Showing papers on "Immobilized enzyme published in 1979"

Immobilization of enzymes and microbial cells using carrageenan as matrix.

Abstract: Conditions for the gelation k-carrageenan, which is a new polymer for immobilization of enzymes and microbial cells, were investigated in detail k-Carrageenan was easily induced to gel by contact with metal ions, amines, amino acid derivatives, and water-miscible organic solvents By using this property of k-carrageenan, the immobilization of enzymes and microbial cells was investigated Several kinds of enzymes and microbial cells were easily immobilized with high enzyme activities Immobilized preparations were easily tailor-made to various shape such as cube, bead, and membrane The obtained immobilized preparations were stable, and columns packed with them were used for continuous enzyme reaction for a long period Their operational stabilities were enhanced by hardening with glutaraldehyde and hexamethylenediamine

Enzyme electrode provided with immobilized enzyme membrane

Abstract: An enzyme electrode comprising an anode, a cathode, an immobilized enzyme membrane, and an electrolyte, which is characterized in that said membrane consists of a dense skin layer having selective hydrogen peroxide permeability and a porous layer wherein the enzyme is immobilized onto the porous layer of the membrane and said immobilized enzyme membrane is arranged in the electrode so that the skin layer is faced to the electrode and the porous layer immobilized with the enzyme is in the position of contacting with the test solution. The enzyme electrode is used for the polarographic determination of a slight amount of components in body fluid, body tissue, foodstuffs, or the like without any undesirable hindrance with low molecular interfering materials without using any other specific means for removing such low molecular interfering materials.

A NEW ANALYTICAL METHOD OF INDIVIDUAL BILE ACIDS USING HIGH PERFORMANCE LIQUID CHROMATOGRAPHY AND IMMOBILIZED 3α-HYDROXYSTEROID DEHYDROGENASE IN COLUMN FORM

Abstract: We studied the use of immobilized enzyme, covalently bound to aminopropyl-CPG, to the analysis on free and conjugated individual bile acids. A microcolumn with the immobilized 3α-hydroxysteroid dehydrogenase was prepared and used in a system of high performance liquid chromatography (HPLC). Reduced NAD+ produced from bile acid by this enzyme was fluorimetrically determined with fluorometer. As the result, the free, glycine- and taurine-conjugated individual bile acids which were separated by the HPLC were determined on high sensitivity and specificity. Clinical trials were done for the serum of patients with hepatobiliary disease.

Support matrices for immobilized enzymes

Abstract: An improved method of preparing support matrices for immobilization of reactive chemical entities, such as enzymes, comprises deposition of a polyamine on a core support, such as an inorganic oxide, contacting the polyamine-coated core support with a bifunctional reagent which cross-links the polyamine and provides pendant functional groups, and recovering the matrix, wherein the improvement comprises deposition of the polyamine as a thin, uniform film from solvents of low surface tension.

Hydrolysis of organophosphate insecticides by an immobilized‐enzyme system

Abstract: An enzyme preparation that could detoxify parathion and eight other organophosphate pesticides was covalently bound to either porous glass or porous silica beads. This immobilized-enzyme system was examined for its use in detoxification of pesticides in production wastewaters. The kinetics of parathion hydrolysis were examined at flow rates up to 96 liter/hr and at influent substrate concentrations ranging from 10--250 mg/liter. The enzyme reactor was able to hydrolyze 95% or more of the parathion added to industrial wastewaters generated during its production, thus reducing the effluent parathion concentration to below 500 ppb. Laboratory continuous-flow experiments were conducted for 70 days with industrial wastewater and indicated no loss in immobilized-enzyme activity. The influence of pH, temperature, solvents, and detergents on enzyme stability and activity and enzyme reactor kinetics will be discussed.

Asymmetrical Coupling of Enzymatic Systems on Collagen Membranes Application to Multienzyme Electrodes

Abstract: An original method of preparation of enzyme collagen membranes has been developed which enables the asymmetrical coupling of multienzymatic systems working in sequence. One or several enzymes are immobilized on one face of the acyl-azide activated membranes, another enzyme being bound on the other face. When the first enzymatic face is exposed to the adequate reaction mixture, the products diffuse through the collagen membrane and act as substrates for the enzyme present on the other side. This possibility has been applied to the design of new and polyvalent multienzyme electrodes.

Immobilization of enzymes on activated carbon: selection and preparation of the carbon support.

Abstract: Based upon its superior catalytic activity for H2O2 decomposition, a bituminous coal-based activated carbon was selected for investigations of pretreatment and enzyme immobilization methods. Pretreatments considered include acid washing, exposure to strong oxidizing agents, contact with concentrated peroxide solutions, nitration and amination, isothiocyanate derivatization, silanization, and stearic acid coating. Effects of these pretreatments on morphology and trace-metal content of the carbon pellets have been studied using scanning electron microscopy and dispersive analysis of x rays. Immobilization of glucoamylase by adsorption, glutaraldehyde crosslinking, and covalent attachment to carbon activated by water-soluble diimide or diazotization have been examined. These different enzyme-carbon catalysts have been characterized by their enzyme loading, enzyme activity, catalytic activity for H2O2 decomposition, or combinations of these measures of performance.

Immobilized hydroxysteroid dehydrogenases for the transformation of steroids in water–organic solvent systems

Abstract: The hydroxysteroid dehydrogenases: beta-HSDH, 20 beta-HSDH, and 3 alpha-HSDH, were immobilized on CNBr-activated Sepharose. The effect of various immobilization conditions on the activity recovery and stability were examined. The presence of cofactor during the immobilization reaction increased the activity recovery (40--60% of the total) and also led to materials highly stable in the presence of organic solvents. For example, beta-HSDH maintained 60% of its original activity two months after continuous use in the water--ethyl-acetate system. Kinetic experiments showed that the increase of the apparent Km values is poor and demonstrated that the organic solvent behaves as a weak inhibitor (ki greater than 0.2M) for the substrate. The immobilized enzymes lyophilized in the presence of sucrose had full activity restored even after several months storage at room temperature. Immobilized hydroxysteroid dehydrogenases were shown to be suitable for preparative transformation of steroids in water--organic solvent systems.

Enzyme immobilization techniques on poly(glycidyl methacrylate-co-ethylene dimethacrylate) carrier with penicillin amidase as model.

Abstract: Two types of bead-form macroporous carriers based on glycidyl methacrylate with ethylene dimethacrylate copolymers were used for the immobilization of penicillin amidase either directly or after chemical modification. Direct binding through oxirane groups, which is equally efficient at pH 4.2 and 7, is relatively slow and brings about an activity loss at low enzyme concentrations. The most efficient immobilization was achieved on glutaraldehyde-activated amino carrier, irrespective of whether the amino groups were formed by ammonia or 1,6-diaminohexane treatment of the original oxirane carrier. Hydrazine treatment gave lower immobilization yields. The same is true of the azide method independent of the length of the spacer. Most enzyme activity was preserved by coupling the carbodiimide-activated enzyme to the carrier with alkyl or arylamino groups at the end of a longer substituent. Immobilization on diazo-modified carrier gave average results. Rapid immobilization by a lysine-modified phosgene-treated carrier resulted in an activity loss. It is suggested that multipoint and very tight attachment of the enzyme molecule to the matrix decreased the activity. The immobilized activity is quite stable in solution and very stable upon lyophilization with sucrose.

The use of immobilized enzymes in the food industry: A review

Abstract: The production of high fructose corn syrups was greatly facilitated by the use of immobilized glucose isomerase. Similarly, in Japan, the fermentation industry proved its processing efficiency for amino acids through the use of immobilized amino acid acylase. This article discusses the use of soluble enzymes in the food industry followed by a section on the various available methods to immobilize enzymes. Once enzymes are immobilized, many of their operational parameters could be altered. Rationale for the determination of the effects of immobilization is provided. A relatively new concept is the use of a single matrix for immobilizing more than one enzyme. Immobilized multi-enzyme systems offer many attractive advantages; however, such a process also raises some interesting questions about kinetics. These questions and their suggested answers are discussed in the penultimate section. The major emphasis of this article is on the use of immobilized enzymes in the food industry. Two systems--amino acylase and glucose isomerase--have been demonstrated to be techno-economically feasible. Immobilization of other enzymes, such as glucoamylase, lactase, protease, and flavor modifying enzymes, has received some attention. The potential of these new systems are also discussed.

Deacylation of benzylpenicillin by immobilized penicillin acylase in a continuous four‐stage stirred‐tank reactor

Abstract: Immobilized penicillin acylase has been used for the deacylation of benzylpenicillin at 37°C in a continuous reactor consisting of four 1 liter stirred tanks connected in series. There was good agreement between the predicted and actual conversions obtained in each tank under steady-state conditions. The operational stability of the immobilized enzyme in the tanks depended on the pH and the rate of addition and concentration of alkali needed to neutralize the acid produced during the reaction. At pH 7 with the addition of 2M NaOH, the half-life for enzyme stability was greater than 400 hr in all tanks. This was over half the value for the immobilized enzyme when stored at 37°C and pH 7.

Use of an enzyme thermistor in continuous measurements and enzyme reactor control.

Abstract: The enzyme thermistor measures the heat produced by the action of an immobilized enzyme on a substrate present in the sample. Its application in analysis of discrete samples, e.g., in clinical chemistry, is well documented, but it has not been used so far for continuous measurements. We decribe here the application of the enzyme thermistor for continuous monitoring and control of enzyme reactors. An enzyme thermistor filled with coimmobilized glucose oxidase and catalase was used to measure the amount of glucose in the outflow from a column reactor containing immobilized lactase acting on a lactose solution pumped through the reactor. The lactose conversion was kept on a constant level, irrespective of the actual enzymatic activity in the reactor, by regulating the flow through the reactor. The experiments were carried out with aqueous solutions of lactose as well as with whey from cow's milk.

Enzyme thermistor analysis in clinical chemistry and biotechnology

Abstract: The combination of calorimetry and immobilized enzymes forms a pair with unique properties. Calorimetry is a general detection principle and specificity is introduced by the enzyme. We discuss here simple "semi-adiabatic' calorimeters and their application in clinical chemistry, enzyme immunoassay, process control and environmental control.

Improved nonporous magnetic supports for immobilized enzymes.

Abstract: Ni powders coated by deposition of TiO2 or controlled oxidation to NiO develop substantial resistance to corrosion. Chymotrypsin immobilized to these coated Ni supports shows very high stability of activity on storage. Chymotrypsin immobilized by adsorption and glutaraldehyde crosslinking was fairly rapidly eluted under operational conditions in the presence of substrate. If 3-aminopropyltriethoxysilane (APS) was used to produce a covalent linkage, desorption of enzyme still occurred because of relatively unstable bonding of the silane to the oxide surface. A more stable attachment was produced by joining together many silane links with a layer of polyglutaraldehyde. The mechanism of action of APS as a coupling agent under these conditions is discussed. γ-Fe2O3, and particularly a Mn-Zn ferrite, are suitable magnetic support materials available with smaller particle sizes. Particles below 1 μm give the expected higher specific activities of immobilized enzymes.

Enzyme immobilization by radiation-induced polymerization of 2-hydroxyethyl methacrylate at low temperatures.

Abstract: Enzyme immobilization by radiation-induced polymerization of hydrophilic glass-forming monomers, such as 2-hydroxyethyl methacrylate, was studied. Enzyme radiation damage could be sufficiently retarded at low temperatures. The immobilized enzyme activity yield was markedly higher at low temperature than at higher temperature polymerization. At low temperatures the polymerized composite had a porous structure owing to ice crystallization which depends on the monomer concentration. It was deduced that the enzyme was partially trapped on the polymer surface, partially isolated in the pore, and partially occluded inside the polymer matrix. A decrease in activity caused by enzyme leakage was observed with repeated use in enzyme reactions where the composites had a large porosity. The activity yield showed a maximum at certain optimum porosities, i.e., at optimum monomer concentrations. Continuous enzyme reaction was preferably carried out using immobilized enzyme columns.

Urease bound to chitin with glutaraldehyde

Abstract: The enzyme urease (urea amidohydrolase, EC 3.5.1.5) prepared from Cajanus indicus, has been immobilized with glutaraldehyde-treated chitin as the solid support. The immobilized enzyme was characterized by determining the pH profiles and optimum temperature. Effect of glutaraldehyde concentration on the binding of enzyme to chitin was studied. The storage stability of the chitin-urease system was determined.

Cross-linked enzyme membrane

Abstract: A cross-linked enzyme membrane is prepared by directly adsorbing enzymes into the pores of a microporous non-fibrous filter membrane made of a silica modified vinylchloride polymer and then cross-linking the enzyme with a bi-functional coupling agent whereby enzyme molecules are cross-linked to each other without chemically bonding the enzyme molecules to the membrane. The cross-linked enzyme membrane is used to carry out enzymatic reactions by passing a solution of substrate for the enzyme through the membrane under a pressure differential.

Immobilized enzyme electrodes for the potentiometric measurement of glucose concentration: immobilization techniques and materials.

Abstract: Glucose oxidase, catalase, and bovine serum albumin were co-immobilized with glutaraldehyde around a platinum screen or around a single platinum—iridium wire. The potential difference between this dual enzyme electrode and a Ag/AgCl reference electrode was proportional to the logarithm of the glucose concentration over the range from 10 to about 150 mg glucose per 100 ml in buffered solution at pH 7.4 and 37°C. The enzyme electrode responded in serum only if coated with a semipermeable film, such as cellulose acetate, to exclude serum macromolecules. The potentiometric results were similar to those obtained with the two enzymes co-immobilized in polyacrylamide gel around a platinum screen or with only one of the enzymes, glucose oxidase, covalently coupled to a platinum screen. The results so far suggest that these potentiometric enzyme electrodes may have sufficient specificity for glucose for development of a continuous in vivo glucose sensor.

Immobilized enzymes in continuous-flow analysis.

Abstract: Glucose oxidase, uricase, and urease were immobilized on the interior surface of activated polyamide tubing. The shelf-life of such enzyme bearing tubes was at least six months. The tubes were used for continuous-flow analysis of glucose, uric acid, and urea with conventional systems and with hybrid micro-scale systems in which modules of different manufacture were combined. The length of enzyme-bearing tube required for each system was ascertained empirically. Each tube could be used for several thousand assays, but glucose oxidase-bearing tubes were more stable than urease- or uricase-bearing tubes. Results for patients' samples correlated well with results obtained by accepted methods.

Properties and uses of immobilized light-emitting enzyme systems from Beneckea harveyi.

Abstract: Bacterial luciferase and NADH:FMN oxidoreductase have been immobilized onto arylamine glass beads. These immobilized enzymes can detect as little as 0.2 pmol of NADH per assay sample. Glucose-6-phosphate dehydrogenase has been co-immobilized with these enzymes, and with this system it is possible to quantitate 1 pmol of glucose 6-phosphate. By co-immobilizing a fourth enzyme, hexokinase, onto the glass beads, the system can reproducibly detect 20 pmol of glucose per liter. These immobilized enzyme systems are potentially superior to soluble enzymes by being reusable and much more stable. We compared the light-emitting properties of the immobilized enzyme systems with that of an equivalent mixture of the soluble enzymes. The most striking difference was the apparently more efficient conversion of NADH or glucose 6-phosphate to light by the immobilized enzymes. We used hydroxysteroid dehydrogenase in developing a soluble coupled system for the assay of androsterone and testosterone. The lower limit of detection was 100 pmol.

Immobilized enzyme membrane

Abstract: An immobilized enzyme membrane for use at the working face of an electrochemical electrode is prepared which comprises an asymmetrical membrane integrally formed from a skin layer substantially incapable of permeating an enzyme therethrough but capable of permeating a gas and a liquid, and a sponge layer having pores containing an enzyme immobilized therein by crosslinking and which pores intercommunicate with one another throughout the sponge layer and provide sufficient porosity for retaining a necessary amount of the enzyme. The immobilized enzyme membrane contains a large amount of enzyme, has good diffusion and permeability, and has stabilized enzyme activity for a prolonged period of time. Additionally, there is obtained a quick response time and good analytical precision when the immobilized enzyme membrane is used at the working face of an electrode of electrochemical measuring instruments.

Fermentation and enzyme technology

Abstract: Outlines the fundamental microbiological, biochemical, genetic, and engineering aspects of fermentation, presenting advanced methods of fermentation and control. Covers the isolation of enzymes, especially those found in intracellular contents of microorganisms. Discusses enzyme immobilization and the factors influencing the use of enzymes in reactors.