Showing papers on "Immobilized enzyme published in 1974"

A theoretical model for enzymatic catalysis using asymmetric hollow fiber membranes

Abstract: The behavior of an immobilized enzyme reactor utilizing asymmetric hollow fibers is simulated using a theoretical model. In this reactor, an enzyme solution contained within the annular open-cell porous support structure of the fiber is separated from a substrate flowing through the fiber lumen by an ultrathin dense membrane impermeable to enzyme but permeable to substrate and product. The coupled set of model equations describing the behavior of this reactor represents an extended Graetz problem in the fiber lumen, with diffusion through the ultrathin fiber skin and reaction in the microporous sponge region. Exact analytic expressions for substrate concentration profiles throughout an idealized fiber which incorporate the membrane and hydrodynamic mass transfer resistances are obtained for a first-order enzyme reaction, and numerical techniques for their evaluation are given. This analysis is extended to yield a numerical finite difference solution for nonlinear Michaelis-Menten reaction kinetics, which is shown to agree with the analytic solution, as Km/C0, the ratio of the Michaelis constant to the initial substrate concentration, becomes large (> 100).

Effect of diffusional limitations on lineweaver‐burk plots for immobilized enzymes

Abstract: Lineweaver-Burk plots of reaction rate data obtained with immobilized enzymes need not be linear even when intrinsic enzyme kinetics follow the simple Michaelis-Menten rate expression. Theoretical calculations show that mass transfer effects may cause curvature which is concave or convex to the abscissa, depending upon experimental conditions. Consequently, graphical procedures commonly employed for analysis of soluble enzyme kinetics may yield misleading results when applied to immobilized enzymes. Three approaches which follow from the behavior of numerical and asymptotic solutions to the problem are proposed for extraction of intrinsic kinetic information.

Simultaneously immobilized glucose oxidase and catalase in controlled‐pore titania

Abstract: The immobilization of glucose oxidase and catalase by adsorption within the pores of controlled-pore titania has yielded a remarkably stable enzyme system. Catalase apparently acts as both a stabilizer and an activator for glucose oxidase within the pores of this material. Hydrogen peroxide concentrations and flow rates have a marked effect upon the apparent activity of the immobilized enzyme system. The carrier parameters were varied to obtain optimum loading and stability information.

Immobilized biochemicals and affinity chromatography.

Abstract: One: Affinity Chromatography.- Affinity Chromatography--Old Problems and New Approaches.- Affinity Chromatography. New Approaches for the Preparation of Spacer Containing Derivatives and for Specific Isolation of Peptides.- Quantitative Parameters in Affinity Chromatography.- Non-Specific Binding of Proteins by Substituted Agaroses.- A Solid Phase Radioimmune Assay for Ornithine Transcarbamylase.- Purification of Acetylcholinesterase by Covalent Affinity Chromatography.- Cooperative Effects of AMP ATP, and Fructose 1,6-Diphosphate on the Specific Elution of Fructose 1,6-Diphosphatase from Cellulose Phosphate.- An Analysis of Affinity Chromatography Using Immobilised Alkyl Nucleotides.- Affinity Chromatography of Kinases and Dehydrogenases on Sephadex and Sepharose Dye Derivatives.- Affinity Chromatography of Thymidylate Synthetases Using 5-Fluoro-2?-Deoxyuridine 5?-Phosphate Derivatives of Sepharose.- The Biosynthesis of Riboflavin: Affinity Chromatography Purification of GTP-Ring-Opening Enzyme.- Purification of Tyrosine-Sensitive 3-Deoxy-D-Arabino-heptulosonate-7-Phosphate and Tyrosyl-tRNA Synthetase on Agarose Carrying Carboxyl-Linked Tyrosine.- Structural Requirement of Ligands for Affinity Chromatography Absorbents: Purification of Aldehyde and Xanthine Oxidases.- Two: Immobilized Biochemicals.- Immobilized Polynucleotides and Nucleic Acids.- Immobilized Cofactors and Multi-Step Enzyme-Systems.- Preparation, Characterization, and Applications of Enzymes Immobilized on Inorganic Supports.- Lactase Immobilized on Stainless Steel and Other Dense Metal and Metal Oxide Supports.- The Use of Membrane-Bound Enzymes in an Immobilized Enzyme Reactor.- The Optimization of Porous Materials for Immobilized Enzyme Systems.- Water Encapsulated Enzymes in an Oil-Continuous Reactor: Kinetics and Reactivity.- Analysis of Reactions Catalyzed by Polysaccharide-Enzyme Derivatives in Packed Beds.- The Preparation of Microenvironments for Bound Enzymes by Solid Phase Peptide Synthesis.- Optimization of Activities of Immobilized Lysozyme, ?-Chymotrypsin, and Lipase.- Chemical Modification of Mushroom Tyrosinase for Stabilization to Reaction Inactivation.- Chain Refolding and Subunit Interactions in Enzyme Molecules Covalently Bound to a Solid Matrix.- Immobilization of Lipase to Cyanogen Bromide Activated Polysaccharide Carriers.- Use of Immobilized Enzymes for Synthetic Purposes.

Influence of intraparticle diffuisional limitation on the observed kinetics of immobilized enzymes and on catalyst design

Abstract: Numerical solutions to the equations describing simultaneous mass transfer and enzymic reaction within porous spherical particles have been used to examine the effect of enzyme content and other parameters on the kinetic behavior of immobilized enzymes. These solutions have also been compared with experimental data for enzymes immobilized to DEAE-cellulose particles. The influence of particle size and enzyme content on catalyst design is discussed.

Immobilized enzymes in food and microbial processes

Abstract: Present and Future Trends in Enzyme Technology and Its Application.- Immobilized Enzyme in Milk Systems.- Preparation and Application of Immobilized ss-Galactosidase of Saccharomyces lactis.- The Use of Tannic Acid and Phenol-Formaldehyde Resins with Glutaraldehyde to Immobilize Enzymes.- The Uses of Precipitated Nylon as an Enzyme Support: An ?-Galactosidase Reactor.- Glucose Isomerase Cells Entrapped in Cellulose Acetates.- Glucose Isomerase: A Case Study of Enzyme-Catalyzed Process Technology.- Immobilized ?-Amylase for Clarification of Colloidal Starch-Clay Suspensions.- Immobilized Glucose Oxidase and Catalase in Controlled Pore Titania.- Collagen as a Carrier for Enzymes: Materials Science and Process Engineering Aspects of Enzyme Engineering.- The Immobilization of Enzymes with Imidoester-Containing Polymers.- Basic Concepts in the Effects of Mass Transfer on Immobilized Enzyme Kinetics.- A Comparison of Proposed Methods for the in vitro Synthesis of Edible Carbohydrates.- Biological Technology-Plea for a New Commitment.- Contributors.

Immobilized enzyme reaction stability: Attrition of the support material

Abstract: One of the main reasons for immobilizing an enzyme is to enable its reuse, or continuous use, in a reactor. Consequently immobilized enzyme stability is an important factor in enzyme reactor design. The performance of the reactor will decrease if during operation the support material disintegrates into smaller particles that pass out of the reactor system. When β-galactosidase is immobilized by covalent attachment to AE-cellulose, the smaller particles have a higher activity. After subjection of the immobilized enxyme to a shear stress the average particle size decreases and the total enzymic activity increases. A loss of small particles from the reactor, although constituting a small weight percent loss of support, will result in a disproportionately large loss in activity. The relevance of these observations to reactor performance is discussed.

Chemically modified nylons as supports for enzyme immobilization. Polyisonitrile-nylon

Abstract: Four-component condensations between amine, carboxyl, isocyanide and aldehyde lead to the formation of N-substituted amides (Ugi, 1962). The present paper describes the use of such condensations for the introduction of chemically reactive groups on to the polyamide backbone of nylon. Polyisonitrile-nylon was synthesized by partial hydrolysis of nylon-6 powder, followed by resealing of the newly formed -CO(2)... NH(2) (-) pairs via a four-component condensation, by using acetaldehyde and 1,6-di-isocyanohexane. Polyisonitrile-nylon could also be converted into a diazotizable arylamino derivative, polyaminoaryl-nylon, by a four-component condensation by using a bifunctional amine, pp'-diaminodiphenylmethane, in the presence of an aldehyde and a carboxylate compound. The versatility of four-component condensations involving the isocyanide functional group of polyisonitrile-nylon allowed coupling of proteins, in an aqueous medium at neutral pH, through either their amino or carboxyl groups. Trypsin and papain were bound to polyisonitrile-nylon through their amino groups by a four-component condensation by using acetaldehyde and acetate; conversely, succinyl-(3-carboxypropionyl-)trypsin, pepsin and papain were coupled through their carboxyl groups in the presence of acetaldehyde and an amine (Tris). Diazotized polyaminoaryl-nylon could be utilized for the immobilization of papain, via the tyrosine residues of the enzyme.

SYMPOSIUM: Immobilized Enzymes in Food Systems

Abstract: Intrinsic kinetics of an immobilized enzyme can be different from that of soluble enzyme due to changes of the macromolecule caused by immobilization. Also transport of substrate to enzymic sites on the inert support creates a hindrance to overall reaction. Such diffusional effects on immobilized enzyme reactions were analyzed in terms of effectiveness factor and film factor employing the theories developed earlier in heterogeneous catalysis. The pore diffusion effect in the design of an integral reactor was analytically treated. A brief discussion was given to the diffusional effects in relation to common kinetic studies of immobilized enzymes.

SYMPOSIUM: Immobilized Enzymes in Food Systems

Abstract: Economics of immobilization and characteristics of immobilized enzymes and substrates which are important in treatment of foods are discussed. Methods of immobilizing enzymes and activity and stability of enzymes which may be used in food processing and analyses are controlled and limited by the properties of foods. Specific immobilized enzymes which have been used or show promise for use in food processing and analysis are described.

Immobilization of enzymes by radiopolymerization of acrylamide

Abstract: A new and simple method for immobilization of enzymes by the aerobic radio-polymerization of acrylamide was developed. Irradiation treatment of acrylamide in the frozen state produces a spongy immobilized enzyme membrane without the addition of carriers. Aerobic polymerization yields of acrylamide in the frozen state were increased by the addition of starch and also by lyophilization. Glucose oxidase (activity recovery was 12.3–33.7%), invertase (69.2%), D-amono acid oxidase (25.0–70.5%), aminoacylase (39.2–43.7%), mold α-amylase (18.0%), malt β-amylase (4.1%), glucoamylase (6.5%), alkaline protease (5.3%), and neutral protease (10.5%) were immobilized by this method. Invertase entrapped by this method had a wider optium pH range and was active at higher temperatures.

An immobilized alpha-galactosidase continuous flow reactor.

Abstract: An α-galactosidase which will hydrolyze the oligosaccharides melibiose, raffinose, and stachyose has been immobilized on nylon microfibrils suitable for use in large flow-through reactors. This catalyst system is stable for many months, both under use and storage conditions. The immobilized enzyme behaves similarly to the enzyme in solution, characteristically exhibiting both product and substrate inhibition. The catalyst is prepared in situ and a large, 8-liter reactor has been made. The catalyst has been used to reduce the raffinose concentration in beet sugar molasses.

Covalent bond between the enzyme amyloglucosidase and a porous glass carrier: The effect of shearing

Abstract: The utility of an immobilized enzyme depends not only on initial loading but also on operational half-life. The loss of activity of an immobilized enzyme in a column reactor may occur in several ways. It is therefore of interest to determine whether the decay in activity is due to the rupture of any of the enzyme-carrier bonds in the composite. In order to do this, it is first necessary to establish whether the enzyme on the composite is in fact covalently bound or adsorbed. In this report we have shown that there are a number of covalent links per protein molecule. We have also shown that the bond energies are sufficient to prevent shearing of the enzyme itself from the composite under any stresses which may occur in a chromatography column or packed-bed reactor during continuous operation. The effect of the siloxane linkage on composite stability is also discussed.

Immobilized enzymes in the production of radiopharmaceutically pure amino acids labeled with 13N.

Abstract: Nitrogen-13 and 11C compounds may be obtained by enzymatic synthesis but the frequent presence of potentially pyrogenic and antigenic enzymes in the final product creates problems of radiopharmaceutical purity. This report describes methods for the synthesis of pharmaceutical-quality 13N-labeled amino acids by binding the required enzymes to a solid-state support. Nitrogen-13-L-glutamic acid was synthesized on stream by using glutamic acid dehydrogenase which had been immobilized on porous derivatized (N-hydroxysuccinimide) silica beads and packed into a column. Nitrogen-13-L-alanine was synthesized by adding pyruvate to 13N-L-glutamic acid and passing this mixture through a second column of similar silica beads containing the immobilized enzyme glutamic-pyruvic transaminase.

Immobilized D-Amino Acid Oxidase Preparation, Some Enzymatic Properties, and Potential Uses

Abstract: Immobilization of d-amino acid oxidase was investigated by covalently binding the enzyme to cyanogen bromide activated polysaccharides. Among polysaccharides tested, Sepharose 6B was found to be the best carrier.Some enzymatic properties of the immobilized enzyme were investigated and compared with those of the native enzyme. The optimum pH of the immobilized enzyme was shifted by 0.5 pH units to the acid side in comparison with that of the native enzyme. With regard to substrate specificity, heat stability and effect of temperature, no significant differences were observed between the immobilized and native enzymes.The immobilized enzyme was conveniently used for a determination of d-amino acids and an analysis of optical purity of l-amino acids.

Characterization and immobilization of E. coli (ATCC-26) Beta-galactosidase.

Abstract: The β-galactosidase from Escherichia coli ATTCC-26 was partially purified and characterized. It was found to be comparable to galactosidases from other E. coli strains in stability, pH and temperature maxima, and activity requirements, but it had a more favorable ratio of activity toward lactose versus synthetic substrates. The galactosidase was immobilized on porous glass beads by three covalent bonding methods. Kinetic data for the free and bound enzymes were determined using natural and synthetic substrates. Activity characteristics of the free and immobilized enzymes were comparable, however, the bound forms were less stable to heat.

SYMPOSIUM: Immobilized Enzymes in Food Systems

Abstract: Immobilized multienzymic systems are responsible for the high efficiency of many physiological reaction sequences carried out in living cells. Chemical and physical means for artificially immobilizing enzymes presents the opportunity for duplicating this efficiency for use in processing operations. Such artificially immobilized enzymes also serve as useful model systems for studying the properties of physiological multi-enzymic sequences. Factors characteristic of the immobilized multi-enzymic state are described. Comparisons are made between immobilized multi- and single-enzyme systems and between immobilized and soluble multi-enzymic ones.

Immobilized enzyme catalysis with reaction‐generated pH change

Abstract: Many enzyme-catalyzed reactions involve the liberation or consumption of hydrogen ions. In this paper a mathematical model is employed to investigate how such reactions behave when the enzyme is immobilized. Shifted pH optima, disappearance of an optimum pH, insensitivity to bulk pH, and very large effectiveness factors are some of the phenomena which appear as a result of pH coupling between the reaction and the enzyme's activity. Several of the qualitative features revealed by the model are consistent with earlier experimental observations. In addition, preliminary guidelines for optimal choice of enzyme support are suggested.

Preparation and properties of asparaginase entrapped in the lattice of polyacrylamide gel.

Abstract: Summary Immobilization of asparaginase (l-asparagine amidohydrolase, EC 3.5.1.1) was investigated by lattice entrapment using hydrophilic polyacrylamide gel. As a result, about a 30% yield of immobilized asparaginase was obtained. The optimum pH of the immobilized asparaginase shifted by 1 pH unit to the acid side in comparison with that of native enzyme. The apparent Michaelis constant of immobilized enzyme was about 200 times higher than that of the native enzyme. The immobilized asparaginase was found to be stable during continuous operation and to be resistant to attack by proteolytic enzymes. l-Asparagine in blood was completely decomposed by the immobilized asparaginase column.

Preparation of immobilized enzymes by N-vinylpyrrolidone and the general properties of the glucoamylase gel.

Abstract: Immobilized glucoamylase, invertase, and β-galactosidase were prepared by using N-vinylpyrrolidone monomer (VP) under γ-ray irradiation. The enzyme-VP solutions were gelled by irradiation with 2.9 Mrad and the added enzymes were almost completely entrapped. Activity losses on entrapping were 55% for the VP-glucoamylase gel, and more than 90% in the case of VP-invertase and VP-β-galactosidase gels. No leakage of enzyme from these gels could be detected within 1 hr. The VP-glucoamylase gel was capable of hydrolyzing dextrin (mol wt 10,400) to glucose and the glucose equivalent was equal to that obtain able with native enzyme. The optimum temperature, heat stability, pH activity curve, and pH stability of VP-glucoamylase gel were slightly inferior to those of native enzyme, while Km was a little larger than that of native enzyme.

Covalent coupling of alkaline bacillus subtilis protease to controlledpore silica with a new simplified coupling technique

Abstract: A controlled-pore silica with an average pore diameter of 510 A° was used as the carrier for the immobilization of alkaline Bacillus subtilis protease. The coupling of the enzyme was accomplished by a simple two-step procedure which involved the aqueous reaction of a bifunctional diazonium salt with the surface of the silica followed by the coupling of the enzyme through the remaining available azo functional group. The stability of the coupled enzyme was compared to that of an adsorbed enzyme produced under similar conditions. Both immobilized enzymes were stored in water at room temperature and were repeatedly assayed at intervals up to 103 days. These protease preparations were assayed with 1% casein solutions at pH 7.8 at 37°C. Under these conditions, the adsorbed enzyme exhibited a half-life of approximately 48 days, while that of the coupled protease was somewhat greater than 80 days.

Poly (urea-urethane) foams containing immobilized active enzymes

Abstract: An active, immobilized enzyme system is formed from an enzyme and a hydrophilic poly (urea-urethane) foam, said foam surrounding, entrapping and supporting the enzyme in an active configuration The hydrophilic foam is formed by the reaction of water with a hydrophilic isocyanate-terminated polyoxyalkylene prepolymer containing at least 50 mole percent of oxyethylene in the prepolymer backbone

Preparation of an immobilized two-enzyme system, Beta-amylase--pullulanase, to an acrylic copolymer for the conversion of starch to maltose. III. Process kinetic studies on continuous reactors.

Abstract: Kinetic studies on the parameters influencing the potential industrial application of an immobilized two-enzyme system of β-amylase and pullulanase for conversion of starch to a product with high maltose content, have been performed. The apparent Michaelis constant, the apparent product inhibitor constant, and the activation energy have been determined for the immobilized preparation and compared to the values for the corresponding soluble enzyme system. The catalytic activity of the immobilized enzymes was studied in a plug-flow reactor and a continuous feed stirred tank reactor. Mathematical models for these reactors have been formulated and adapted to fit the experimental data. Comparisons of the reactor efficiencies were made and the conditions were found to be such as to favor the plug-flow reactor. Results on operational stability tests at different temperatures and substrate concentrations are given.