Showing papers on "Immobilized enzyme published in 2001"
TL;DR: In this article, a transesterification reaction was performed using triglycerides and short-chain alcohol by immobilized lipase in non-aqueous conditions and the long-chain fatty acid ester, which is the product of this reaction, can be used as a diesel fuel that does not produce sulfur oxide and minimize the soot particulate.
Abstract: Transesterification reaction was performed using triglycerides and short-chain alcohol by immobilized lipase in non-aqueous conditions. The long-chain fatty acid ester, which is the product of this reaction, can be used as a diesel fuel that does not produce sulfur oxide and minimize the soot particulate. Immobilized Pseudomonas fluorescens lipase showed the highest activity in this reaction. Immobilization of lipase was carried out using porous kaolinite particle as a carrier. When methanol and ethanol were used as alcohol, organic solvent like 1,4-dioxane was required. The reaction could be performed in absence of solvent when 1-propanol and 1-butanol were used as short-chain alcohol. The activity of immobilized lipase was highly increased in comparison with free lipase because its activity sites became more effective. Immobilized enzyme could be repeatedly used without troublesome method of separation and the decrease in its activity was not largely observed.
487 citations
TL;DR: The nanoparticle surface biochemical functionalization demonstrates the feasibility of using nanoparticles for biosensing and biomarking applications and shows that the silica nanoparticles are a good biocompatible solid support for enzyme immobilization.
Abstract: In this report, we demonstrate the biochemical modification of silica based nanoparticles. Both pure and dye-doped silica nanoparticles were prepared, and their surfaces were modified with enzymes and biocompatible chemical reagents that allow them to function as biosensors and biomarkers. The nanoparticles produced in this work are uniform in size with a 1.6% relative standard deviation. They have a pure silica surface and can thus be modified easily with many biomolecules for added biochemical functionality. Specifically, we have modified the nanoparticle surfaces with enzyme molecules (glutamate dehydrogenase (GDH) and lactate dehydrogenase (LDH)) and a biocompatible reagent for cell membrane staining. Experimental results show that the silica nanoparticles are a good biocompatible solid support for enzyme immobilization. The immobilized enzyme molecules on the nanoparticle surface have shown excellent enzymatic activity in their respective enzymatic reactions. The nanoparticle surface biochemical functionalization demonstrates the feasibility of using nanoparticles for biosensing and biomarking applications.
442 citations
TL;DR: In this article, the same authors used a hexagonal mesoporous SBA-15-type molecular sieves with pore sizes in the range 51-56 A for immobilization of trypsin using non-ionic block copolymers.
Abstract: Functionalised hexagonal mesoporous SBA-15-type molecular sieves with pore sizes in the range 51–56 A have been prepared using non-ionic block copolymers and used for immobilisation of the enzyme trypsin. Thiol, chloride, amine, and carboxylic acid functional groups were attached by siloxypropane tethers to the siliceous surface of SBA-15 via two methods, post-synthesis grafting and in situ synthesis. Phenylsiloxane groups were also incorporated using these two methods. The resulting solids were rendered porous and used to immobilise trypsin, giving variable but in general higher retention of the enzyme molecules than was observed on unfunctionalised, purely siliceous SBA-15. The resulting supported enzyme catalysts were shown to be active and stable catalysts for the hydrolysis of N -α-benzoyl-DL-arginine-4-nitroanilide (BAPNA). The solids prepared by supporting the enzyme on thiol-functionalised SBA-15 prepared by in situ synthesis were found to be the most promising. Trypsin supported on thiol-functionalised SBA-15 was shown to be recyclable.
311 citations
01 Jan 2001
TL;DR: Functionalised hexagonal mesoporous SBA-15-type molecular sieves with pore sizes in the range 51–56 A have been prepared using non-ionic block copolymers and used for immobilisation of the enzyme trypsin, showing the most promising solids prepared by supporting the enzyme on thiol-functionalised Sba-15 prepared by in situ synthesis.
Abstract: Abstract Functionalised hexagonal mesoporous SBA-15-type molecular sieves with pore sizes in the range 51–56 A have been prepared using non-ionic block copolymers and used for immobilisation of the enzyme trypsin. Thiol, chloride, amine, and carboxylic acid functional groups were attached by siloxypropane tethers to the siliceous surface of SBA-15 via two methods, post-synthesis grafting and in situ synthesis. Phenylsiloxane groups were also incorporated using these two methods. The resulting solids were rendered porous and used to immobilise trypsin, giving variable but in general higher retention of the enzyme molecules than was observed on unfunctionalised, purely siliceous SBA-15. The resulting supported enzyme catalysts were shown to be active and stable catalysts for the hydrolysis of N -α-benzoyl-DL-arginine-4-nitroanilide (BAPNA). The solids prepared by supporting the enzyme on thiol-functionalised SBA-15 prepared by in situ synthesis were found to be the most promising. Trypsin supported on thiol-functionalised SBA-15 was shown to be recyclable.
291 citations
TL;DR: In this paper, the size of mesopores was controlled by means of the combination of the alkyl chain lengths of surfactants and a swelling agent (triisopropyl benzene).
266 citations
TL;DR: The effects of important reaction parameters for enhancing isoamyl acetate formation through lipase-catalyzed esterification ofisoamyl alcohol were investigated and the operational stability of lipase was also observed to be reasonably high enabling ten reuses of the biocatalyst.
236 citations
TL;DR: It appears that the glass-enzyme complex developed in the present work can be used as a high-performance biocatalyst for various chemical processing applications, particularly in organic media.
Abstract: A unique nanoporous sol-gel glass possessing a highly ordered porous structure (with a pore size of 153 A in diameter) was examined for use as a support material for enzyme immobilization. A model enzyme, α-chymotrypsin, was efficiently bound onto the glass via a bifunctional ligand, trimethoxysilylpropanal, with an active enzyme loading of 0.54 wt%. The glass-bound chymotrypsin exhibited greatly enhanced stability both in aqueous solution and organic solvents. The half-life of the glass-bound α-chymotrypsin was >1000-fold higher than that of the native enzyme, as measured either in aqueous buffer or anhydrous methanol. The enhanced stability in methanol, which excludes the possibility of enzyme autolysis, particularly reflected that the covalent binding provides effective protection against enzyme inactivation caused by structural denaturation. In addition, the activity of the immobilized α-chymotrypsin was also much higher than that of the native enzyme in various organic solvents. From these results, it appears that the glass–enzyme complex developed in the present work can be used as a high-performance biocatalyst for various chemical processing applications, particularly in organic media. Published by John Wiley & Sons Biotechnol Bioeng 74: 249–255, 2001.
197 citations
TL;DR: The use of mesoporous molecular sieves in enzyme immobilisation has been studied in this article, where three different types of sieves (MCM-41, MCM-48 and SBA-15) were selected because of the differences in their pore dimensions and structures.
194 citations
TL;DR: Results indicated a structural change of YADH with a decrease in affinity for NADH and 2-butanone after immobilization compared to the free enzyme.
Abstract: Yeast alcohol dehydrogenase (YADH) was immobilized covalently on Fe3O4 magnetic nanoparticles (10.6 nm) via carbodiimide activation. The immobilization process did not affect the size and structure of magnetic nanoparticles. The YADH-immobilized magnetic nanoparticles were superparamagnetic with a saturation magnetization of 61 emu g−1, only slightly lower than that of the naked ones (63 emu g−1). Compared to the free enzyme, the immobilized YADH retained 62% activity and showed a 10-fold increased stability and a 2.7-fold increased activity at pH 5. For the reduction of 2-butanone by immobilized YADH, the activation energies within 25–45 °C, the maximum specific activity, and the Michaelis constants for NADH and 2-butanone were 27 J mol−1, 0.23 mol min−1 mg−1, 0.62 mM, and 0.43 M, respectively. These results indicated a structural change of YADH with a decrease in affinity for NADH and 2-butanone after immobilization compared to the free enzyme.
190 citations
TL;DR: The uptake of glucose oxidase (GOx) onto a polycationic redox polymer (PAA-Os)-modified surface, by adsorption from dilute aqueous GOx solutions, was followed by the quartz crystal microbalance (QCM) and shows double exponential kinetics.
Abstract: The uptake of glucose oxidase (GOx) onto a polycationic redox polymer (PAA−Os)-modified surface, by adsorption from dilute aqueous GOx solutions, was followed by the quartz crystal microbalance (QCM) and shows double exponential kinetics. The electrochemistry of the layer-by-layer-deposited redox-active polymer was followed by cyclic voltammetry in glucose-free solutions, and the enzyme catalysis mediated by the redox polymer was studied in β-d-glucose-containing solutions. AFM studies of the different layers showed the existence of large two dimension enzyme aggregates on the osmium polymer for 1 μM GOx and less aggregation for 50 nM GOx solutions. When the short alkanethiol, 2,2'-diaminoethyldisulfide was preadsorbed onto gold, a monoexponential adsorption law was observed, and single GOx enzyme molecules could be seen on the surface where the enzyme was adsorbed from 50 nM GOx in water.
184 citations
TL;DR: The cross-linking immobilization of β-galactosidase from Kluyveromyces lactis on graphite surfaces was investigated, and the deactivation rate was found to follow the Arrhenius law with the activation energy of about 200 kJ mol −1 for the de activation of the immobilized enzyme.
TL;DR: Thermal and storage stabilities were found to increase with immobilization, and the optimum operational temperature was 5°C higher for immobilized enzyme than that of the free enzyme.
TL;DR: Fungal laccase was immobilized on carbon-fiber electrodes using classical methods: physical adsorption, glutaraldehyde, carbodiimide and carbodiIMide/glutaraldehyde to obtain a biosensor that showed an optimum response at pH 5.0 and at an applied potential of -100 mV.
TL;DR: A novel method for the immobilization of penicillin G acylase involves the physical aggregation of the enzyme, followed by chemical cross-linking to form insoluble cross-linked enzyme aggregates (CLEAs) that possess a high specific activity as well as a high productivity and synthesis/hydrolysis ratio in the synthesis of semi-synthetic antibiotics in aqueous media.
Abstract: A novel method for the immobilization of penicillin G acylase (penicillin amidohydrolase, EC 35111) is reported It involves the physical aggregation of the enzyme, followed by chemical cross-linking to form insoluble cross-linked enzyme aggregates (CLEAs) Compared with conventionally immobilized penicillin G acylases, these CLEAs possess a high specific activity as well as a high productivity and synthesis/hydrolysis (S/H) ratio in the synthesis of semi-synthetic antibiotics in aqueous media Moreover, they are active in a broad range of polar and apolar organic solvents
TL;DR: In this article, the immobilization of lactate dehydrogenase (LDH) on electrochemically polymerized polypyrrole-polyvinylsulphonate (PPY-PVS) films has been accomplished via cross-linking technique using glutaraldehyde.
TL;DR: The effect of solvents and solvent mixtures on the synthesis of myristic acid esters of different carbohydrates with an immobilized lipase from C. antarctica was investigated and no synthesis activity was observed with maltotriose, cellobiose, sucrose, and lactose as substrate.
Abstract: The effect of solvents and solvent mixtures on the synthesis of myristic acid esters of different carbohydrates with an immobilized lipase from C. antarctica was investigated. The rate of myristyl glucose synthesized by the enzyme was increased from 3.7 to 20.2 μmol min−1 g−1 by changing the solvent from pure tert-butanol to a mixture of tert-butanol:pyridine (55:45 v/v), by increasing the temperature from 45°C to 60°C, and by optimizing the relative amounts of glucose, myristic acid, and the enzyme preparation. Addition of more than 2% DMSO to the tert-butanol:pyridine system resulted in a reduction of enzyme activity. Lowering the water content of the enzyme preparation below 0.85% (w/w) resulted in significant decreases in enzyme activity, while increasing the water content up to 2.17% (w/w) did not significantly affect the enzyme activity. The highest yields of myristyl glucose were obtained when an excess of unsolubilized glucose was present in the reaction system. In this case, all of the initially solubilized and a significant amount of the initially unsolubilized glucose was converted to the ester within 24 h of incubation, resulting in a myristyl glucose concentration of 34 mg/mL−1. Myristic acid esters of fructose (22.3 μmol min−1 g−1), α-D-methyl-glucopyranoside (26.9 μmol min−1 g−1) and maltose (1.9 μmol min−1 g−1) could also be prepared using the tert-butanol:pyridine solvent system. No synthesis activity was observed with maltotriose, cellobiose, sucrose, and lactose as substrate. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 74: 483–491, 2001.
TL;DR: In this article, the authors investigated the activity and reusability of a commercial pectinase named as Pectinex Ultra SP-L on carrot puree and found that the activity loss was only 20% after nine batches run model system studies and the average yield increment was 30.23% with respect to the yield obtained from non-enzymic processed carrot juice.
TL;DR: The immobilized catalase preparation from a newly isolated Bacillus sp.
TL;DR: In this article, a Saccharomyces cerevisiae invertase was immobilized in alginate capsules and the immobilization resulted in 87% relative activity and activity for 36 days without decrease in activity.
TL;DR: It was shown that the amounts of solute adsorption and the immobilization capacity of acid phosphatase on cross-linked chitosan beads were substantially affected by their degree of cross-linking.
TL;DR: Electrodeposition was used for the codeposition of glucose oxidase enzyme and a gold nanoparticle-silicate network onto an indium tin oxide (ITO) glass electrode, which imparts biocatalytic activity to the film.
Abstract: Electrodeposition was used for the codeposition of glucose oxidase enzyme and a gold nanoparticle–silicate network onto an indium tin oxide (ITO) glass electrode. This co-entrapment of glucose oxidase enzyme in a gold nanoparticle–silicate network imparts biocatalytic activity to the film. The gold nanoparticles in the network catalyse the oxidation and reduction of H2O2, the by-product of the enzymatic reaction. The low operating potential of the sensor eliminates the interference from common interferents, such as acetaminophen, ascorbic acid, dopamine, etc.
TL;DR: Electron paramagnetic resonance, in conjunction with active-site specific spin labels, kinetic analyses, and membrane properties are used to characterize these systems and provide greater efficiency of biocatalysis, bioseparations, and bioanalysis.
TL;DR: Taking advantage of the selectivity properties of the novel support, a new kind of multifunctional epoxy support was developed, which was able to immobilize up to 30 mg of protein per gram of modified Eupergit 250 using either pure enzyme or a very crude enzyme extract.
Abstract: Epoxy supports covalently immobilize proteins following a two-step mechanism; that is, the protein is physically adsorbed and then the covalent reaction takes place. This mechanism has been exploited to combine the selectivity of metal chelate affinity chromatography with the covalent immobilization capacity of epoxy supports. In this way, it has been possible to accomplish, in a simple manner, the purification, immobilization, and stabilization of a poly-His-tagged protein. To fulfill this objective we developed a new kind of multifunctional epoxy support (chelate epoxy support [CES]), which was tested using a poly-His-tagged glutaryl acylase as a model protein (an alphabeta-heterodimeric enzyme of significant industrial interest). The selectivity of the immobilization in CES toward poly-His-tagged proteins was dependent to a large extent on the density and nature of the chelated metal. The highest selectivity was achieved by using low-density chelate groups (e.g., 5 micromol/g) and metals with a low affinity (e.g., Co). However, the rate of covalent immobilization of the protein by its reaction with the epoxy groups on the support significantly increased at alkaline pH values. The multipoint attachment to the CES also depended on the reaction time. The immobilization of both glutaryl acylase subunits was achieved by incubation of the enzyme derivative at pH 10 for 24 h, with the best enzyme derivative 100-fold more stable than the soluble enzyme. By taking advantage of the selectivity properties of the novel support, we were able to immobilize up to 30 mg of protein per gram of modified Eupergit 250 using either pure enzyme or a very crude enzyme extract.
TL;DR: A flow injection amperometric biosensor for the determination of organophosphate nerve agents was developed and the applicability of the biosensor to monitor paraoxon and methyl parathion in distilled water and simulated well water was demonstrated.
Abstract: A flow injection amperometric biosensor for the deter mination of organophosphate nerve agents was developed. The biosensor incorporated an immobilized enzyme reactor that contains the enzyme organ...
TL;DR: This type of derivative may be appropriate for extracorporeal devices in the clinical treatment of acute leukemia and might thus bring about inherent advantages in that all subunits are covalently bound to the support, with a longer half‐life and a virtually nil risk of subunit release into the circulating blood stream.
Abstract: A new protocol for the stabilization of the quaternary structure of multimeric enzymes has been attempted using as model enzyme (tetrameric) L-asparaginase from Escherichia coli. Such strategy is based upon multisubunit covalent immobilization of the enzyme onto activated supports (agarose-glutaraldehyde). Supports activated with different densities of reactive groups were used; the higher the density of groups, the higher the stabilization attained. However, because of the complexity of that enzyme, even the use of the highest densities of reactive groups was not enough to encompass all four subunits in the immobilization process. Therefore, a further chemical intersubunit cross-linking with aldehyde-dextran was pursued; these derivatives displayed a fully stabilized multimeric structure. In fact, boiling the modified enzyme derivative in the presence of sodium dodecyl sulfate and beta-mercaptoethanol did not lead to release of any enzyme subunit into the medium. Such a derivative, prepared under optimal conditions, retained ca. 40% of the intrinsic activity of the free enzyme and was also functionally stabilized, with thermostabilization enhancements of ca. 3 orders of magnitude when compared with its soluble counterpart. This type of derivative may be appropriate for extracorporeal devices in the clinical treatment of acute leukemia and might thus bring about inherent advantages in that all subunits are covalently bound to the support, with a longer half-life and a virtually nil risk of subunit release into the circulating blood stream.
TL;DR: To investigate the concentration effects of immobilized biosignal molecules by microscopic observation, a gradient micropattern immobilization technique using a photomask was devised and growth enhancement was observed only in dense EGF derivative immobilized regions.
TL;DR: In this article, β-galactosidase from Kluyveromyces lactis was immobilized onto graphite surface using glutaraldehyde as the cross-linking reagent with the specific activity yield of 17% and 25%, while the enzyme loading was 1.8 and 1.1 U/cm2, respectively.
TL;DR: Immobilized trypsin shows a high thermal and pH stability and can be stored for several months without loosing its activity, and was characterized more thoroughly by testing the activity behaviour after repetitive use and various storage conditions.
TL;DR: Enzyme solution-oxidized porous silicon-crystalline silicon structure was used to detect changes in pH during the hydrolysis of tributyrin as a shift in the capacitance-voltage (C-V) characteristics.
TL;DR: The ability of the enzyme organophosphorus acid anhydrolase (OPAA) to selectively hydrolyze the PF bond of fluorine containing organophorus compounds has been used to develop a biosensor for specific detection of these compounds as mentioned in this paper.