Showing papers on "Immobilized enzyme published in 1994"
TL;DR: Glucose oxidase or lactate oxidase were immobilized in an osmium-based three-dimensional redox hydrogel that electrically connected the enzyme's redox centers to electrodes that correlated well with a substrate calibration in phosphate buffer.
Abstract: Glucose oxidase (GOX) or lactate oxidase (LOX) were immobilized in an osmium-based three-dimensional redox hydrogel that electrically connected the enzyme's redox centers to electrodes The enzyme "wiring" hydrogel was formed by cross-linking poly(1-vinylimidazole) (PVI) complexed with Os-(4,4'-dimethylbpy)2Cl (termed PVI15-dmeOs) with poly(ethylene glycol) diglycidyl ether (peg) Glucose and lactate sensors exhibited typical limiting current densities of 250 and 500 microA/cm2, respectively When the electrodes were poised at 200 mV (SCE), the currents resulting from electrooxidation of ascorbate, urate, acetaminophen, and L-cysteine were negligible When a Nafion film was employed, the linear range was extended from 6 to 30 mM glucose and from 4 to 7 mM lactate The redox potential of the gel-forming polymer was 95 mV (SCE) Glucose and lactate measurements performed in bovine calf serum correlated well with a substrate calibration in phosphate buffer
468 citations
TL;DR: A template-based synthetic method is developed that yields hollow polymeric microcapsules of uniform diameter and length that function as enzymatic bioreactors in both aqueous solution and organic solvents.
Abstract: Current methods for immobilizing enzymes for use in bioreactors and biosensors include adsorption on or covalent attachment to a support, micro-encapsulation, and entrapment within a membrane/film or gel. The ideal immobilization method should employ mild chemical conditions, allow for large quantities of enzyme to be immobilized, provide a large surface area for enzyme-substrate contact within a small total volume, minimize barriers to mass transport of substrate and product, and provide a chemically and mechanically robust system. Here we describe a method for enzyme immobilization that satisfies all of these criteria. We have developed a template-based synthetic method that yields hollow polymeric microcapsules of uniform diameter and length. These microcapsules are arranged in a high-density array in which the individual capsules protrude from a surface like the bristles of a brush. We have developed procedures for filling these microcapsules with high concentrations of enzymes. The enzyme-loaded microcapsule arrays function as enzymatic bioreactors in both aqueous solution and organic solvents.
307 citations
TL;DR: The structure and reactivity of glutaraldehyde protein immobilization is detailed, which shows a complex chemistry that is transparent to most practitioners of immobilization.
Abstract: Immobilization of proteins to solid matrices has been performed for the last thirty years and has provided numerous examples of successful preparations with use in enzyme reactors, sensor preparation and immunodiagnostics. Among the arsenal of coupling reagents and procedures, glutaraldehyde plays a critical role due to its reliability and ease of use. It displays a complex chemistry that is transparent to most practitioners of immobilization. In this article we detail the structure and reactivity of glutaraldehyde protein immobilization.
262 citations
TL;DR: In this paper, the development of an amperometric sensor utilizing the NAD(P) + -cofactor-dependent enzyme, malic enzyme, was described using a quinone-enzyme monolayer modified electrode.
Abstract: The development of an amperometric sensor utilizing the NAD(P) + -cofactor-dependent enzyme, malic enzyme, is described using a quinone-enzyme monolayer-modified electrode. Pyrroloquinolinequinone (PQQ, 1) was covalently linked to a self-assembled monlayer of cysteamine on an Au electrode. The resulting PQQ-monolayer electrode (PQQ surface coverage 1.98×10 -10 mol-cm -2 ) catalyzes the electrooxidation of NADPH and NADH. The developed anodic currents are controlled by NAD(P)H concentrations and provide an amperometric sensor for the cofactor
180 citations
TL;DR: It appeared that immobilization enhanced the stability of the enzyme and increased the number of usable organic solvents, which will be useful for the detection of pesticide residues present in water and food at very low levels.
137 citations
TL;DR: This review summarizes selected studies that have used immobilization to tailor the catalytic properties of enzymes, and highlights the application of immobilizing to the rational design of biocatalysts.
133 citations
TL;DR: The in situ immobilised tannase can be used for removing tannins responsible for unwanted effects in food or cosmetology processing and is officially approved in numerous countries for enzyme production in the food industry.
123 citations
TL;DR: In this article, the use of β-glucosidase for synthesizing octyl-β-glocoside was studied and the best results were obtained using a high initial water activity which was decreased during the course of the reaction.
117 citations
TL;DR: In this article, the electron transfer rate is governed by the potential difference, the reorganisation energy and, most significantly for the development of enzyme electrodes, the distance between the involved redox centers.
108 citations
TL;DR: In this article, a generic biosensor concept is proposed, which involves immobilization of an appropriate enzyme in the conducting polymer matrix, which results in a change in redox potential and/or pH of the micro environment in the polymer matrix.
107 citations
TL;DR: A new biocomposite, based on the incorporation of the enzyme tyrosinase into a graphite-epoxy resin matrix, was used for the effective biosensing of phenolic compounds and offers convenient quantification for various phenolic substrates.
Abstract: A new biocomposite, based on the incorporation of the enzyme tyrosinase into a graphite-epoxy resin matrix, was used for the effective biosensing of phenolic compounds. The enzyme retains its bioactivity on confinement in the epoxy resin environment. This renewable (polishable) and rigid bioprobe offers convenient quantification for various phenolic substrates. The fast response (steady-state time = 25 s) accrues from the close proximity of the enzyme and graphite sites. The influence of various experimental variables was explored for optimum biosensing performance. Flow-injection monitoring of phenolic compounds at a rate of 50 samples h–1 yielded a detection limit of 1×10–6 mol l–1 and a relative standard deviation of 1.4%(n= 40).
TL;DR: In this paper, the electropolymerization of a nitrate reductase-amphiphilic pyrrole viologen mixture preadsorbed on the electrode surface provides the immobilization of the enzyme in a N-substituted viologens polypymole film.
Abstract: The electropolymerization of a nitrate reductase-amphiphilic pyrrole viologen mixture preadsorbed on the electrode surface provide the immobilization of the enzyme in a N-substituted viologen polypymole film. Furthermore, the electrogenerated redox polymer simultaneously entraps the enzyme and connects it electrically with the electrode. The immobilized enzyme catalyzes the reduction of nitrate to nitrite mediated by the viologen redox couple (V 2+/.+ ). This enzyme immobilization has been accomplished on electrode surface previously coated by a polypyrrole-viologen film, leading to a bilayer electrode configuration. The sensitivity and the detection light of this biosensor are 13.8 mA M -1 cm -2 and 4×10 - M, respectively
TL;DR: Glucose isomerase has been successfully immobilized in films and in porous p-trimethylamine-polystyrene beads by using a new method, viz. deposition and immobilization of enzyme multilayers based on cationic-anionic attraction as driving force.
Abstract: Glucose isomerase has been successfully immobilized in films and in porous p-trimethylamine-polystyrene beads by using a new method, viz. deposition and immobilization of enzyme multilayers based on cationic-anionic attraction as driving force. The activity of the enzyme is maintained in the multilayers
TL;DR: In this paper, the enzyme transistors were prepared by connecting two carbon band electrodes (∼10 μm wide, 4.5 mm long separated by a 20μm gap) with an anodically grown film of poly(aniline).
Abstract: Microelectrochemical enzyme transistors, or enzyme switches, were prepared by connecting two carbon band electrodes (∼10 μm wide, 4.5 mm long separated by a 20-μm gap) with an anodically grown film of poly(aniline). Glucose oxidase (GOx, EC 1.1.3.4) was immobilized onto the poly(aniline) film in an insulating poly(1,2-diaminobenzene) polymer grown electro-chemically on top of the poly(aniline) film to complete the device
TL;DR: A potential use of this procedure for enzyme immobilization by entrapment in copper alginate gel for the construction of bioreactors to be used in the detoxification of polluted waste waters is indicated.
TL;DR: In this article, the biochemical properties of a commercial glycosidase from Aspergillus niger (Cytolase PCL5, Genencor) were investigated.
TL;DR: The influence of polyhydric alcohols and carbohydrates on the thermostability of Bacillus licheniformis α‐amylase was studied in the temperature range 96° to 130°C to show biphasic then first‐order inactivation kinetics, depending on the additive concentration and temperature.
Abstract: The influence of polyhydric alcohols and carbohydrates on the thermostability, i.e., the heat inactivation kinetics, of Bacillus licheniformis α-amylase was studied in the temperature range 96° to 130°C. High concentrations (from 9 to 60 weight percent) of glycerol, sorbitol, mannitol, sucrose, or starch can markedly decrease the inactivation rate constant, k, and in the studied cases, this stabilizing effect grows stronger with increasing additive concentration. Statements about stabilization should, however, be specified carefully with respect to temperature, because EA is mostly altered likewise. For dissolved enzyme EA was almost always decreased in the presence of polyol or carbohydrate, whereas for immobilized enzyme it was augmented in each studied instance. The inactivation of dissolved enzyme can, in all the studied cases, be adequately described as a firstorder process. Immobilized enzyme, however, shows biphasic then first-order inactivation kinetics, depending on the additive concentration and temperature. © 1994 John Wiley & Sons, Inc.
TL;DR: In this paper, the β-galactosidase immobilization on chitosan beads of 2.2 mm diameter was performed at 37 o C with a 1% glutaraldehyde concentration and the addition of galactose.
Abstract: The β-galactosidase immobilization on chitosan was studied. Enzymatic activity and stability were determined for the different conditions used. The best results were obtained on chitosan beads of 2.2 mm diameter where the immobilization process was carried out at 37 o C with a 1% glutaraldehyde concentration and the addition of galactose. The higher activity values of the immobilized enzyme compared with those of the free β-galactosidase could be obtained over a larger pH range and were at higher pH's and temperatures, but the best activity is only 10.7% of the free enzyme values
TL;DR: An on-line tubular electrode system is described which provides real time measurements of glutamate in brain microdialysate and is optimized for use with a microdialysis probe implanted in the brain and perfused at 2 microL/min.
Abstract: An on-line tubular electrode system is described which provides real time measurements of glutamate in brain microdialysate. It is based on the enzyme glutamate oxidase (EC 1.4.3.11) and the detection of H2O2 on a platinum electrode at 600 mV vs Ag/AgCl. The enzyme is immobilized in a layer of o-phenylenediamine electropolymerized at 750 mV in a phosphate buffer, pH 7.0. The layer is 10-15 nm thick and enables good enzyme loading and fast response time. The ability of the polymer to block out electroactive interferents like ascorbate and uric acid combined with a preoxidation system run at 600 mV resulted in a virtually interference free glutamate assay with a lower detection limit of 0.3 microM in the presence of physiological levels of interferents, a sensitivity of 4 nA/microM, and a linear region up to 30 microM. The system is optimized for use with a microdialysis probe implanted in the brain and perfused at 2 microL/min. It provides a time resolution of < 1 min and has been tested in vivo.
TL;DR: Non-cross-linked and also cross-linked poly(ether amine quinone)s were prepared and tested for their efficiency as electron-transfer relay systems in amperometric glucose biosensors and showed efficiently mediated electron transfer from reduced glucose oxidase to a conventional carbon paste electrode.
Abstract: Non-cross-linked and also cross-linked poly(ether amine quinone)s were prepared and tested for their efficiency as electron-transfer relay systems in amperometric glucose biosensors. Cyclic voltammetry and constant applied potential measurements showed that poly(ether amine quinone) relay systems efficiently mediated electron transfer from reduced glucose oxidase to a conventional carbon paste electrode. Sensors containing these relay systems respond rapidly to low (< 0.1 mM) glucose concentrations and reach steady-state current responses in less than 1 min. Electrodes constructed with cross-linked polymer and glucose oxidase were stable, indicating that the glucose oxidase was trapped in the polymer matrix and did not freely diffuse away from the electrode surface into aqueous solutions. The cross-linked polymer is a large molecular system which facilitates a flow of electrons from enzyme to the electrode, acting as an electron-transfer relay system and not as a diffusional mediator.
TL;DR: Oxidative treatment of juices with laccase was shown by HPLC to specifically remove these phenolic constituents measured in the two enzyme assays.
Abstract: The properties of a frugal laccase have been exploited to develop two enzyme assays for the phenolic content of natural beverages: first, by monitoring the fall in oxygen tension after addition of enzyme to a diluted juice sample in an end-point batch assay; second, by monitoring the current due to reduction of products after injection of sample into a flow injection analysis (FIA) system which incorporates the immobilized enzyme. Using apple juices, a linear (R>0.9) correlation was observable between these enzyme assays and the Folin-Ciocalteu wet chemical assay for total phenol content. Using purified substrates, a study has been made of those phenolic constituents measured in the two enzyme assays. Oxidative treatment of juices with laccase was shown by HPLC to specifically remove these phenolics
TL;DR: Amperometry in stirred solution has been used for the systematic evaluation of modified screen-printed carbon electrodes (SPCEs) with a view to developing a reagentless biosensor for uric acid, suggesting that the process is diffusion-controlled and not governed by the kinetics of the enzyme.
Abstract: Amperometry in stirred solution has been used for the systematic evaluation of modified screen-printed carbon electrodes (SPCEs) with a view to developing a reagentless biosensor for uric acid. The developed system consists of a base cobalt phthalocyanine (CoPC) electrode tailored to the electrocatalytic oxidation of H2O2 by means of a cellulose acetate (CA)–uricase bilayer. Uricase was immobilized by drop-coating the enzyme onto the CA membrane covering the CoPC-SPCE. The device exploits the near-universal H2O2-generating propensity of oxidases, the permselectivity of the CA film towards H2O2 and the electrocatalytic oxidation of this product at the CoPC-SPCE. The electrochemical oxidation of the resulting Co+ species was used as the analytical signal, facilitating the application of a greatly reduced operating potential when compared with that required for direct oxidation of H2O2 at unmodified electrodes. The time required to achieve 95% of the steady-state current (t95iSS) was 44 s [relative standard deviation = 7.5%(n= 10)]. Amperometric calibrations were linear over the range from 13 × 10–6 to 1 × 10–3 mol dm–3, with the former representing the limit of detection. The CA membrane extended the linear range of the biosensor by over two orders of magnitude, when apparent Michaelis–Menten constants (Km′) of immobilized and free enzymes are compared. This suggests that the process is diffusion-controlled and not governed by the kinetics of the enzyme. The precision of electrode fabrication was determined by cyclic voltammetry to be 4.9%(n= 6). Successive amperometric calibrations (n= 7) over 7 d using a single sensor revealed only a 14.0% diminution in sensitivity from the original response. Sensor stability and the dependence of the steady-state current on the pH, ionic strength and temperature of the supporting electrolyte were studied and the results are presented. The functioning of the biosensor is indifferent to a wide range of potential interferences studied in a synthetic sample and results correlate favourably with those of a standard hospital method.
TL;DR: In this article, a gold electrode surface was modified with a chemisorbed monolayer of cystamine, and the amino groups were used for covalent immobilization of pyrroloquinoline quinone (PQQ).
TL;DR: A method is described to obtain a new type of microcapsules for immobilization of enzymes or living cells that can be better controlled when the membrane is built up by several consecutive steps, each being optimized with respect to a special property, for example, permeability or mechanical strength.
Abstract: A method is described to obtain a new type of microcapsules for immobilization of enzymes or living cells. The wall of these capsules consists of several layers of poly(ethyleneimine) and poly(acrylic acid). The idea is that diverging properties of the whole assembly can be better controlled when the membrane is built up by several consecutive steps, each being optimized with respect to a special property, for example, permeability or mechanical strength. The encapsulation of acidic phosphatase and the cleavage of p-nitrophenyl phosphate was used as a model system. The charged capsules were characterized by their enzymatic activity, as a function of membrane composition (number and sequence of layers) and storing time. The permeability for the substrate and the retaining ability for the enzyme were also measured.
TL;DR: Preliminary in vitro studies of the biosensor show stable performance during several recharge cycles (of 14 days each) over a period of 4 months, and calibration curves of the amperometric signal show linearity over a wide range of glucose concentrations.
Abstract: A potentially implantable glucose biosensor for continuous monitoring of glucose levels in diabetic patients has been developed. The glucose biosensor is based on an amperometric oxygen electrode and glucose oxidase immobilized on carbon powder held in a form of a liquid suspension. The enzyme material can be replaced (the sensor recharged) without sensor disassembly. Recharging of the biosensor is achieved by injecting fresh immobilized enzyme into the sensor using a septum. Diffusion membranes made of silastic latex-rubber coatings over a microporous polycarbonate membrane are used. Calibration curves of the amperometric signal show linearity over a wide range of glucose concentrations-up to 500 mg/dL (28 mM), covering hypoglycemic, normoglycemic, and hyperglycemic conditions. Preliminary in vitro studies of the biosensor show stable performance during several recharge cycles (of 14 days each) over a period of 4 months. (c) 1994 John Wiley & Sons, Inc.
TL;DR: The chitosan-xanthan complex was used to immobilize two enzymes either as single enzymes or as a binary system, and the immobilized xylanase activity was significantly greater with respect to the free enzyme while the binary enzyme system promoted protease activity.
Abstract: A method for the preparation of hydrogels from the complexation of chitosan and xanthan is reported. Stable hydrogels capable of retaining be tween 65 and 95% weight water were prepared. The water retention and prop erties of the hydrogels were studied as a function of the degree of acetylation of chitosan and the ratio chitosan/xanthan used in the preparation of the gel. Spectroscopic FTIR was used to confirm complexation between the amine (chi tosan) and carboxylic (xanthan) groups. Electron micrographs (SEM and TEM) show the formation of a fibrillar structure with characteristic pore sizes be tween 100 and 1000 nm and fibril diameters between 50 and 100 nm. The diffu sion coefficient of 4-O-methyl-d-glucurono-D-xylan Remazol Brilliant Blue R (RBB-xylan) in the complex chitosan-xanthan was 2.02 × 10-12 m 2s-1 at 30°C. The chitosan-xanthan complex was used to immobilize two enzymes (endo-1,4- β-xylanase and protease) either as single enzymes or as a binary system. Immo bilization varied between 85 and 98...
TL;DR: Evidence indicating that the catalytic reduction of H 2 O 2 at the CCP/EPG electrode occurs via the intact enzyme and not via some form of «free» heme is discussed.
Abstract: Cytochrome c peroxidase (CCP) adsorbed at (sub)monolager surface coverage on edge-oriented pyrolytic graphite (EPG) is being used as a model system for determining the effects of immobilization on the kinetic properties of an enzyme and for developing molecular insight into enzymatic electrocatalysis. Evidence indicating that the catalytic reduction of H 2 O 2 at the CCP/EPG electrode occurs via the intact enzyme and not via some form of «free» heme is discussed. Rotating disk electrode (RDE) voltammetry has been used to determine a Michaelis constant of 48 μM and a minimum turnover number of 110 s -1 for the adsorbed enzyme at 3 o C
TL;DR: Oxygen saturation, flow-rate and immobilised enzyme amount were the main parameters used to study phenolic oxidation in immobilised Laccase.
Abstract: The possibility of the use of immobilised Laccase (EC 1.10.3.1.) for the enzymatic removal of phenolic compounds from must and wine has been attempted on laboratory scale. The immobilised enzyme was packed in column and model solutions of catechin or white grape must were eluted through. Oxygen saturation, flow-rate and immobilised enzyme amount were the main parameters used to study phenolic oxidation.
TL;DR: A key chiral intermediate for the semi‐synthesis of paclitaxel (taxol; 5), an anti‐cancer compound, was prepared by an enzymic process and a lipase fermentation using Pseudomonas sp.
Abstract: A key chiral intermediate [(3R-cis)-3-(acetyloxy)-4-phenyl-2-azetidinone (2)] for the semi-synthesis of paclitaxel (taxol; 5), an anti-cancer compound, was prepared by an enzymic process. The stereoselective enzymic hydrolysis of cis-3-(acetyloxy)-4-phenyl-2-azetidinone (1) to the corresponding (S)-(-)-alcohol (3) was carried out using various lipases. Lipase PS-30 (Pseudomonas cepacia) and BMS (Bristol-Myers Squibb) lipase (Pseudomonas sp. SC13856) catalysed hydrolysis of the undesired enantiomer of racemic compound 1, producing the (S)-(-)-alcohol (3) and the desired (R)-(+)-acetate (2). Reaction yields of > 96% and optical purities of > 99.5% were obtained. For a very efficient enzyme source (BMS lipase), a lipase fermentation using Pseudomonas sp. SC13856 was developed. In a fed-batch process using soybean oil, the fermentation resulted in 1500 units of extracellular lipase activity/ml. Crude BMS lipase (1.7 kg, containing 140,000 units/g) was recovered from the filtrate by ethanol precipitation. BMS lipase and commercially available lipase PS-30 were independently immobilized on Accurel polypropylene. These immobilized lipases were re-used (ten cycles) without loss of enzyme activity, productivity or optical purity of the product. The enzymic reaction process was scaled up to 75 and 150 litres using immobilized BMS lipase and lipase PS-30 respectively. From the reaction mixture, compound 2 was isolated in 88-90 mol% yield and 99.5% optical purity. A purity of 99.9 (area %) was demonstrated by g.c. for isolated compound 2.