Showing papers on "Immobilized enzyme published in 1987"
TL;DR: In this article, a dimethylferrocene-mediated enzyme electrode for glucose was evaluated for application to microbial fermentation monitoring and control, and the deficiencies revealed - insufficient stability, progressive increase in response time and progressive decrease in linear range - were investigated using electrochemical and radiochemical techniques.
248 citations
TL;DR: This paper discusses recent work on the immobilisation of glucose oxidase in polypyrrole, poly-N-methylpyr role, polyaniline and polyphenol films electrochemically grown at the electrode surface and on the covalent attachment of redox mediators to glucose oxidation in order to achieve direct electron transfer to the electrode.
215 citations
TL;DR: Control of the amount of water in the reaction system was found to be a valuable tool for the orientation of the reaction process and for the determination of the final reaction products.
Abstract: Immobilized lipase activity is studied in organic solvent systems of controlled water content under the influence of a variety of reaction parameters, such as temperature, relative humidity, substrate concentrations, and type of fatty acid used. Control of the amount of water in the reaction system was found to be a valuable tool for the orientation of the reaction process and for the determination of the final reaction products. The properties of the immobilized lipase were studied using the interesterification of triolein and palmitic acid as the model system.
193 citations
TL;DR: The observations are summarized in the conclusion that more hydrophobic solvents are preferable to lesshydrophobic ones and the log P value gives a good guidance when selecting an organic solvent for enzymatic conversions.
Abstract: The influence of solvents on enzymatic activity and stability was investigated. As a model reaction the α-chymotrypsin-catalyzed esterification of N-acetyl-l-phenylalanine with ethanol was used. The enzyme was adsorbed on porous glass beads and used in various solvents. Small amounts of water were added to increase the enzymatic activity. These enzyme preparations obeyed. Michaelis-Menten kinetics. K
m,app decreased slightly with the log P value of the solvent while V
app increased markedly with the log P value. Log P values were also useful for generalizing the influence of solvents on enzyme stability. The enzyme preparations showed a markedly higher thermostability in dry solvents having log P values >0.7 than in less hydrophobic solvents. Also the operational stability was better in the more hydrophobic solvents. The amount of water added to the enzyme preparations greatly influenced the initial reaction rates. For some solvents optimal water contents were determined. The thermostability decreased with increasing water content. The observations are summarized in the conclusion that more hydrophobic solvents are preferable to less hydrophobic ones. The log P value gives a good guidance when selecting an organic solvent for enzymatic conversions.
185 citations
TL;DR: The novel use of magnetic particles isolated from magnetotactic bacteria collected from enriched sludge by use of a magnetic harvesting apparatus and glucose oxidase and uricase coupled with biogenic magnetic particles retained their activities when they were reused 5 times.
Abstract: We report the novel use of magnetic particles isolated from magnetotactic bacteria. Magnetotactic bacteria were collected from enriched sludge by use of a magnetic harvesting apparatus. Magnetic particles separated from magnetotactic bacteria were shown to be pure magnetite. Glucose oxidase and uricase were immobilized on magnetic particles. The activity of glucose oxidase immobilized on biogenic magnetites was 40 times that immobilized on artificial magnetites or Zn-ferrite particles. Both glucose oxidase and uricase coupled with biogenic magnetic particles retained their activities when they were reused 5 times.
170 citations
TL;DR: This paper reviews the results that have been reported on ISFET based enzyme sensors and introduces a coulometric system that compensates for the analyte buffer capacity.
166 citations
TL;DR: No single immobilization method is best for all enzymes or all applications of any given enzyme, so a survey of enzyme coupling techniques and enzyme immobilization, with emphasis on the carder matrices, is discussed.
Abstract: Publisher Summary This chapter discusses a survey of enzyme coupling techniques and enzyme immobilization, with emphasis on the carder matrices. Immobilized enzymes have been used to prepare enzyme electrodes and transistors for diagnostic purposes, as supports in high-performance liquid chromatography (HPLC) separations and as therapeutic agents, for chemical synthesis. The majority of immobilized enzymes used in these applications have been prepared using a small group of matrices. The most prevalent and most widely applicable of the commercially available matrices for covalent coupling enzymes are the polysaccharides, agarose and cellulose, and their derivatives. The most widely employed method of immobilizing antibodies in solid phase, heterogeneous, immunoassay is absorption on polystyrene, either as beads or as styrene tubes. However, no single immobilization method is best for all enzymes or all applications of any given enzyme.
141 citations
TL;DR: Numerical simulations were developed that incorporate spatially heterogeneous catalytic and inactivation processes that are consistent with the reaction of hydrogen peroxide and certain catalytic intermediates of both enzymes.
Abstract: Homogeneous membranes containing immobilized glucose oxidase and catalase were stored in buffered solutions at 37 degrees C to determine the mechanisms and rates of catalyst inactivation. The experiments were designed so that inactivation occurred homogeneously throughout the membrane, thereby simplifying the analysis. The mechanism of inactivation is consistent with the reaction of hydrogen peroxide and certain catalytic intermediates of both enzymes. Based on this information, numerical simulations were developed that incorporate spatially heterogeneous catalytic and inactivation processes.
133 citations
TL;DR: Scale-up production of ethyl butyrate was examined in a packed column with recycling of n-hexane containing substrates and increased substrate concentrations were stimulatory up to a point after which inhibition and enzyme destabilization in repeated runs occurred.
Abstract: Candidacylindracea lipase adsorbed to silica gel produced a variety of flavor esters when hydrated and shaken in n-heptane containing substrates. Scale-up production of ethyl butyrate was examined in a packed column with recycling of n-hexane containing substrates. Increased substrate concentrations were stimulatory up to a point after which inhibition and enzyme destabilization in repeated runs occurred.
109 citations
106 citations
TL;DR: The αs1-casein film obtained showed a high tensile strength (105 g/cm2) and strain (72%), but it was insoluble in water, 10% 2-mercaptoethanol, 6.6 m urea and 6 m guanidine hydrochloride, even if it was diluted with water and then heated at 100°C for 10 min this paper.
Abstract: Transglutaminase is a Ca2 +-dependent enzyme that covalently polymerizes proteins through the formation of e-(γ-glutamyl)lysyl cross-links. We have reported that highly concentrated protein solutions were firmly gelatinized by transglutaminase. The technique of gelation of an αs1-casein solution with transglutaminase was applied to the preparation of an αs1-casein film. The αs1-casein film obtained showed a high tensile strength (105 g/cm2) and strain (72%). It was insoluble in water, 10% 2-mercaptoethanol, 6.6 m urea, 10% SDS and 6 m guanidine hydrochloride. Even if it was diluted with water and then heated at 100°C for 10 min, it remained insoluble. However, it was gradually hydrolyzed by chymotrypsin. These results suggest the usefulness of the αs1-casein film as a supporting material of immobilized enzymes, a medical polymer and an edible film.
TL;DR: The preparation and performance of two enzyme-based fiber-optic sensors (optrodes) capable of detecting penicillin are described and may be considered complementary alternatives in future enzyme optrode applications.
Abstract: The preparation and performance of two enzyme-based fiber-optic sensors (optrodes) capable of detecting penicillin are described. Each sensor consists of a polymer membrane that is covalently attached to the tip of a glass optical fiber. The membrane contains the enzyme penicillinase and a pH-sensitive fluorescent dye. A signal is produced when the enzyme catalyzes the cleavage of the ..beta..-lactam ring of penicillin to produce penicilloic acid and, consequently, a pH change in the microenvironment of the membrane. The sensors differ in the way the polymer membrane is constructed and in the type of pH indicator dye used. Both optrodes exhibit response times (40-60 s) significantly lower than those of the corresponding enzyme electrodes (2 min). Each gives a linear response over the concentration range of 0.00025 to 0.01 M penicillin G, when measured in a 0.005 M phosphate buffer. The data indicate that these immobilization strategies produce similar results and may be considered complementary alternatives in future enzyme optrode applications.
TL;DR: It was observed that upon immobilization the affinity of GOD towards glucose was substantially decreased, and increasing the GOD content of the membrane adversely affected the activity, and Membranes with the highest enzyme activity were found to be the most permeable.
TL;DR: An immobilized lipase suitable for fat interesterification has been prepared by precipitation with acetone of a commercial lipase from Rhizopus arrhizus onto diatomaceous earth and little activity was lost processing shea oleine, but slow poisoning of the bed occurred when shea oil was fed to the column.
Abstract: An immobilized lipase suitable for fat interesterification has been prepared by precipitation with acetone of a commercial lipase from Rhizopus arrhizus onto diatomaceous earth. As observed previously with a less active enzyme from Aspergillus sp., the interesterification activity was enhanced by addition of purified lipase or by high loadings of commercial enzyme. The interesterification activities reached maximum values in both cases. For immobilized preparations with purified enzyme, interesterification activity was also enhanced by the presence of a precoat of glutaraldehyde cross-linked commercial lipase. A 2.9-L column of immobilized lipase was used to interesterify batches of shea oleine (67 kg) and shea oil (40 kg). Little activity was lost processing shea oleine, but slow poisoning of the bed occurred when shea oil was fed to the column.
TL;DR: The cells immobilized in polyacrylamide provided a convenient small‐scale laboratory model system and it was found that the Citrobacter sp.
Abstract: Polyacrylamide gel-immobilized cells of a Citrobacter sp. removed cadmium from flows supplemented with glycerol 2-phosphate, the metal uptake mechanism being mediated by the activity of a cell-bound phosphatase that precipitates liberated inorganic phosphate with heavy metals at the cell surface. The constraints of elevated flow rate and temperature were investigated and the results discussed in terms of the kinetics of immobilized enzymes. Loss in activity with respect to cadmium accumulation but not inorganic phosphate liberation was observed at acid pH and was attributed to the pH-dependent solubility of cadmium photsphate. Similarly high concentrations of chloride ions, and traces of cyanide inhibited cadmium uptake and this was attributed to the ability of these anions to complex heavy metals, especially the ability of CN(-) to form complex anions with Cd(2+). The data are discussed in terms of the known chemistry of chloride and cyanide-cadmium complexes and the relevance of these factors in the treatment of metal-containing liquid wastes is discussed. The cells immobilized in polyacrylamide provided a convenient small-scale laboratory model system. It was found that the Citrobacter sp. could be immobilized on glass supports with no chemical treatment or modification necessary. Such cells were also effective in metal accumulation and a prototype system more applicable to the treatment of metal-containing streams on a larger scale is described.
TL;DR: Using immobilized β-glucosidase, purity and yield of genipin, which is a hydrolyzate ofgeniposide, was improved comparing with the native enzyme.
Abstract: The reaction velocity of immobilized β-glucosidase was approximated by the first-order reaction kinetics. A plug flow reactor was used for continuous hydrolysis of geniposide with this immobilized enzyme. The activity of this immobilized enzyme was retained 100% for 600 h. The amount of genipin formed by using the immobilized enzyme was 17 fold that formed using the native enzyme without reuse. Using immobilized enzyme, purity and yield of genipin, which is a hydrolyzate of geniposide, was improved comparing with the native enzyme.
TL;DR: Chymotrypsin immobilized by the preparation of soluble–insoluble immobilized enzyme catalyzed the effective peptide synthesis in a heterogeneous reaction system containing water‐miscible organic solvent.
Abstract: In order to carry out an effective enzyme reaction, the preparation of soluble-insoluble immobilized enzyme was investigated. Proteases were selected as model enzymes, and their immobilization was carried out by using an enteric coating polymer as a carrier. Among the polymers tested, methacrylic acid-methylacrylate-methylmethacrylate copolymer (MPM-06) gave the most active soluble-insoluble immobilized papain. This immobilized papain showed insoluble from below pH 4.8 and soluble form above pH 5.8; it was also soluble in water-miscible organic solvent. It was reusable and more stable with heat and water-miscible organic solvents than native proteases. Furthermore, various proteases could be immobilized by using MPM-06 with high activity. Chymotrypsin immobilized by this method catalyzed the effective peptide synthesis in a heterogeneous reaction system containing water-miscible organic solvent.
TL;DR: Sulfonate esters are suitable for affinity ligand and enzyme immobilization to hydroxyl group carrying supports, such as agarose, cellulose, glycophase glass, and glycerylpropyl-silica, and tresylated supports allow efficient immobilization at near neutral pH and at 4°.
Abstract: Publisher Summary As enzymes are costly and quite unstable molecules, it is desirable that the method used for immobilization be efficient and mild, giving a high yield of bound active enzyme even at neutral pH and at low temperature. For engineering purposes, it is advantageous if the enzyme is stabilized by its immobilization. The method used should be easy to reproduce and should give consistent couplings. Sulfonate esters are suitable for affinity ligand and enzyme immobilization to hydroxyl group carrying supports, such as agarose, cellulose, glycophase glass, and glycerylpropyl-silica. Tresylated supports allow efficient immobilization at near neutral pH and at 4°. Such activated supports are now commercially available. Apart from the activation of agarose and glycerylpropyl-silica, cellulose, hydroxyethyl methacrylate, and glycophase glass have also been activated with tresyl chloride.
TL;DR: An enzyme sensor system for the determination of phosphate ions was constructed using immobilized enzymes and an oxygen electrode using nucleoside phosphorylase catalyzed reaction for which the presence of inorganic phosphorus is indispensable.
TL;DR: 100% selectivity was obtained and there was no significant decrease in the fractional conversion of PC to PG (98%–99%) in the first three batch cycles, while in the cases of a packed-bed reactor and a continuous stirred-tank reactor used for continuous synthesis of PG with the immobilized PLD, the operational stabilities were almost the same.
Abstract: The repeated batch and continuous operations for transphosphatidylation reaction were carried out for phosphatidylglycerol (PG) synthesis from phosphatidylcholine (PC) with the help of immobilized cabbage phospholipase D (PLD) in the presence of glycerol. The biphasic reaction system was used which included the aqueous phase containing immobilized PLD along with high concentrations of glycerol (30%–50%) and buffer, whereas the main part of substrate (PC) and products (mainly PG) formed were in the organic phase (diethyl ether).
TL;DR: The data on natural and synthetic carriers used for enzyme immobilisation, chemistry of enzyme binding, in vitro and in vivo properties of immobilised enzymes as well as their clinical use are reviewed.
TL;DR: In this study, the overall rate of reaction of immobilized glucose oxidase is investigated theoretically and a novel steady‐state model based on a ping‐pong kinetic mechanism for glucose oxidation is developed.
Abstract: Determination of glucose concentrations in fluids frequently requires the application of immobilized glucose oxidase. An accurate description of the immobilized enzyme kinetics is critical for such applications. In this study, the overall rate of reaction of immobilized glucose oxidase is investigated theoretically. A novel steady-state model based on a ping-pong kinetic mechanism for glucose oxidase is developed. Numerical studies are used to examine the parameter sensitivity of this model. The enzyme loading, matrix thickness and geometric configuration are found to have a significant influence on substrate uptake by insolubilized glucose oxidase. Additionally, this new model is compared with a previously developed model based on an alternative ping-pong kinetic mechanism. Under steady-state conditions, no significant difference between the two models is apparent when appropriate kinetic parameters are applied to each of the models. The model developed herein is also compared with models utilizing the simplifying assumption of Michaelis-Mented kinetics for substrate reaction. Numerical studies indicate that under most realizable biological conditions, a model based on ping-pong kinetics should be applied to accurately describe substrate uptake by immobilized glucose oxidase.
TL;DR: Using commercial immobilized glucose isomerase (SWETASE®, Nagase Co.), the effect of substrate protection on enzyme deactivation has been studied in a batch manner and the protection factor was proposed to elucidate the dependence of the degree of substrateprotection.
Abstract: Using commercial immobilized glucose isomerase (SWETASE(R), Nagase Co.), the effect of substrate protection on enzyme deactivation has been studied in a batch manner. The data analysis was carried out based on Briggs-Haldane kinetics in which enzyme deactivation accompanying the protection of substrates was also considered. The protection factor was proposed to elucidate the dependence of the degree of substrate protection. The existence of the protection of glucose isomerase by the substrates has been verified experimentally. Also, the enzyme-substrate complex deactivates with a decay constant which is one-half that of the free enzyme. Theoretical analysis of enzyme deactivation with substrate protection offers an effective understanding which is essential for enzyme replacement and process optimization.
TL;DR: The immobilized α-galactosidase was continuously used for 30 days to hydrolyze raffinose in beet sugar molases and was active at pH 3–6 and at 70–80°C, and stable between pH 3 and 9, and below 70°C.
TL;DR: In this paper, the total reaction rate in an electrochemical cell containing free enoate reductase is described and their use in determining optimal cell and electrode dimensions is discussed.
TL;DR: The hemodialysis-like procedure proved to be without side effects, specific for phenylalanine, and suitable in the management of pregnant women with phenylketonuria and late-onset hyperphenylalaninemia, and represents a new, safe, and effective therapeutic modality.
Abstract: Multitubular enzyme reactors with immobilized enzymes were developed to achieve depletion of circulating substrate by extracorporeal means. To act as prototypes, reactors were prepared wit...
TL;DR: In this article, L-asparaginase was conjugated with N-succinimidyl acrylate and analyzed via isoelectric focusing showed that a molar ratio of 9.5 free monomers per enzyme was needed during the conjunction for each vinyl group bound.
Abstract: Monomer conjugation of an enzyme followed by copolymerization with free monomer is a useful method of enzyme immobilization. L-asparaginase was conjugated with N-succinimidyl acrylate. Analysis of the conjugated enzyme via isoelectric focusing showed that a molar ratio of 9.5 free monomers per enzyme was needed during the conjunction for each vinyl group bound. Only 3% of the enzyme activity was lost per vinyl group added, and conjugation of an average of four monomers per enzyme thermally destabilized the enzyme only at temperatures above 50‡C. Activity of the enzyme at physiological temperatures was relatively unaffected.
Patent•
06 Oct 1987
TL;DR: Immobilized enzyme-polysaccharide graft copolymer products are prepared by hydrolyzing polysaccharides and then contacting the enzyme with an enzyme to immobilize the enzyme on the copolymers as mentioned in this paper.
Abstract: Immobilized enzyme-polysaccharide graft copolymer products are prepared by hydrolyzing a polysaccharide graft copolymer and then contacting the hydrolyzed copolymer with an enzyme to immobilize the enzyme on the copolymer. The copolymer-enzyme products retain large quantities of active enzyme, and activity of the enzyme is retained over long periods of use. In the case of glucoamylase immobilized in accordance with the method of the invention, improved yield of glucose from starch is obtained.
TL;DR: In this paper, the authors presented two possibilities for using immobilized thermoase, i.e., continuous column operation and batchwise operation, in order to clarify the mechanism of deactivation by organic solvents and to find strategies to overcome it.
Abstract: The enzymatic method for the production of aspartame has many advantages over chemical methods, e.g., cheap racemic phenylalanine can be used and no β-aspartame is produced. The former leads to a lowering in the cost of raw materials and the latter to a simplified purification procedure. The disadvantages as compared with chemical methods are that the racemization of unreacted d -Phe-OMe is necessary and that an expensive catalyst is used. The racemization of d -Phe-OMe can be done easily as stated before, but the recovery and recycling of the delicate biocatalyst are not easy, although thermoase is a very stable enzyme. Here we have presented two possibilities for using immobilized thermoase, i.e., continuous column operation and batchwise operation. In both procedures, the use of an organic cosolvent is required, which leads to deactivation of the catalyst. Therefore it is most important to clarify the mechanism of deactivation by organic solvents and to find strategies to overcome it, perhaps by development of more sophisticated immobilization methods. In an industrial operation using the batchwise mode, deterioration of the immobilized enzyme occurs by the friction caused by stirring. Furthermore, separation of the immobilized enzyme is necessary after batchwise reaction, which is troublesome and costly. In the continuous column operation, channeling of the organic and aqueous layers was observed in the packed column, thus rendering the efficiency of the catalyst low, and in addition, deactivation tended to occur more rapidly than during batchwise operation. Nevertheless, the continuous column operation seems to be more advantageous than the batchwise operation from the viewpoint of industrial production needs.