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Showing papers on "Immobilized enzyme published in 1985"


Journal Article
TL;DR: In this paper, the authors present several enzyme immobilization methods which are abundant in the literature, depending on many factors: the nature of substrate, use of the final product, need for sanitary conditions, and, of course, capital and processing costs.

292 citations


Journal ArticleDOI
TL;DR: The hydrolysis seemed to be limited by diffusion of fat or fatty acid through the micropores of the membrane at higher interfacial enzyme concentrations, and the lipase was stabilized significantly by glycerol added to the buffer solution.
Abstract: Continuous hydrolysis of olive oil byCandida cylindracea’s lipase was studied in a microporous hydrophobic membrane bioreactor. Olive oil and buffer solution, fed continuously through two compartments partitioned by membrane, caused reaction at the interface of lipase-adsorbed membrane and buffer solution. Fatty acid was obtained in a single phase without being mixed with components of other phases. At all mean residence times, countercurrent flow mode was superior to cocurrent one. The lipase was adsorbed onto the membrane, and its adsorption was suggested to be partially specific from the experiments with enzymes having various levels of purity. The percent hydrolysis depended hyperbolically on the interfacial enzyme concentration. The hydrolysis seemed to be limited by diffusion of fat or fatty acid through the micropores of the membrane at higher interfacial enzyme concentrations. The lipase was stabilized significantly by glycerol added to the buffer solution. Satisfactory performance of the membrane bioreactor was obtained in a longterm continuous operation which lasted for 24 days by feeding buffer-glycerol (18.0%) solution over the adsorbed lipase. The operational half-life of the adsorbed enzyme was 15 days at 40 C.

150 citations


Journal ArticleDOI
TL;DR: A simple and efficient preparation of neuronal tissue is described using an optional prepurification step on Sephadex G‐10 columns, offering the possibility to detect choline and acetylcholine as well as catecholamines and their related metabolites in the same tissue sample.
Abstract: A simple, efficient, economic, and sensitive method is presented for the detection of choline and acetylcholine in neuronal tissue using HPLC, a postcolumn enzyme reactor with immobilized enzyme, and electrochemical detection. The method is based on a separation of choline and acetylcholine by cation exchange HPLC followed by passage of the effluent through a postcolumn reactor containing a mixture of acetylcholinesterase and choline oxidase; the latter enzyme converts choline to betaine and hydrogen peroxide, the former enzyme hydrolyzes acetylcholine to acetate and choline. The hydrogen peroxide produced is electrochemically detected. A simple and efficient preparation of neuronal tissue is described using an optional prepurification step on Sephadex G-10 columns, offering the possibility to detect choline and acetylcholine as well as catecholamines and their related metabolites in the same tissue sample. The sensitivity of the assay system is 250 fmol for choline and 500 fmol for acetylcholine.

138 citations


Journal ArticleDOI
TL;DR: In this paper, a p-benzoquinone-carbon paste electrode was used as a glucose sensor that is relatively insensitive to variations of oxygen tension in sample solutions, and it showed high current response to glucose, and was stable for more than a week.
Abstract: Glucose oxidase was immobilized on the surface of a p-benzoquinone-carbon paste electrode by coating the enzyme-loaded surface with a nitrocellulose film. The electrode was able to oxidize glucose electrocatalytically. It showed high current response to glucose, and was stable for more than a week. The electrode can be used as a glucose sensor that is relatively insensitive to variations of oxygen tension in sample solutions.

125 citations


Journal ArticleDOI
TL;DR: A membrane-free glucose sensor was made by covalent immobilization of glucose oxidase on graphite followed by adsorption of N-methyl-phenazinium ion (PMS+) and the mediator was found to be necessary for the electron transfer between the enzyme and the electrode.

118 citations


Patent
Yasusi Niiyama1, Kenshi Sugahara1
25 Nov 1985
TL;DR: In this article, an electrochemical sensor is formed having a working electrode for detecting hydrogen peroxide surrounded by a cylinder portion, and with an enzymecontaining membrane at its tip.
Abstract: An electrochemical sensor is formed having a working electrode for detecting hydrogen peroxide surrounded by a cylinder portion, and with an enzymecontaining membrane at its tip. The membrane has a porous layer permeable to hydrogen peroxide between a layer containing an immobilized enzyme capable of decomposing hydrogen peroxide and a layer containing an immobilized enzyme capable of decomposing a substrate to form hydrogen peroxide. The cylinder portion is embedded in the layer containing the hydrogen peroxide decomposing enzyme and surrounds the working electrode such that the electrode is in contact with the porous layer but is not in contact with the layer containing the hydrogen peroxide decomposing enzyme. The layer containing the hydrogen peroxide forming enzyme is on a side of the porous layer opposite the electrode so as not to contact the electrode. Activity of the hydrogen peroxide decomposing enzyme is no more than one-fourth of the activity of the hydrogen peroxide forming enzyme. The electrochemical sensor reduces base line elevation after measurement action has been discontinued and measurement of a next sample is restarted.

117 citations


Journal ArticleDOI
TL;DR: In this article, the fabrication process and characteristics of monolithically integrated urea and glucose FETs are described, and an insoluble membrane with enzyme entrapped in it is formed, which is used for the preparation of multifunctional and one-chip biosensors with various other pairs of immobilized enzymes.

101 citations


Book ChapterDOI
01 Jan 1985
TL;DR: The concept of an enzyme electrode was introduced by Hicks and Updike as discussed by the authors, which is a thin enzyme layer held in close proximity to the active surface, and is used to measure the rate of enzyme catalyzed reaction in solution.
Abstract: The rapid growth in the number of enzymes and the reactions associated with them, ensures that a wide variety of enzyme catalyzed analysis procedures are now available. The development of immobilized enzymes probes (I.E.P.) is related to advances in both immobilization technology and in the improvement of numerous sensing devices. Potentiometric, amperometric, enthalpimetric, and chemiluminescent sensors have been employed as transducers for enzyme electrodes. Most of them have been designed for organic and biological substrates for which simple analyses were not available. Nevertheless, few electrodes have been constructed for inhibitor determination. Other immobilized enzyme systems developed for analysis of enzyme inhibitors will be treated under reactor systems since they are generally used in a flow-through reactor configuration. On the other hand, if some type of electrode is used to measure the rate of an enzyme catalyzed reaction which takes place in solution, this is not a true enzyme electrode. The concept of an enzyme electrode was introduced by Hicks and Updike. The basic features of an I.E.P. are a thin enzyme layer held in close proximity to the active surface...

101 citations


Journal ArticleDOI
TL;DR: A detailed study of the phase transition effect on thermal stability of the enzymes and protein-protein interactions has been carried out and found that change of the catalytic activity and Thermal stability of N-PEC-bound penicillin amidase is fully reversible and reproducible.
Abstract: Penicillin amidase, α-chymotrypsin and urease have been immobilized in water-soluble nonstoichiometric polyelectrolyte complexes (N-PEC). N-PEC are formed by modified poly(N-ethyl-4-vinyl-pyridinium bromide) (polycation) and excess poly(methylacrylic acid) (polyanion). N-PEC are a new class of polymers capable, characteristically, of phase transitions solution ⇄ precipitate induced by slight change in pH or ionic strength. Neither the chemical structure of the carrier nor the number of cross-linkages between an enzyme and a carrier change on phase transition. That gives an unique opportunity to elucidate the difference between enzymes immobilized on water-soluble and water-insoluble supports. A detailed study of the phase transition effect on thermal stability of the enzymes and protein-protein interactions has been carried out. The following effects were found. 1 Pronounced thermal stabilization of penicillin amidase and urease may be achieved on two conditions: (a) the enzyme is in the precipitate; (b) the-enzyme is linked to the N-PEC nucleus. Then the thermal stability of N-PEC-bound penicillin amidase increases 7-fold at pH 5.7, 60°C, and 300-fold at pH 3.1, 25°C, compared to the native enzyme. For urease, the thermal stabilization increases 20-fold at pH 5.0, 70°C. 2 The localization of enzyme on N-PEC has been established by titration of α-chymotrypsin bound to a polycation or polyanion with basic pancreatic trypsin inhibitor. Both in solution (pH 6.1) and in N-PEC precipitate (pH 5.7), an α-chymotrypsin molecule bound to a polyanion is fully exposed to the solution. If the enzyme is bound to a polycation, only 20% of α-chymotrypsin molecules in the precipitate and 40% in solution retain their ability for protein-protein interactions. This means that a polycation-bound enzyme is localized in the hydrophobic nucleus of the complex, whereas the polyanion-bound enzyme sits on the hydrophilic shell of the complex. 3 On pH-induced phase transition (pH decreases from 6.1 to 5.7), there occurs a stepwise decrease in penicillin amidase activity which is due to a 9.8-fold increase in the Km for 2-nitro-4-phenylacetamidobenzoic acid. 4 Change of the catalytic activity and thermal stability of N-PEC-bound penicillin amidase is fully reversible and reproducible. Such soluble-insoluble immobilized enzymes with controllable thermal stability and activity may be used for simulating events in vivo and in biotechnology.

100 citations


Journal ArticleDOI
TL;DR: In this paper, an enzyme electrode with a chemically-amplified response for l -lactate is constructed from an oxygen electrode and a layer containing immobilized lactate oxidase, to oxidize l - lactate, and lactate dehydrogenase to regenerate the l − lactic acid.

98 citations


Journal ArticleDOI
TL;DR: Application a un systeme modele utilisant la phosphatase alcaline comme enzyme immobilisee, le p-nitrophenylphosphate comme substrat and the p-Nitrophenoxyde comme produit detecte.
Abstract: Application a un systeme modele utilisant la phosphatase alcaline comme enzyme immobilisee, le p-nitrophenylphosphate comme substrat et le p-nitrophenoxyde comme produit detecte

Journal ArticleDOI
TL;DR: Ethyl acetate was found to be the most effective organic solvent for the synthesis of this precursor of the synthetic sweetener aspartame in an organic solvent with immobilized thermolysin.
Abstract: N - (benzyloxycarbonyl) - L - aspartyl - L - phenylalanine methyl ester, the precursor of the synthetic sweetener aspartame, was continuously synthesized in an organic solvent with immobilized thermolysin. Ethyl acetate was found to be the most effective organic solvent for the synthesis of this precursor. The plug flow type reactor was found unsuitable, because the immobilized enzyme in it was gradually inactivated even if calcium, the essential stabilizing factor for thermolysin, was added to the substrate. In addition, a severe channeling of the flow occurred in a column. A stirred tank reactor was successfully operated for over 300 hours with a yield of approximately 90 percent and a large space velocity.

Journal ArticleDOI
TL;DR: Phenylalanine ammonia‐lyase immobilized within semipermeable microcapsules has an assayed enzyme activity which is 20% ± 4% of the enzyme in free solution, and enzyme activity is higher for the immobilized enzyme at the lower pH range.

Journal ArticleDOI
TL;DR: A simple method for the effective, covalent, immobilization of porcine liver esterase ( PLE) is described and the application of this reagent for the preparation of chiral building blocks on a 50 – 500 mmol scale is demonstrated.

Journal ArticleDOI
TL;DR: Dried spheres made from an alginate solution containing magnetite particles have excellent potential as a support for enzyme immobilization and chromatographic applications and were found to be much stronger than gels such as polyacrylamide and dextran, indicating that high flow rates and pressures could be used in column separations.
Abstract: Dried spheres made from an alginate solution containing magnetite particles have excellent potential as a support for enzyme immobilization and chromatographic applications. The beads were found to be much stronger than gels such as polyacrylamide and dextran, indicating that high flow rates and pressures could be used in column separations. The support withstood not only temperatures of up to 120 degrees C, but also most pH values and common solvents. While some solutions, such as phosphate buffers, dissolved the spheres, stabilization with Tyzor TE(R) eliminated this problem. The physical properties of the beads include a glasslike density of 2.2 g/mL, excellent sphericity, low porosity, and a narrow size distribution. The magnetite present in the support allows the beads to be used for magnetic separations such as high gradient magnetic filtration. Their high degree of microroughness provides a large exposed surface area for enzyme and ligand binding. Mixed Actinomyces fradiae proteases and Aspergillus niger alpha-amylase, two enzymes representative of classes which attack large substrates, were immobilized on the bead's surface with high activity and stability. A cyanuric dye which can be used in chromatographic applications (Cibacron Blue F3GA(R)) was also readily coupled to the surface of this support with good yield. The support should have a wide range of applications in bioseparation and immobilized biochemical technology.

Journal ArticleDOI
TL;DR: Results show that even for adsorption processes lasting almost 10 h, the majority of the enzyme is confined to the outer half of the support and, for high initial enzyme concentrations in the bath, this loading takes place as a slowly moving front.
Abstract: Enzyme adsorption from a finite bath (batch adsorption) onto porous spherical supports is investigated both experimentally and theoretically using β-galactosidase and Duolite ion-exchange resin as a model system. Efficient numerical techniques are presented that have been used in conjunction with a parameter estimation routine to evaluate adsorption isotherm constants. Results show that even for adsorption processes lasting almost 10 h, the majority of the enzyme is confined to the outer half of the support and, for high initial enzyme concentrations in the bath, this loading takes place as a slowly moving front. Information on the enzyme distribution has practical importance in the design of immobilized enzyme reactors that in previous works have almost always been analyzed assuming a uniform catalyst distribution.

Journal ArticleDOI
TL;DR: The major components of cellulase and d-xylanase complexes have been immobilized on glass beads activated by 3-aminopropyltriethoxysilane or 3-glycidoxypropyltrimethoxisilane to spread their optimum pH range and the major reaction products were identified as a d-glucose andd-xylose respectively.

Patent
26 Aug 1985
TL;DR: The peptidyl-glycine alpha-amidating monooxygenase (PA-MAOgenase) is an enzyme extractable from medullary thyroid carcinoma cell lines and tissue samples, having a molecular mass of about 60,000 to 65,000 daltons.
Abstract: Peptidyl-glycine alpha-amidating monooxygenase is an enzyme extractable from medullary thyroid carcinoma cell lines and tissue samples, having a molecular mass of about 60,000 to 65,000 daltons. It has been purified so as to exhibit a single, homogeneous, well-defined band using electrophoretic procedures performed on SDS-polyacrylamide gels, and has a specific enzymatic activity of at least 50mU per mg protein. The free or immobilized enzyme, in the presence of Cu+2 ions, ascorbate, and oxygen, can be used to prepare an alpha-amidated protein from a polypeptide substrate possessing a carboxyl-terminal glycine residue. The purified enzyme can be used as an antigen in order to produce enzyme-specific monoclonal antibodies, and can provide the information necessary to design and construct prokaryotes or other appropriate unicellular organisms or host cells isolated from multicellular organisms which possess peptidyl-glycine alpha-amidating capability.

Journal ArticleDOI
TL;DR: Acetylcholinesterase and choline oxidase were co-immobilized by reaction with glutaraldehyde onto alkylamino-bonded silica, which was incorporated as the enzyme reactor in an h.p.l.c. system for the determination of acetylcholine and Choline.

Journal ArticleDOI
TL;DR: Ascorbate oxidase from zucchini squash was immobilized onto CH-Sepharose via carbodiimide and the properties of the immobilized enzyme were found to be similar to those of the free ascorbate oxid enzyme.

Journal ArticleDOI
TL;DR: An ion exchange HPLC procedure was used to purify the galactose oxidase, in particular from catalase, and the kinetics and the selectivity of a reactor containing the immobilized enzyme was investigated.
Abstract: A flow injection manifold containing a dialyzer and reactors with immobilized galactose oxidase and peroxidase was used for the determination of galactose in urine, lactose in milk and dihydroxyacetone in a biotechnological reaction medium. The hydrogen peroxide which is formed by the galactose oxidase reaction was detected by amperometric reduction of a mediator. The latter had been produced from hydrogen peroxide in a peroxidase catalyzed reaction. The hydrogen peroxide detection step was studied with several mediators and hexacyanoferrate (II) was selected. An ion exchange HPLC procedure was used to purify the galactose oxidase, in particular from catalase, and the kinetics and the selectivity of a reactor containing the immobilized enzyme was investigated. Columns for removal of certain interferents such as ascorbic acid were used in the determination of galactose in urine. The response to galactose standards was linear from the detection limit of 2 μM to 60 mM. The throughput was 45 samples ...

Journal ArticleDOI
TL;DR: The interesterification activity of immobilized preparations was enhanced by the use of higher concentrations of the crude lipase or, more substantially, by admixture of purified lipase.

Journal ArticleDOI
Howard H. Weetal1
TL;DR: Studies indicate that maximum esterification of gallic acid occurs with amyl alcohol, and the enzyme shows broad alcohol specificity, however, the enzyme exhibits absolute specificity for the acid portion of the ester.
Abstract: Gallic acid esters of n-propyl and amyl alcohols have been produced by enzymatic synthesis in organic solvents using immobilized tannase. Studies indicate that maximum esterification of gallic acid occurs with amyl alcohol. The enzyme shows broad alcohol specificity. However, the enzyme exhibits absolute specificity for the acid portion of the ester. Studies were carried out on K(m), V(max), pH, and temperature optima.

Journal ArticleDOI
TL;DR: FireflyLuciferase from Photinus pyralis has been covalently bound to a collagen strip via an acylazide activation process and the results were found in good agreement with those obtained by soluble luciferase.
Abstract: Firefly luciferase from Photinus pyralis has been covalently bound to a collagen strip via an acylazide activation process. Immobilization performed in the presence of both substrates ATP and luciferin allows to increase the activity retained on the strip. The best activity exhibited by immobilized luciferase was obtained in a 0.05M Tris-acetate buffer, pH 7.75. The pH optimum and the activation energy of luciferase have been found unchanged after immobilization. In the chosen stirring conditions, no diffusional limitations of substrates appear. ATP measurements can be performed with collagen-bound luciferase in the range 1.10−11M−3.10−6M. It was possible to store the strips at 4°C in a dehydrated form; then, the bound enzyme retains 20% of its initial activity after eight months. Human blood ATP was measured with this collagen-bound luciferase and the results were found in good agreement with those obtained by soluble luciferase.

Journal ArticleDOI
Arturo Manjón1, Josefa Bastida1, C. Romero1, A. Jimeno1, J.L. Iborra1 
TL;DR: The efficient kinetic parameters shown by the most active and stable derivative enables it to be used for debittering of naringin-containing juices.
Abstract: Naringinase from Penicillium sp was covalently linked to Glycophasecoated controlled-pore glass The parameters of the immobilization process were characterized with respect to both the coupling method as well as to support pore size The efficient kinetic parameters shown by the most active and stable derivative enables it to be used for debittering of naringin-containing juices

Patent
30 Mar 1985
TL;DR: In this paper, the authors used amorphous, approximately spherical silica particles obtained from synthetic calcium silicate to bound enzymes to an inorganic carrier by an amino group through a bifunctional spacer.
Abstract: Immobilized enzymes covalently bound to an inorganic carrier by an amino group through a bifunctional spacer have improved properties when the carrier is formed of amorphous, approximately spherical silica particles obtained from synthetic calcium silicate. The silica particles have an average particle size of from 15 to 80 μm, an apparent particle volume of from 1.3 to 3 cm3 /g, and a specific surface area of from 250 to 800 m2 /g.

Journal ArticleDOI
01 Jan 1985-Analyst
TL;DR: Whole blood glucose measurements were correlated with a spectrophotometric method based on glucose oxidase and the derivatised signal allowed temporal of the enzyme-mediated signal from electrochemical interference.
Abstract: Glucose dehydrogenase (E.C. 1.1.99.17) has been used to produce an amperometric enzyme electrode for glucose. With phenazine ethosulphate as the electron mediator, a linear response for up to 0.8 mM glucose was obtained with response times of 3–5 min. 2,6-Dichlorophenolindophenol as mediator gave long (> 30 min) response times, but the measurement time was reduced to 30 s by monitoring the first-order differential of the response; the derivatised signal allowed temporal separation of the enzyme-mediated signal from electrochemical interference. Whole blood glucose measurements were correlated with a spectrophotometric method based on glucose oxidase (y= 0.84x + 0.67, r= 0.963, n= 36).

Journal ArticleDOI
TL;DR: A novel method for amperometric determination of substrates of hydrolytic enzymes has been developed and the pH dependence of electrochemical oxidation of hydrazine in the Tafel region was combined with the urease catalysed splitting of urea to construct an amPerometric membrane electrode for urea.


Journal ArticleDOI
Howard H. Weetall1
TL;DR: Enzymes immobilized on inorganic supports by covalent attachment show unique pH optimums, thermal profiles and kinetics, and are characterized and used for several applications.