polyaniline silicone support.

Science.gov (United States)

Nadruz, W; Marques, E T; Azevedo, W M; Lima-Filho, J L; Carvalho, L B

1996-03-01

A polyaniline silicone support to immobilize xanthine oxidase is proposed as a reactor coil to monitor the action of xanthine oxidase on hypoxanthine, xanthine and 6-mercaptopurine. A purified xanthine oxidase immobilized on this support lost 80% of the initial activity after 12 min of use. Co-immobilization of superoxide dismutase and catalase increased the stability of immobilized xanthine oxidase so that the derivative maintained 79% of its initial activity after 4.6 h of continuous use in which 1.5 mumol purine bases were converted by the immobilized enzyme system. There is no evidence of either polyaniline or protein leaching from the coil during 3 h of continuous use. When solutions (10 ml) of hypoxanthine, xanthine and 6-mercaptopurine were circulated individually through the xanthine oxidase-superoxide dismutase-catalase-polyaniline coil (1 mm internal diameter and 3 m in length, 3 ml internal volume) activities of 8.12, 11.17 and 1.09 nmol min-1 coil-1, respectively, were obtained. The advantages of the reactor configuration and the redox properties of the polymer, particularly with respect to immobilized oxidoreductases, make this methodology attractive for similar enzyme systems. This immobilized enzyme system using polyaniline-silicone as support converted 6-mercaptopurine to 6-thiouric acid with equal efficiency as resins based on polyacrylamide and polyamide 11.

Elimination of methane in exhaust gas from biogas upgrading process by immobilized methane-oxidizing bacteria.

Science.gov (United States)

Wu, Ya-Min; Yang, Jing; Fan, Xiao-Lei; Fu, Shan-Fei; Sun, Meng-Ting; Guo, Rong-Bo

2017-05-01

Biogas upgrading is essential for the comprehensive utilization of biogas as substitute of natural gas. However, the methane in the biogas can be fully recovered during the upgrading process of biogas, and the exhaust gas produced during biogas upgrading may contain a very low concentration of methane. If the exhaust gas with low concentration methane releases to atmosphere, it will be harmful to environment. In addition, the utilization of large amounts of digestate produced from biogas plant is another important issue for the development of biogas industry. In this study, solid digestate was used to produce active carbon, which was subsequently used as immobilized material for methane-oxidizing bacteria (MOB) in biofilter. Biofilter with MOB immobilized on active carbon was used to eliminate the methane in exhaust gas from biogas upgrading process. Results showed porous active carbon was successfully made from solid digestate. The final methane elimination capacity of immobilized MOB reached about 13molh -1 m -3 , which was more 4 times higher than that of MOB without immobilization. Copyright © 2017 Elsevier Ltd. All rights reserved.

Dietary Immunogen® modulated digestive enzyme activity and immune gene expression in Litopenaeus vannamei post larvae.

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Miandare, Hamed Kolangi; Mirghaed, Ali Taheri; Hosseini, Marjan; Mazloumi, Nastaran; Zargar, Ashkan; Nazari, Sajad

2017-11-01

Pacific white shrimp Litopenaeus vannamei (Boone, 1931) is an important economical shrimp species worldwide, especially in the Middle East region, and farming activities of this species have been largely affected by diseases, mostly viral and bacterial diseases. Scientists have started to use prebiotics for bolstering the immune status of the animal. This study aimed to investigate the influence of Immunogen ® on growth, digestive enzyme activity and immune related gene expression of Litopenaeus vannamei post-larvae. All post-larvae were acclimated to the laboratory condition for 14 days. Upon acclimation, shrimps were fed on different levels of Immunogen ® (0, 0.5, 1 and 1.5 g kg -1 ) for 60 days. No significant differences were detected in weight gain, specific growth rate (SGR) and food conversion ratio (FCR) in shrimp post-larvae in which fed with different levels of Immunogen ® and control diet. The results showed that digestive enzymes activity including protease and lipase increased with different amounts of Immunogen ® in the shrimp diet. Protease activity increased with 1.5 g kg -1 Immunogen ® after 60 days and lipase activity increased with 1 and 1.5 g kg -1 Immunogen ® after 30 and 60 days of the trial respectively (P  0.05). The expression of immune related genes including, prophenoloxidase, crustin and g-type lysozyme increased with diet 1.5 g kg -1 Immunogen ® (P < 0.05) while expression of penaeidin gene increased only with experimental diet 1 g kg -1 of Immunogen ® . These results indicated that increase in digestive enzymes activity and expression of immune related genes could modulate the Immunogen ® in the innate immune system in L. vannamei in this study. Copyright © 2017. Published by Elsevier Ltd.

Immobilization of indigenous holocellulase on iron oxide (Fe2O3) nanoparticles enhanced hydrolysis of alkali pretreated paddy straw.

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Kumar, Ajay; Singh, Surender; Tiwari, Rameshwar; Goel, Renu; Nain, Lata

2017-03-01

The holocellulase from Aspergillus niger SH3 was characterized and found to contain 125 proteins including cellulases (26), hemicellulases (21), chitinases (10), esterases (6), amylases (4) and hypothetical protein (32). The crude enzyme was immobilized on five different nanoparticles (NPs) via physical adsorption and covalent coupling methods. The enzyme-nanoparticle complexes (ENC) were screened for protein binding, enzymatic activities and immobilization efficiency. Magnetic enzyme-nanoparticle complexes (MENC) showed higher immobilization efficiency (60-80%) for most of the enzymes. MENC also showed better catalytic efficiencies in term of higher V max and lower K m than free enzyme. Saccharification yields from alkali treated paddy straw were higher (375.39mg/gds) for covalently immobilized MENC than free enzyme (339.99mg/gds). The immobilized enzyme was used for two cycles of saccharification with 55% enzyme recovery. Hence, this study for the first time demonstrated the immobilization of indigenous enzyme and its utilization for saccharification of paddy straw. Copyright © 2016 Elsevier B.V. All rights reserved.

NOVEL APPLICATION OF POROUS AND CELLULAR MATERIALS FOR COVALENT IMMOBILIZATION OF PEPSIN

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K. Szałapata

Full Text Available Abstract Pepsin was immobilized via covalent bonds on different carriers: a silica gel carrier, acrylic beads, and a cellulose-based carrier - Granocel. All carriers were functionalized through the presence of -OH, -COOH, -NH2, or glycidyl groups on their surfaces. Three different cross-linkers were used for activation thereof. The results showed that Granocel activated by glutaraldehyde or carbodiimide and silica gel activated by glutaraldehyde were suitable carriers for the expression of enzyme activity. The optimum pH range for the native enzyme was 2.5-3.5 and this range was extended to the value 6.5 in the case of enzyme immobilized on the silica gel carrier and on Granocel. The optimum temperature values for the native and immobilized enzyme were in the range 37-40 °C and 40-50 °C, respectively. The activity of the immobilized pepsin at different values of pH and temperature was higher in comparison with the activity of the free enzyme.

Sodium bicarbonate-gelled chitosan beads as mechanically stable carriers for the covalent immobilization of enzymes.

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Wahba, Marwa I

2018-03-01

The poor mechanical stability of chitosan has long impeded its industrial utilization as an immobilization carrier. In this study, the mechanical properties of chitosan beads were greatly improved through utilizing the slow rate of the sodium bicarbonate-induced chitosan gelation and combining it with the chemical cross-linking action of glutaraldehyde (GA). The GA-treated sodium bicarbonate-gelled chitosan beads exhibited much better mechanical properties and up to 2.45-fold higher observed activity of the immobilized enzyme (β-D-galactosidase (β-gal)) when compared to the GA-treated sodium tripolyphosphate (TPP)-gelled chitosan beads. The differences between the sodium bicarbonate-gelled and the TPP-gelled chitosan beads were proven visually and also via scanning electron microscopy, elemental analysis, and differential scanning calorimetry. Moreover, the optimum pH, the optimum temperature, the apparent K m , and the apparent V max of the β-gals immobilized onto the two aforementioned types of chitosan beads were determined and compared. A reusability study was also performed. This study proved the superiority of the sodium bicarbonate-gelled chitosan beads as they retained 72.22 ± 4.57% of their initial observed activity during the 13 th reusability cycle whereas the TPP-gelled beads lost their activity during the first four reusability cycles, owing to their fragmentation. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:347-361, 2018. © 2017 American Institute of Chemical Engineers.

Optimization of Adsorptive Immobilization of Alcohol Dehydrogenases

NARCIS (Netherlands)

Trivedi, Archana; Heinemann, Matthias; Spiess, Antje C.; Daussmann, Thomas; Büchs, Jochen

2005-01-01

In this work, a systematic examination of various parameters of adsorptive immobilization of alcohol dehydrogenases (ADHs) on solid support is performed and the impact of these parameters on immobilization efficiency is studied. Depending on the source of the enzymes, these parameters differently

Covalent immobilization of lipase from Candida rugosa on Eupergit®

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Bezbradica Dejan I.

2005-01-01

Full Text Available An approach is presented for the stable covalent immobilization of Upase from Candida rugosa on Eupergit® with a high retention of hydrolytic activity. It comprises covalent bonding via lipase carbohydrate moiety previously modified by periodate oxidation, allowing a reduction in the involvement of the enzyme functional groups that are probably important in the catalytic mechanism. The hydrolytic activities of the lipase immobilized on Eupergif1 by two conventional methods (via oxirane group and via glutaralde-hyde and with periodate method were compared. Results of lipase assays suggest that periodate method is superior for lipase immobilization on Eupergit® among methods applied in this study with respect to both, yield of immobilization and hydrolytic activity of the immobilized enzyme.

Immobilization of α-amylase and amyloglucosidase onto ion-exchange resin beads and hydrolysis of natural starch at high concentration.

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Gupta, Kapish; Jana, Asim Kumar; Kumar, Sandeep; Maiti, Mithu

2013-11-01

α-Amylase was immobilized on Dowex MAC-3 with 88 % yield and amyloglucosidase on Amberlite IRA-400 ion-exchange resin beads with 54 % yield by adsorption process. Immobilized enzymes were characterized to measure the kinetic parameters and optimal operational parameters. Optimum substrate concentration and temperature were higher for immobilized enzymes. The thermal stability of the enzymes enhanced after the immobilization. Immobilized enzymes were used in the hydrolysis of the natural starch at high concentration (35 % w/v). The time required for liquefaction of starch to 10 dextrose equivalent (DE) and saccharification of liquefied starch to 96 DE increased. Immobilized enzymes showed the potential for use in starch hydrolysis as done in industry.

Immobilization of glucoamylase on ceramic membrane surfaces modified with a new method of treatment utilizing SPCP-CVD.

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Ida; Matsuyama; Yamamoto

2000-07-01

Glucoamylase, as a model enzyme, was immobilized on a ceramic membrane modified by surface corona discharge induced plasma chemical process-chemical vapor deposition (SPCP-CVD). Characterizations of the immobilized enzyme were then discussed. Three kinds of ceramic membranes with different amounts of amino groups on the surface were prepared utilizing the SPCP-CVD method. Each with 1-time, 3-times and 5-times surface modification treatments and used for supports in glucoamylase immobilization. The amount of immobilized glucoamylase increased with the increase in the number of surface modification treatments and saturated to a certain maximum value estimated by a two-dimensional random packing. The operational stability of the immobilized glucoamylase also increased with the increase in the number of the surface treatment. It was almost the same as the conventional method, while the activity of immobilized enzyme was higher. The results indicated the possibility of designing the performance of the immobilized enzyme by controlling the amount of amino groups. The above results showed that the completely new surface modification method using SPCP was effective in modifying ceramic membranes for enzyme immobilization.

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

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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 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.

Immobilization/Stabilization of Ficin Extract on Glutaraldehyde-Activated Agarose Beads. Variables That Control the Final Stability and Activity in Protein Hydrolyses

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El-Hocine Siar

2018-04-01

Full Text Available Ficin extract has been immobilized on different 4% aminated-agarose beads. Using just ion exchange, immobilization yield was poor and expressed activity did not surpass 10% of the offered enzyme, with no significant effects on enzyme stability. The treatment with glutaraldehyde of this ionically exchanged enzyme produced an almost full enzyme inactivation. Using aminated supports activated with glutaraldehyde, immobilization was optimal at pH 7 (at pH 5 immobilization yield was 80%, while at pH 9, the immobilized enzyme became inactivated. At pH 7, full immobilization was accomplished maintaining 40% activity versus a small synthetic substrate and 30% versus casein. Ficin stabilization upon immobilization could be observed but it depended on the inactivation pH and the substrate employed, suggesting the complexity of the mechanism of inactivation of the immobilized enzyme. The maximum enzyme loading on the support was determined to be around 70 mg/g. The loading has no significant effect on the enzyme stability or enzyme activity using the synthetic substrate but it had a significant effect on the activity using casein; the biocatalysts activity greatly decreased using more than 30 mg/g, suggesting that the near presence of other immobilized enzyme molecules may generate some steric hindrances for the casein hydrolysis.

Immobilization of glucose oxidase on sepharose by UV-initiated graft copolymerization

International Nuclear Information System (INIS)

D'Angiuro, L.; Cremonesi, P.

1982-01-01

The performance of a new method of enzyme immobilization based on photochemically initiated direct graft copolymerization was recently investigated. The immobilization reaction can be carried out in a simple way and by carefully selecting the reaction conditions, the enzyme-graft copolymer can be obtained as the main reaction product. Coupling efficiency of glucose oxidase has been found to depend only on the amount of photocatalyst (FeCl 3 ) fixed on Sepharose used as polysaccharide support. Small quantities of glycidylmethacrylate (GMA) (0.25 g/g dry Sepharose) are sufficient but necessary to achieve the best enzyme coupling efficiency (20-40%). Enzyme immobilization occurs very rapidly and the entire reaction occurs within 60 min. Reaction patterns and physicochemical characteristics of the obtained enzyme-graft copolymers exclude the glucose oxidase entrapment: therefore a covalent attachment mechanism may be proposed. The kinetic parameters of immobilized glucose oxidase (K/sub m/' = 2.0 x 10 -2 M) are quite similar to those of free enzyme (K/sub m/ = 1.93 x 10 -2 M), and no diffusion limitation phenomena are evidenced in samples having different enzyme or polymer content. Lyophilization, thermostability, and long-term continuous operation also have been investigated. The advantages of this method over that using vinylenzyme copolymerization are discussed

The effect of aerobic exercise training on growth performance, digestive enzyme activities and postprandial metabolic response in juvenile qingbo (Spinibarbus sinensis).

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Li, Xiu-Ming; Yu, Li-Juan; Wang, Chuan; Zeng, Ling-Qing; Cao, Zhen-Dong; Fu, Shi-Jian; Zhang, Yao-Guang

2013-09-01

Continual swimming exercise usually promotes growth in fish at a moderate water velocity. We hypothesized that the improvement in growth in exercise-trained fish may be accompanied by increases in digestive enzyme activity, respiratory capacity and, hence, postprandial metabolism. Juvenile qingbo fish (Spinibarbus sinensis) were subjected to aerobic training for 8weeks at a water velocity of control (3cms(-1)), 1, 2 and 4 body length (bl)s(-1) at a constant temperature of 25°C. The feed intake (FI), food conversion rate (FCR), specific growth rate (SGR), whole-body composition, trypsin and lipase activities, maximal oxygen consumption (M˙O2max) and postprandial M˙O2 response were measured at the end of the training period. Aerobic exercise training induced a significant increase in FI compared with the control group, while the FCR of the 4bls(-1) group was significantly lower than for the other three groups (PFI after long-term training; (3) and aerobic exercise training boosted the activity of digestive enzymes and maximum digestive metabolism, which could favor fast digestion and growth in juvenile S. sinensis. Copyright © 2013 Elsevier Inc. All rights reserved.

Stabilization of penicillin G acylase by immobilization on glutaraldehyde-activated chitosan

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W. S. Adriano

2005-12-01

Full Text Available The objective of this work was to study enzyme immobilization on chitosan activated with glutaraldehyde, aiming to produce a cheap biocatalyst. Two different immobilization strategies were studied: one-point and multipoint covalent attachment to the solid matrix. The multipoint covalent attachment derivative had an 82% immobilization yield. It was 4.9-fold more stable than the free enzyme at 50°C and 4.5-fold more stable than soluble enzyme at pH 10.0. The one-point derivative had an 85% immobilization yield. It was 2.7-fold more stable than the free enzyme at 50°C and 3.8-fold more stable than soluble PGA at pH 10.0. Results indicated that chitosan can be loaded with PGA above 330 IU/g. Intraparticle diffusive effects, however, limited hydrolysis of penicillin G catalyzed by those derivatives at 37°C and 25°C. Operational stability assays were performed and the multipoint derivative exhibited a half-life of 40 hours.

A simple enzyme assay for dry matter digestibility and its value in studying food selection by generalist herbivores.

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Choo, Gillian M; Waterman, Peter G; McKey, Doyle B; Gartlan, J Stephen

1981-05-01

The dry matter digestibility of 94 species of leaf was assayed by a simple method involving sequential treatment with pepsin and fungal cellulase enzymes. It was demonstrated that for foliage from rainforest trees of a wide range of dicotyledonous plant families the assay showed high positive correlation with estimates of dry matter digestibility obtained using rumenliquor from a fistulated steer. Both assays were found to reflect negative correlates of digestibility, notably fibre and condensed tannin, rather than the nutritional value of an item. The higher dry matter digestibility of immature leaves relative to mature leaves appeared to be accounted for by their lower fibre content. It is suggested that the pepsin/cellulase assay offers a cheap, quick, routine method of gaining information on the effects of some types of plant secondary compounds (digestibility reducers) on the 'food potential' of different kinds of foliage to herbivores. Its use in studies of herbivory in rainforest areas in relation to analyses for plant secondary compounds and food selection by herbivores is discussed.

The effect of chaya (Cnidoscolus aconitifolius) leaf meal and of exogenous enzymes on amino acid digestibility in broilers.

Science.gov (United States)

Sarmiento-Franco, L; McNab, J M; Pearson, A; Belmar-Casso, R

2003-07-01

1. The apparent ileal nitrogen (N) and amino acid digestibilities in chaya leaf meal (CLM) (Cnidoscolus aconitifolius) with added enzymes, and the same variables in diets containing different amounts of CLM were studied in chickens. 2. In the first experiment pectinase, beta-glucanase, and pectinase + beta-glucanase were added to CLM. In the second experiment, there were three diets based on maize and soybean: 0, 150 and 250 g/kg CLM. 3. Pectinase significantly increased both lysine and overall amino acid digestibilities in CLM. 4. In experiment 2, the amino acid digestibility in birds fed on CLM250 was lower than that from birds fed on either control or CLM150. Only the digestibilities of alanine, arginine and proline were lower in birds fed on CLM150 than in those fed on the control diet. Nitrogen digestibility was lower in birds fed on the CLM250 diet than on either control or CLM150 diets. These findings were attributed to the increasing concentration of fibre with increasing dietary CLM.

Effects of dietary fish oil replacement by vegetable oil on the digestive enzymes activity and intestinal morphology in Meagre, Argyrosomus regius (Asso, 1801

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Fernando Antunes Magalhães

2014-07-01

The results were analyzed by three way factorial. Amylase activity was bigger in FO when compared with VO (Table 1. The same result was observed in chymotrypsin activity. On the other hand, lipase activity was higher in VO. Regarding the levels of lipids, diets with 17% had higher amylase activity than diets with 12%. The inverse was observed in chymotrypsin activity. In relation to lipase activity, no differences were observed on the two levels of lipids studied. No differences in digestive enzymes activities were observed when diets were supplemented with selenium. Epithelium architecture of the posterior intestine was slightly affected by dietary treatments. Higher levels of lipids seem to induce enterocyte vacuolization, and vacuoles seem to be larger when a blend of vegetable oils was used instead of fish oil. No clear role can be attributed to selenium regarding intestinal morphology. In conclusion, our study showed that the source and levels of lipid in diets for meagre have influence in activity of digestible enzymes like amylase, lipase and chymotrypsin. Furthermore, levels of selenium do not cause an alteration in studied digestible enzymes.

Novel immobilization process of a thermophilic catalase: efficient purification by heat treatment and subsequent immobilization at high temperature.

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Xu, Juan; Luo, Hui; López, Claudia; Xiao, Jing; Chang, Yanhong

2015-10-01

The main goal of the present work is to investigate a novel process of purification and immobilization of a thermophilic catalase at high temperatures. The catalase, originated from Bacillus sp., was overexpressed in a recombinant Escherichia coli BL21(DE3)/pET28-CATHis and efficiently purified by heat treatment, achieving a threefold purification. The purified catalase was then immobilized onto an epoxy support at different temperatures (25, 40, and 55 °C). The immobilizate obtained at higher temperatures reached its maximum activity in a shorter time than that obtained at lower temperatures. Furthermore, immobilization at higher temperatures required a lower ionic strength than immobilization at lower temperatures. The characteristics of immobilized enzymes prepared at different temperatures were investigated. The high-temperature immobilizate (55 °C) showed the highest thermal stability, followed by the 40 °C immobilizate. And the high-temperature immobilizate (55 °C) had slightly higher operational stability than the 25 °C immobilizate. All of the immobilized catalase preparations showed higher stability than the free enzyme at alkaline pH 10.0, while the alkali resistance of the 25 °C immobilizate was slightly better than that of the 40 and 55 °C immobilizates.

One-year monitoring of core biomarker and digestive enzyme responses in transplanted zebra mussels (Dreissena polymorpha).

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Palais, F; Dedourge-Geffard, O; Beaudon, A; Pain-Devin, S; Trapp, J; Geffard, O; Noury, P; Gourlay-Francé, C; Uher, E; Mouneyrac, C; Biagianti-Risbourg, S; Geffard, A

2012-04-01

A 12-month active biomonitoring study was performed in 2008-2009 on the Vesle river basin (Champagne-Ardenne, France) using the freshwater mussel Dreissena polymorpha as a sentinel species; allochthonous mussels originating from a reference site (Commercy) were exposed at four sites (Bouy, Sept-Saulx, Fismes, Ardre) within the Vesle river basin. Selected core biomarkers (acetylcholinesterase (AChE) activity, glutathione-S transferase (GST) activity, metallothionein concentration), along with digestive enzyme activities (amylase, endocellulase) and energy reserve concentrations (glycogen, lipids), were monitored throughout the study in exposed mussels. At the Fismes and Ardre sites (downstream basin), metallic and organic contamination levels were low but still high enough to elicit AChE and GST activity induction in exposed mussels (chemical stress); besides, chemical pollutants had no apparent deleterious effects on mussel condition. At the Bouy and Sept-Saulx sites (upstream basin), mussels obviously suffered from adverse food conditions which seriously impaired individual physiological state and survival (nutritional stress); food scarcity had however no apparent effects on core biomarker responses. Digestive enzyme activities responded to both chemical and nutritional stresses, the increase in energy outputs (general adaptation syndrome-downstream sites) or the decrease in energy inputs (food scarcity-upstream sites) leading to mid- or long-term induction of digestive carbohydrase activities in exposed mussels (energy optimizing strategy). Complex regulation patterns of these activities require nevertheless the use of a multi-marker approach to allow data interpretation. Besides, their sensitivity to natural confounding environmental factors remains to be precised.

Silver nanoparticle (AgNPs) doped gum acacia-gelatin-silica nanohybrid: an effective support for diastase immobilization.

Science.gov (United States)

Singh, Vandana; Ahmed, Shakeel

2012-03-01

An effective carrier matrix for diastase alpha amylase immobilization has been fabricated by gum acacia-gelatin dual templated polymerization of tetramethoxysilane. Silver nanoparticle (AgNp) doping to this hybrid could significantly enhance the shelf life of the impregnated enzyme while retaining its full bio-catalytic activity. The doped nanohybrid has been characterized as a thermally stable porous material which also showed multipeak photoluminescence under UV excitation. The immobilized diastase alpha amylase has been used to optimize the conditions for soluble starch hydrolysis in comparison to the free enzyme. The optimum pH for both immobilized and free enzyme hydrolysis was found to be same (pH=5), indicating that the immobilization made no major change in enzyme conformation. The immobilized enzyme showed good performance in wide temperature range (from 303 to 323 K), 323 K being the optimum value. The kinetic parameters for the immobilized, (K(m)=10.30 mg/mL, V(max)=4.36 μmol mL(-1)min(-1)) and free enzyme (K(m)=8.85 mg/mL, V(max)=2.81 μmol mL(-1)min(-1)) indicated that the immobilization improved the overall stability and catalytic property of the enzyme. The immobilized enzyme remained usable for repeated cycles and did not lose its activity even after 30 days storage at 40°C, while identically synthesized and stored silver undoped hybrid lost its ~31% activity in 48 h. Present study revealed the hybrids to be potentially useful for biomedical and optical applications. Copyright © 2011 Elsevier B.V. All rights reserved.

Effect of dietary genistein on growth performance, digestive enzyme activity, and body composition of Nile tilapia Oreochromis niloticus

Science.gov (United States)

Chen, Dong; Wang, Wei; Ru, Shaoguo

2015-01-01

An 8-week feeding experiment was performed to evaluate the effect of dietary genistein on growth performance, body composition, and digestive enzymes activity of juvenile Nile tilapia ( Oreochromis niloticus). Four isonitrogenous and isoenergetic diets were formulated containing four graded supplements of genistein: 0, 30, 300, and 3 000 μg/g. Each diet was randomly assigned in triplicate to tanks stocked with 15 juvenile tilapia (10.47±1.24 g). The results show that 30 and 300 μg/g dietary genistein had no significant effect on growth performance of Nile tilapia, but the higher level of genistein (3 000 μg/g) significantly depressed the final body weight and specific growth rate. There was no significant difference in survival rate, feed intake, feed efficiency ratio or whole body composition among all dietary treatments. An assay of digestive enzymes showed that the diet containing 3 000 μg/ggenistein decreased stomach and hepatopancreas protease activity, and amylase activity in the liver and intestine, while a dietary level of 300 μg/g genistein depressed stomach protease and intestine amylase activities. However, no significant difference in stomach amylase activity was found among dietary treatments. Overall, the results of the present study indicate that a high level of dietary genistein (3 000 μg/g, or above) would significantly reduce the growth of Nile tilapia, partly because of its inhibitory effect on the activity of major digestive enzymes. Accordingly, the detrimental effects of genistein, as found in soybean products, should not be ignored when applied as an alternative ingredient source in aquaculture.

Covalent immobilization of α-amylase on magnetic particles as catalyst for hydrolysis of high-amylose starch.

Science.gov (United States)

Guo, Hui; Tang, Yi; Yu, Yang; Xue, Lu; Qian, Jun-Qing

2016-06-01

Enzyme immobilized on magnetic particles can be used as efficient recoverable biocatalysts under strong magnetic response. To enable re-use of enzyme, modified Fe3O4 particles were used as carrier to immobilize α-amylase in this paper. Firstly, the surface of Fe3O4 particles were coated with amino groups by direct using TEOS (tetraethoxysilane) followed by treatment with APTES (3-aminopropyltriethoxysilane) and then carboxylated by reacting it with succinic anhydride. In addition, the effect of the immobilization condition on enzyme activity recovery and immobilization efficiency were investigated. The results showed that the optimal immobilization occurred under following conditions: pH 5.5, 40°C, enzyme concentration of 20mgmL(-1), reaction time for 36h. Using immobilized α-amylase as biocatalyst, the optimum pH and temperature for hydrolysis were observed to be 6.5 and 60°C. The kinetics of hydrolysis reaction were studied using Michaelis-Menten equation. The affinity constant (Km) and maximum reaction rate (vmax) of magnetic particles immobilization α-amylase (MPIA) was 0.543mgmL(-1) and 1.321mgmin(-1) compared to those of 0.377mgmL(-1) and 6.859mgmin(-1) of free enzyme. After immobilization, enzymatic activity, storage stability, thermo-stability, and reusability of MPIA were found superior to those of the free one. MPIA maintained 86% enzyme activity after 30 days and maintained 78% enzyme activity after recycling six times. Copyright © 2016 Elsevier B.V. All rights reserved.

Expression of an antimicrobial peptide, digestive enzymes and nutrient transporters in the intestine of E. praecox-infected chickens

Science.gov (United States)

Coccidiosis is a major intestinal disease of poultry, caused by several species of the protozoan Eimeria. The objective of this study was to examine changes in expression of digestive enzymes, nutrient transporters and an antimicrobial peptide following an Eimeria praecox challenge of chickens at d...

Effects of traditional Chinese medicine formula on ruminal fermentation, enzyme activities and nutrient digestibility of beef cattle.

Science.gov (United States)

Zhu, Zhi; Song, Zhen-Hui; Cao, Li-Ting; Wang, Yong; Zhou, Wen-Zhang; Zhou, Pei; Zuo, Fu-Yuan

2018-04-01

This study was conducted to evaluate effects of traditional Chinese medicine formula (TCMF) combined with several herbs on ruminal fermentation, enzyme activities and nutrient digestibility. Twenty finishing bulls were assigned to control or different TCMFs (Yufeisan-1, -2, -3; 2.5% dry matter (DM) in concentrate). Results showed that DM intake was higher (P < 0.05) in the Yufeisan-3 group than others. Compared to control, apparent digestibility of crude protein and neutral detergent fiber were increased (P < 0.05) by Yufeisan-3. No changes were observed in ruminal pH, concentrations of ammonia-N, microbial crude protein and total volatile fatty acid, whereas ratio of acetate to propionate was lower (P < 0.05) and propionate proportion tended to be higher (P < 0.1) in three TCMFs than control. Ruminal xylanase (P = 0.061) and carboxymethylcellulase (P < 0.05) activities were higher in Yufeisan-3 than control. No changes were observed in abundance of total bacteria, fungi and protozoa, whereas Fibrobacter succinogenes (P = 0.062) and Ruminococcus flavefaciens (P < 0.05) were increased and total methanogens was reduced (P = 0.069) by Yufeisan-3 compared to control. Yufeisan-3 improved nutrient digestibility and ruminal enzyme activity, and modified fermentation and microbial community, maybe due to the presence of Herba agastaches, Cortex phellodendri and Gypsum fibrosum. © 2018 Japanese Society of Animal Science.

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

Science.gov (United States)

Güleç, Hacı Ali

2013-04-01

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

Radiation-induced polymerization for the immobilization of penicillin acylase

International Nuclear Information System (INIS)

Boccu, E.; Carenza, M.; Lora, S.; Palma, G.; Veronese, F.M.

1987-01-01

The immobilization of Escherichia coli penicillin acylase was investigated by radiation-induced polymerization of 2-hydroxyethyl methacrylate at low temperature. A leak-proof composite that does not swell in water was obtained by adding the cross-linking agent trimethylolpropane trimethacrylate to the monomer-aqueous enzyme mixture. Penicillin acylase, which was immobilized with greater than 70% yield, possessed a higher Km value toward the substrate 6-nitro-3-phenylacetamidobenzoic acid than the free enzyme form (Km = 1.7 X 10(-5) and 1 X 10(-5) M, respectively). The structural stability of immobilized penicillin acylase, as assessed by heat, guanidinium chloride, and pH denaturation profiles, was very similar to that of the free-enzyme form, thus suggesting that penicillin acylase was entrapped in its native state into aqueous free spaces of the polymer matrix

Immobilization of microbial cells on cellulose-polymer surfaces by radiation polymerization

International Nuclear Information System (INIS)

Kumakura, M.; Kaetsu, I.

1983-01-01

Streptomyces phaeochromogens cells were immobilized on cellulose-polymer surfaces by radiation polymerization using hydrophilic monomers and paper. The enzyme activity of immobilized cell sheets was higher than that of immobilized cell composites obtained by the usual radiation polymerization technique. The enzyme activity of the sheets was affected by monomer concentration, the thickness of paper, and the degree of polymerization of paper. The copolymerization of hydroxyethyl methacrylate and methoxytetraethyleneglycol methacrylate in the sheets led to a further increase of the enzyme activity due to the increase of the hydrophilicity of the polymer matrix. The Michaelis constant of the sheets from low monomer concentration was close to that of intact cells

Alginate Immobilization of Metabolic Enzymes (AIME) for High ...

Science.gov (United States)

Alginate Immobilization of Metabolic Enzymes (AIME) for High-Throughput Screening Assays DE DeGroot, RS Thomas, and SO SimmonsNational Center for Computational Toxicology, US EPA, Research Triangle Park, NC USAThe EPA’s ToxCast program utilizes a wide variety of high-throughput screening (HTS) assays to assess chemical perturbations of molecular and cellular endpoints. A key criticism of using HTS assays for toxicity assessment is the lack of xenobiotic metabolism (XM) which precludes both metabolic detoxification as well as bioactivation of chemicals tested in vitro thereby mischaracterizing the potential risk posed by these chemicals. To address this deficiency, we have developed an extracellular platform to retrofit existing HTS assays with XM activity. This platform utilizes the S9 fraction of liver homogenate encapsulated in an alginate gel network which reduces the cytotoxicity caused by direct addition of S9 to cells in culture. Alginate microspheres containing encapsulated human liver S9 were cross-linked to solid supports extending from a 96-well plate lid and were assayed using a pro-luciferin substrate specific for CYP3A4 (IPA). We demonstrate that S9 was successfully encapsulated and remained enzymatically active post-encapsulation with 5-10X the CYP3A4 activity as compared to 1 µg solubilized human liver S9. Ketoconazole, a known inhibitor of human CYP3A4, inhibited CYP3A4 activity in a concentration-dependent manner (IC50: 0.27 µM) and inhibiti

Immobilization of Glycoside Hydrolase Families GH1, GH13, and GH70: State of the Art and Perspectives

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Natália G. Graebin

2016-08-01

Full Text Available Glycoside hydrolases (GH are enzymes capable to hydrolyze the glycosidic bond between two carbohydrates or even between a carbohydrate and a non-carbohydrate moiety. Because of the increasing interest for industrial applications of these enzymes, the immobilization of GH has become an important development in order to improve its activity, stability, as well as the possibility of its reuse in batch reactions and in continuous processes. In this review, we focus on the broad aspects of immobilization of enzymes from the specific GH families. A brief introduction on methods of enzyme immobilization is presented, discussing some advantages and drawbacks of this technology. We then review the state of the art of enzyme immobilization of families GH1, GH13, and GH70, with special attention on the enzymes β-glucosidase, α-amylase, cyclodextrin glycosyltransferase, and dextransucrase. In each case, the immobilization protocols are evaluated considering their positive and negative aspects. Finally, the perspectives on new immobilization methods are briefly presented.

Radiation technology for immobilization of bioactive materials

International Nuclear Information System (INIS)

1988-12-01

Within the framework of the Agency's coordinated research programme on ''Application of Radiation Technology in Immobilization of Bioactive Materials'', the third and final research coordination meeting was held at Beijing University, Beijing, People's Republic of China, 15-18 June 1987. The present publication compiles all presentations made at the meeting. Fundamental processes for the immobilization of enzymes, antibodies, cells and drugs were developed and established using gamma radiation, electron beams and plasma discharge. Applications of various biofunctional components, immobilized by radiation techniques in different processes, were studied. A range of backbone polymers has been examined together with various monomers. Coupling procedures have been developed which are relevant to our particular requirements. Enzymes of various types and characteristics have been immobilized with considerable efficiency. The immobilized biocatalysts have been shown to possess significant activity and retention of activity on storage. There appears to be a high degree of specificity associated with the properties of the immobilised biocatalysts, their activity and the ease of their preparation. Novel additives which lower the total radiation dose in grafting have been discovered and their value in immobilization processes assessed. Potential applications include: medical (diagnostic, therapeutic), and industrial processes (fermentation, bioseparation, etc.). Refs, figs and tabs

Digestive enzyme activities are higher in the shortfin mako shark, Isurus oxyrinchus, than in ectothermic sharks as a result of visceral endothermy.

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Newton, Kyle C; Wraith, James; Dickson, Kathryn A

2015-08-01

Lamnid sharks are regionally endothermic fishes that maintain visceral temperatures elevated above the ambient water temperature. Visceral endothermy is thought to increase rates of digestion and food processing and allow thermal niche expansion. We tested the hypothesis that, at in vivo temperatures, the endothermic shortfin mako shark, Isurus oxyrinchus, has higher specific activities of three digestive enzymes-gastric pepsin and pancreatic trypsin and lipase-than the thresher shark, Alopias vulpinus, and the blue shark, Prionace glauca, neither of which can maintain elevated visceral temperatures. Homogenized stomach or pancreas tissue obtained from sharks collected by pelagic longline was incubated at both 15 and 25 °C, at saturating substrate concentrations, to quantify tissue enzymatic activity. The mako had significantly higher enzyme activities at 25 °C than did the thresher and blue sharks at 15 °C. This difference was not a simple temperature effect, because at 25 °C the mako had higher trypsin activity than the blue shark and higher activities for all enzymes than the thresher shark. We also hypothesized that the thermal coefficient, or Q 10 value, would be higher for the mako shark than for the thresher and blue sharks because of its more stable visceral temperature. However, the mako and thresher sharks had similar Q 10 values for all enzymes, perhaps because of their closer phylogenetic relationship. The higher in vivo digestive enzyme activities in the mako shark should result in higher rates of food processing and may represent a selective advantage of regional visceral endothermy.

Vibratory Reaction Unit for the Rapid Analysis of Proteins and Glycochains

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Yukie Sasakura

2007-01-01

Full Text Available A protein digestion system using immobilized enzymes for protein identification and glycochain analyses has been developed, and a vibration reaction unit for micro-scale sample convection on an enzyme-immobilized solid surface was constructed. BSA as a model substrate was digested by this unit, and was successfully identified by mass spectrometry (MS analyses. Compared to the conventional liquid-phase digestion, the reaction unit increased the number of matched peptides from 9 to 26, protein score from 455 to 1247, and sequence coverage from 21% to 48%. Glycopeptidase F (NGF, an enzyme that cleaves N-glycans from glycoproteins, was also immobilized and used to remove the glycochains from human immunoglobulin G (IgG. Trypsin and NGF were immobilized on the same solid surface and used to remove glycochains from IgG in single-step. Glycochains were labeled with fluorescent reagent and analyzed by HPLC. Several peaks corresponding to the glycochains of IgG were detected. These results suggested that the single-step digestion system, by immobilized multiple enzymes (trypsin and NGF would be effective for the rapid structural analysis of glycoproteins.Abbreviations: BSA: bovine serum albumin; MS: mass spectrometry; NGF: glycopeptidase F; IgG: immunoglobulin G; PTM: post-translational modification; HPLC: high-performance liquid chromatography; PBS: phosphate-buffered saline; EDTA: ethylenediaminetetraacetic acid; DTT: dithiothreitol; RT: retention time; ABOE: p-aminobenzoic acid octyl ester; PDMS: polydimethylsiloxane; ArgC: endoprotease Arginine C.

Immobilization of oxidases and their analytical applications

International Nuclear Information System (INIS)

Yasinzai, M.

2007-01-01

Immobilized enzymes are replacing their soluble counter-parts in nearly every field of application. These enzyme modifications have evolved from a research curiosity into an entire branch of Biotechnology. An immobilization method for flavin containing oxidases and their use in flow injection system is described. An electrochemical detector for H/sub 2/O/sub 2/ is assembled which is used effectively for the determination of glucose using more common glucose oxidase and the simultaneous determination of sugars. The combination of oxidases with hydrolases have been used for the determination of maltose and starch. (author)

The effect of rolled barley, sodium hydroxide-treated wheat or maize cob silage on digestive enzymes activity in the alimentary tract of dairy cows

DEFF Research Database (Denmark)

Moharrey, A.; Hymøller, Lone; Weisbjerg, Martin Riis

2017-01-01

In the present study digestive enzyme activities were studied in the rumen, intestine and faeces of dairy cows fed rations differing in starch source. Three total mixed rations were prepared for dairy cows with maize cob silage (MCS), sodium hydroxide-treated wheat (SHW) or rolled barley as starch...... DM (2.61 vs 2.91 and 3.15%) and a higher ash content (30.99 vs 29.24 and 24.31%) in the ruminal fluid without affecting enzyme activities. Positive correlation between lipolytic and amylolitic activities in ruminal fluid was stated, which supported the hypothesis that amylolytic bacteria provide...... energy for lipolytic bacteria. So, the enzymes activities in the different parts of the digestive tract were not affected by the different starch sources....

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

Science.gov (United States)

Torabizadeh, Homa; Mahmoudi, Asieh

2018-03-01

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

Amperometric Determination of Glucose at Parts per Million Levels with Immobilized Glucose Oxidase.

Science.gov (United States)

Sittampalam, G.; Wilson, G. S.

1982-01-01

An experiment on the operation and utility of an amperometric immobilized enzyme electrode (or probe) is described, including advantages of the experiment, equipment, reagents, preparation of phosphate buffer, enzyme immobilization techniques, laboratory procedures, precautions, and discussion of experimental results. (SK)

A novel and efficient method for the immobilization of thermolysin using sodium chloride salting-in and consecutive microwave irradiation.

Science.gov (United States)

Chen, Feifei; Zhang, Fangkai; Du, Fangchuan; Wang, Anming; Gao, Weifang; Wang, Qiuyan; Yin, Xiaopu; Xie, Tian

2012-07-01

Sodium chloride salting-in and microwave irradiation were combined to drive thermolysin molecules into mesoporous support to obtain efficiently immobilized enzyme. When the concentration of sodium chloride was 3 M and microwave power was 40 W, 93.2% of the enzyme was coupled to the support by 3 min, and the maximum specific activity of the immobilized enzyme was 17,925.1 U mg(-1). This was a 4.5-fold increase in activity versus enzyme immobilized using conventional techniques, and a 1.6-fold increase versus free enzyme. Additionally, the thermal stability of the immobilized thermolysin was significantly improved. When incubated at 70°C, there was no reduction in activity by 3.5h, whereas free thermolysin lost most of its activity by 3h. Immobilization also protected the thermolysin against organic solvent denaturation. The microwave-assisted immobilization technique, combined with sodium chloride salting-in, could be applied to other sparsely soluble enzymes immobilization because of its simplicity and high efficiency. Copyright © 2011 Elsevier Ltd. All rights reserved.

Protein-based inverse opals: A novel support for enzyme immobilization.

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Jiang, Yanjun; Sun, Wenya; Wang, Yaping; Wang, Lihui; Zhou, Liya; Gao, Jing; He, Ying; Ma, Li; Zhang, Xu

2017-01-01

In this study, protein-based inverse opals were prepared for the first time by using the colloidal crystal templating method. The preparation process involved three steps including filling the templates with protein molecules, crosslinking, and template removal. The obtained inverse opals were used to immobilize Penicillin G acylase (PGA) because of its intrinsic biocompatible property. The immobilization process was optimized and the properties of the immobilized PGA (PGA@IO) were investigated. PGA@IO exhibited improved thermal and pH stability compared with its free counterpart. After reusing nine times, it retained 70% of the initial activity. Besides, the PGA@IO retained high activity during the hydrolysis reactions in continuous catalysis in packed-bed reactor (PBR) after 15 days. Copyright © 2016 Elsevier Inc. All rights reserved.

Immobilization of cellulase on functionalized cobalt ferrite nanoparticles

International Nuclear Information System (INIS)

Bohara, Raghvendra Ashok; Thorat, Nanasaheb Devappa; Pawar, Shivaji Hariba

2016-01-01

Amine functionalized cobalt ferrite (AF-CoFe 2 O 4 ) magnetic nanoparticles (MNPs) were used for immobilization of cellulase enzyme via 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDS) and N-hydroxysuccinimide (NHS) coupling reaction. The structural, morphological and magnetic properties of AF-CoFe 2 O 4 were determined. TEM micrograph revealed a mean diameter of -8 nm and showed that the AF-CoFe 2 O 4 remain distinct with no significant change in size after binding with cellulase. Fourier transform infrared (FT-IR) spectroscopy confirmed the binding of cellulase to AF-CoFe 2 O 4 . The properties of immobilized cellulase were investigated by optimizing binding efficiency, pH, temperature and reusability. The results showed that the immobilized cellulase has higher thermal stability than free cellulase, which might be due to covalent interaction between cellulase and AF-CoFe 2 O 4 surface. The immobilized cellulase also showed good reusability after recovery. Therefore, AF-CoFe 2 O 4 MNPs can be considered as promising candidate for enzyme immobilization.

MUCOADHESIVE GEL WITH IMMOBILIZED LYSOZYME: PREPARATION AND PROPERTIES

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Dekina S. S.

2015-08-01

Full Text Available The study of non-covalent immobilized lysozyme, as well as physico-chemical and biochemical properties of obtained mucoadhesive gel was the aim of the research. Lysozyme activity was determined by bacteriolytic method (Micrococcus lysodeikticus cells acetone powder was a substrate. Lysozyme immobilization was conducted by the method of entrapment in gel. Enzyme carrier interaction was studied by viscometric, spectrophotometric and spectrofluorimetric methods. Mucoadhesive gel with immobilized lysozyme, possessing antiinflammatory and antimicrobial activities, was prepared. Due to immobilization, protein-polymer complex with the original enzymatic activity was formed. The product is characterized by high mucoadhesive properties, quantitative retaining of protein and bacteriolytic activity, prolonged release of the enzyme, improved biochemical characteristics (extended pH-activity profile, stability in acidic medium and during storage for 2 years, and it is perspective for further studies. The proposed method for lysozyme immobilization in the carboxymethyl cellulose sodium salt gel allows to obtain a stable, highly efficient product, with high adhesive properties for attachment to the mucous membranes, that is promising for use in biomedicine.

Effects of Hanseniaspora opuntiae C21 on the growth and digestive enzyme activity of juvenile sea cucumber Apostichopus japonicas

Science.gov (United States)

Ma, Yuexin; Liu, Zhiming; Yang, Zhiping; Bao, Pengyun; Zhang, Congyao; Ding, Jianfeng

2014-07-01

The effects of a diet containing Hanseniaspora opuntiae C21 on growth and digestive enzyme activity were estimated in juvenile Apostichopus japonicus. Groups of sea cucumbers were fed diets containing H. opuntiae C21 at 0 (control), 104, 105, and 106 CFU (colony-forming units)/g feed. Results showed that after 45 d the specific growth rate (SGR) of sea cucumbers fed a C21-supplemented diet at 10 4 CFU/g feed was significantly higher than that of the control ( P sea cucumbers. In addition, after feeding the C21-supplemented diets for 15 d, the sea cucumbers were switched to an unsupplemented diet and C21 was confirmed to be capable of colonizing the intestine for at least 31 d after cessation of feeding. In conclusion, C21 was shown to successfully colonize the intestine of juvenile A. japonicus via dietary supplementation, and improve growth and digestive enzyme activity.

Enzyme-MOF (metal-organic framework) composites.

Science.gov (United States)

Lian, Xizhen; Fang, Yu; Joseph, Elizabeth; Wang, Qi; Li, Jialuo; Banerjee, Sayan; Lollar, Christina; Wang, Xuan; Zhou, Hong-Cai

2017-06-06

The ex vivo application of enzymes in various processes, especially via enzyme immobilization techniques, has been extensively studied in recent years in order to enhance the recyclability of enzymes, to minimize enzyme contamination in the product, and to explore novel horizons for enzymes in biomedical applications. Possessing remarkable amenability in structural design of the frameworks as well as almost unparalelled surface tunability, Metal-Organic Frameworks (MOFs) have been gaining popularity as candidates for enzyme immobilization platforms. Many MOF-enzyme composites have achieved unprecedented results, far outperforming free enzymes in many aspects. This review summarizes recent developments of MOF-enzyme composites with special emphasis on preparative techniques and the synergistic effects of enzymes and MOFs. The applications of MOF-enzyme composites, primarily in transferation, catalysis and sensing, are presented as well. The enhancement of enzymatic activity of the composites over free enzymes in biologically incompatible conditions is emphasized in many cases.

Immobilization of yeast inulinase on chitosan beads for the hydrolysis of inulin in a batch system.

Science.gov (United States)

Singh, R S; Singh, R P; Kennedy, J F

2017-02-01

An extracellular inulinase was partially purified by ethanol precipitation and gel exclusion chromatography from a cell free extract of Kluyveromyces marxianus. Partially purified inulinase exhibited 420 IU/mg specific activity and it was immobilized on chitosan beads. Activity yield of immobilized inulinase was optimized with glutaraldehyde concentration (1-5%), glutaraldehyde treatment time (30-240min), enzyme coupling-time (2-16h) and enzyme loading (5-30 IU) as functions. Under the optimized conditions maximum yield 65.5% of immobilized inulinase was obtained. Maximum hydrolysis of inulin 84.5% and 78.2% was observed at 125rpm after 4h by immobilized and free enzyme, respectively. A retention-time of 4h and 5h was found optimal for the hydrolysis of inulin under agitation (125rpm) by free and immobilized enzyme, respectively. The recycling of the developed immobilized biocatalyst was carried out after 5h of inulin hydrolysis in a batch system. The developed immobilized biocatalyst was successfully used for the hydrolysis of inulin for 14 batches. This is the first report on the immobilization of yeast inulinase on chitosan beads for the hydrolysis of inulin in a batch system. Copyright © 2016 Elsevier B.V. All rights reserved.

New method for immobilization of biomolecules using preirradiation grafting at low temperature

Energy Technology Data Exchange (ETDEWEB)

Liang Chang Dong; Hoffman, A.S.

1986-01-01

A new method of biomolecule immobilization is described in which a monomer-conjugated enzyme (asparaginase, Asp) is grafted together with free monomer (acrylamide, AAm) onto a cellulose sheet which had been preirradiated in a /sup 60/Co source. The preirradiation and grafting steps are carried out in air at - 78/sup 0/C and in vacuum at 0/sup 0/C respectively. The grafting is probably caused by trapped radicals. The immobilized enzyme retains significant activity and is stable to storage. The technique is applicable to immobilization of a wide variety of biomolecules, such as enzymes, antibodies and drugs. The products may be used for therapeutic or diagnostic applications.

Immobilized cells of Candida rugosa possessing fumarase activity

Energy Technology Data Exchange (ETDEWEB)

Yang, L.; Zhone, L.

1980-01-01

Immobilized cells of C. rugosa that possessed fumarase activity were prepared by different methods; the most active immobilized cells were entrapped in polyacrylamide gels. The effects of pH temperature, and divalent cations on the fumarase activity of both immobilized and native cells were the same. Mn/sup 2 +/, Mg/sup 2 +/, Ca/sup 2 +/, and Fe/sup 2 +/ did not protect the immobilized enzyme against thermal inactivation. The activity of immobilized fumarase remained constant during 91 days of storage of 4-6 degrees. The immobilized cell column was used for the continuous production of L-malic acid from 1M fumarate at 30 degrees and pH 8.5. The immobilized column operated steadily for 2 months. Half life of the immobilized fumarase at 30 degrees was 95 days.

Preparation of Glutaraldehyde Cross-Linked Chitosan Beads Under Microwave Irradiation and Properties of Urease Immobilized onto the Beads

Institute of Scientific and Technical Information of China (English)

LIANG Zupei; FENG Yaqing; MENG Shuxian; ZHANG Weihong

2005-01-01

The glutaraldehyde cross-linked chitosan beads were prepared under microwave irradiation and urease was immobilized onto the beads. The activity and the yield of enzyme activity of the immobilized urease were 10.83 U/g carrier and 47.7%, respectively. The optimum conditions of immobilization were 1% of glutaraldehyde volume fraction, 10 mg/g of urease/beads weight ratio, 24 h of the processing time and pH 6.5 of the reaction medium for immobilization. The properties of the immobilized urease were investigated and compared with those of the free enzyme. The optimum pH values were 6.5 and 7.0 for the immobilized and free urease, respectively. The optimum temperature was 60 ℃ for the free urease, while it shifted to 65 ℃ for the immobilized enzyme. The Michaelis constant K m was 9.1 mmol/L for the immobilized and 12.5 mmol/L for the free urease. The immobilized urease retained 40% of its initial enzyme activity even after 10 repeated uses. The immobilized urease stored at 4 ℃ retained 46% of its initial activity even after 35 d.

Hydrolysis of whey lactose by immobilized β-galactosidase in a bioreactor with a spirally wound membrane.

Science.gov (United States)

Vasileva, Nastya; Ivanov, Yavor; Damyanova, Stanka; Kostova, Iliana; Godjevargova, Tzonka

2016-01-01

The β-galactosidase was covalently immobilized onto a modified polypropylene membrane, using glutaraldehyde. The optimal conditions for hydrolysis of lactose (4.7%) by immobilized β-galactosidase in a batch process were determined 13.6 U enzyme activity, 40°C, pH 6.8 and 10h. The obtained degree of hydrolysis was compared with results received by a free enzyme. It was found, that the lactose hydrolysis by an immobilized enzyme was 1.6 times more effective than the lactose hydrolysis by a free enzyme. It was determined that the stability of the immobilized enzyme was 2 times higher in comparison with the stability of free enzyme. The obtained immobilized system β-galactosidase/polypropylene membrane was applied to produce glucose-galactose syrup from waste whey. The whey characteristics and the different preliminary treatments of the whey were investigated. Then the whey lactose hydrolysis in a bioreactor by an immobilized enzyme on a spirally wound membrane was performed. The optimal membrane surface and the optimal flow rate of the whey through the membrane module were determined, respectively 100 cm(2) and 1.0 mL min(-1). After 10h, the degree of lactose hydrolysis was increased to 91%. The operation stability was studied. After 20th cycle the yield of bioreactor was 69.7%. Copyright © 2015 Elsevier B.V. All rights reserved.

A Urea Biosensor from Stacked Sol-Gel Films with Immobilized Nile Blue Chromoionophore and Urease Enzyme

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Musa Ahmad

2007-10-01

Full Text Available An optical urea biosensor was fabricated by stacking several layers of sol-gelfilms. The stacking of the sol-gel films allowed the immobilization of a Nile Bluechromoionophore (ETH 5294 and urease enzyme separately without the need of anychemical attachment procedure. The absorbance response of the biosensor was monitoredat 550 nm, i.e. the deprotonation of the chromoionophore. This multi-layer sol-gel filmformat enabled higher enzyme loading in the biosensor to be achieved. The urea opticalbiosensor constructed from three layers of sol-gel films that contained urease demonstrateda much wider linear response range of up to 100 mM urea when compared with biosensorsthat constructed from 1-2 layers of films. Analysis of urea in urine samples with thisoptical urea biosensor yielded results similar to that determined by a spectrophotometricmethod using the reagent p-dimethylaminobenzaldehyde (R2 = 0.982, n = 6. The averagerecovery of urea from urine samples using this urea biosensor is approximately 103%.

A Urea Biosensor from Stacked Sol-Gel Films with Immobilized Nile Blue Chromoionophore and Urease Enzyme.

Science.gov (United States)

Alqasaimeh, Muawia Salameh; Heng, Lee Yook; Ahmad, Musa

2007-10-11

An optical urea biosensor was fabricated by stacking several layers of sol-gelfilms. The stacking of the sol-gel films allowed the immobilization of a Nile Bluechromoionophore (ETH 5294) and urease enzyme separately without the need of anychemical attachment procedure. The absorbance response of the biosensor was monitoredat 550 nm, i.e. the deprotonation of the chromoionophore. This multi-layer sol-gel filmformat enabled higher enzyme loading in the biosensor to be achieved. The urea opticalbiosensor constructed from three layers of sol-gel films that contained urease demonstrateda much wider linear response range of up to 100 mM urea when compared with biosensorsthat constructed from 1-2 layers of films. Analysis of urea in urine samples with thisoptical urea biosensor yielded results similar to that determined by a spectrophotometricmethod using the reagent p-dimethylaminobenzaldehyde (R² = 0.982, n = 6). The averagerecovery of urea from urine samples using this urea biosensor is approximately 103%.

Optimization of Phospholipase A1 Immobilization on Plasma Surface Modified Chitosan Nanofibrous Mat

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