Showing papers on "Immobilized enzyme published in 1978"

Enzyme immobilization with pullulan gel

Abstract: Enzymes are immobilized by a process wherein the carrier used for immobilizing is a hydrophilic pullulan gel having a three-dimensionally reticulated structure obtained by the reaction between pullulan and a bifunctional compound capable of forming an ether linkage with the hydroxyl group contained in the glucose unit of pullulan, or an ionic pullulan gel obtained by the reaction between said hydrophilic pullulan gel and a compound having at one end an ionizable group and at the other end a functional group capable of forming an ether linkage with the hydroxyl group in the pullulan gel in the presence of an alkaline coumpound. Immobilized enzymes prepared according to this process have a high activity and good retention of the activity.

Immobilization of Aspergillus beta-glucosidase on chitosan.

Abstract: beta-Glucosidase of Aspergillus phoenicis QM 329 was immobilized on chitosan, using the bifunctional agent glutaraldehyde. The most active preparation based on the amount of support contained a 1:2.5 enzyme-to-chitosan ratio (wt/wt). However, the specific activity of the bound enzyme decreased from 10 to 1% with increasing enzyme-to-chitosan ratio. Compared with free beta-glucosidase, the immobilized enzyme exhibited: (i) a similar pH optimum but more activity at lower pH values; (ii) improved thermal stability; (iii) a similar response to inhibition by glucose; and (iv) mass transfer limitations as reflected by higher apparent Km and lower energy of activation.

Enzyme leakage and multipoint attachment of agarose-bound enzyme preparations.

Abstract: The solvolytic detachment of leucine aminopeptidase from Sepharose-enzyme conjugates with multiple and single anchoring bonds has been studied under a variety of conditions by radiochemical and enzymological methods. The release of the single-point-fixed conjugate could be described by a leakage function, derived previously, yielding the first-order rate constant of the cleavage of the enzyme-matrix bond. The nucleophile hydroxylamine increased the detachment rate considerably. The release of the immobilized enzyme was incomplete in all experiments even after prolonged times. The enzyme leakage from multipoint-attached conjugates was still high enough to prohibit a long-term use of such preparations in routine work at room temperature.

Industrial applications of immobilized biocatalysts

Abstract: The isomerization of glucose to fructose, the production of amino acids, and the hydrolysis of penicillins to 6-aminopenicillanic acid are some examples of the industrial processes, based on immobilized biocatalysts, that are operative at present These and other processes utilized in the food and the pharmaceutical industries, as well as a number of potentially useful immobilized biocatalysts, which have been investigated on pilot plant or laboratory scale, are discussed in this review A general development of the engineering aspects of the methods will precede the description of specific applications of immobilized enzymes and microbial cells

Phenylalanine depletion for the management of phenylketonuria: use of enzyme reactors with immobilized enzymes

Abstract: Multitubular enzyme reactors with immobilized phenylalanine ammonia lyase were tested in vitro and in vivo for depletion of phenylalanine in circulating blood. Sustained reduction of phenylalanine was achieved in less than 30 minutes. A 50% decrease of phenylalanine was obtained with a 2-hour application of enzyme reactors and was maintained for more than 2 days. Similar enzyme reactors have therapeutic potential for temporary management of phenylketonuric patients when their circulating phenylalanine becomes exceedingly high because of infection, fever, or pregnancy.

Preparation and Properties of Immobilized Naringinase Using Tannin-aminohexyl Cellulose

Abstract: Naringinase from Aspergillus niger was immobilized with 58 to 100% yield by adsorbing the enzyme to tannin-aminohexyl cellulose prepared by reaction of aminohexyl cellulose and cyanogen bromide activated Chinese gallotannin.Some enzymatic properties of the immobilized naringinase were investigated and compared with those of the native enzyme. The optimal pH (4~5) and optimal temperature (50°C) for enzyme reaction by the immobilized enzyme were the same as those of the native enzyme. The heat stability of the immobilized enzyme was better than that of the native naringinase. No significant difference of the Michaelis constant was detected.The operational stability of the immobilized naringinase column was investigated at 25 °C and 37°C by passing a naringin solution and Natsudaidai juice continuously through it. In the case of a naringin solution, the activity of the immobilized enzyme column was very stable and its half-life was estimated to be 198 days at 25°C and 88 days at 37°C. However, in the case of...

Parameter evaluation and performance studies in a fluidized‐bed immobilized enzyme reactor

Abstract: Dispersion and mass-transfer characteristics and fluidization parameters influencing the performance of a small pilot-plant immobilized enzyme reactor are evaluated. The suitability of a dispersed plug-flow model to predict the conversions obtained in the enzymatic reaction (starch → glucose) catalyzed by amyloglucosidase immobilized to solid and porous carriers is assessed. The performance of a fluidized-bed reactor is compared on the basis of a normalized residence time with that of a fixed bed and found to be superior.

Enzyme-bound thermistor as an enthalpimetric sensor

Abstract: The construction of a new type of immobilized enzyme sensor for the determination of chemical species is described. The sensitive part of a thermistor is coated with an artificial enzyme membrane obtained by the cross-linking of the enzyme together with an inert protein, using glutaraldehyde as a bi-functional reagent. The enthalpy change during the enzymatic reaction induces a production or absorption of calories which can be taken as a reaction product and depends then upon the reaction parameters (Km, Vm, [S]). This type of enzyme-bound thermistor is used for the determination of hydrogen peroxide, glucose, and urea. The pH and temperature dependence of the hydrolysis of urea by urease given by this sensor are compared to the results previously obtained by other methods.

Preparation and properties of immobilized methanol oxidase

Abstract: Methanol oxidase produced by the yeast Hansenula polymorpha DL-1 was used for the enzymatic oxidation of methanol to formaldehyde. The kinetics of enzyme and protein release during cell desruption were studied at the laboratory scale with a Braun homogenizer and the pilot plant scale with a Manton–Gaulin homogenizer. Conditions were defined for maximum release and retention of high activity in cell-free extracts. Methanol oxidase was immobilized by adsorption on DEAE-cellulose from enzymes in cell-free extracts or from ammonium sulfate purified purified fractions. The kinetics of formaldehyde formation with both soluble and immobilized enzyme was studied in batch and continuous reactors.

Immobilized Xanthine Oxidase:Kinetics, (In)stability and Stabilization by Coimmobilization with Superoxide Dismutase and Catalase

Abstract: Milk xanthine oxidase was immobilized by covalent attachment to CNBr-activated Sepharose 4B and by adsorption to n-octylamine-substituted Sepharose 4B. The amounts of activity immobilized for the two preparations were 30 and 90%, respectively. The pH optima for free and adsorbed xanthine oxidase were at 8.6 and 8.2, respectively. Both free and immobilized xanthine oxidase show substrate inhibition. The apparent inhibition constant (Ki′) found for adsorbed xanthine oxidase with xanthine as substrate was higher than the Ki for the free enzyme, which was shown to be due to substrate diffusion limitation in the pores of the carrier beads (internal diffusion limitation). Higher substrate concentrations, as desirable for practical application in organic synthesis, can therefore be used with the immobilized enzyme without decreasing the rate. As a result of the internal diffusion limitation the apparent Michaelis constant (Km′) for adsorbed xanthine oxidase was also higher than the Km for the free enzyme. Immobilized xanthine oxidase was more stable than the free enzyme during storage at 4 and 30°C. Both forms rapidly lost activity during catalysis. The loss was proportional to the amount of substrate converted. Coimmobilization of xanthine oxidase with superoxide dismutase and catalase improved the operational stability, suggesting that O2− and H2O2 side-products of the enzymatic reaction were involved in the inactivation. Coimmobilization with albumin also had some stabilizing effect. Complete surrounding of xanthine oxidase by protein, however, by means of etrapment in a glutaraldehyde-crosslinked gelatin matrix, considerably enhanced the operational half-life. This system was less efficient than the Sepharose preparations either because much activity was lost during the immobilization procedure and/or because it had poor flow properties. Xanthine (15 mg)was converted by an adsorbed xanthine oxidase preparation and product (uric acid) was isolated in high yield (84%).

Immobilized-enzyme nylon-tube reactors for routine determination of urea and citrulline in serum.

Abstract: A continuous-flow clinical analyzer for the routine estimation of urea is described that makes use of an immobilized-enzyme nylon-tube reactor as part of a flow-through system (a Technicon AutoAnalyzer I). Results of blood-urea analyses by use of the immobilized urease are compared with determinations made with the diacetyl monoxime method and the urease solution method. Clinical trials carried out routinely with the immobilized enzyme nylon tube reactor give reliable and reproducible results with high precision and low cost. The reactors are stable to intermittent or continued use for at least four months or for 2000 tests. A method is described in which differential colorimetry is used for determining citrulline in blood and which makes use of the immobilized urease, albeit indirectly.

Industrial application of immobilized enzyme systems

Abstract: Industrial applications of immobilized enzymes and immobilized microbial cells for the production of L-amino acids and organic acids are presented. Continuous optical resolution of acyl-DL-amino acid is efficiently carried out using an immobilized aminoacylase column. L-Aspartic acid and L-malic acid are continuously produced using columns of immobilized Escherichia coli and immobilized Brevi-bacterium ammoniageneses , respectively. These continuous production systems are superior to the coventional batch-wise processes using soluble enzymes or intact cells.

Process for preparing immobilized enzymes

Abstract: The activity and stability of immobilized enzymes may be improved by treating an immobilized enzyme comprising an enzyme covalently bound to polymeric material which is absorbed on an inorganic porous support material with a bifunctional monomeric material and a substrate followed by additional treatment with an enzyme and thereafter again treating the complex with additional bifunctional monomer and substrate.

Immobilization of β-Glucosidase in Fibroin Membrane

Abstract: Fibroin membrane was used as a support for immobilized β-gIucosidase. The immobilized enzyme was prepared by drying fibroin-enzyme solution on a horizontal plate, followed by ethyl alcohol treatment which was an essential process for immobilization. The immobilized β-glucosidase was characterized enzymatically compared with soluble enzyme, using p-nitrophenyl-β-D-glucopyranoside as a substrate. The immobilized enzyme showed 47% of the activity of soluble enzyme and little decrease of the activity was observed both on re-use and storage. There were no significant differences in pH dependency between immobilized and soluble enzyme activity. Activation energy was slightly larger with immobilized enzyme than soluble enzyme. The enzyme was considerably enhanced by immobilization in stabilities against heating, electrodialysis and protease treatment. Apparent affinity for the substrate decreased as membrane thickness increased. Enzyme affinity for substrate in the membrane was discussed.

Affinity purification of cardiac adenylate cyclase: dependence on prior hydrophobic resolution

Abstract: The interaction of cardiac adenylate cyclase [ATP pyrophosphate-lyase (cyclizing); EC 4.6.1.1] with a variety of nucleotide affinity resins was systematically investigated. None of these resins effectively bound the native, detergent-solubilized enzyme. However, after hydrophobic resolution on an uncharged resin consisting of long-chain alkyl groups linked to agarose via ether bonds, 40% of the adenylate cyclase activity biospecifically adsorbed to an ATP affinity resin. Gel filtration without detergent after hydrophobic chromatography demonstrated that the enzyme eluted in the identical position as the native enzyme chromatographed in the presence of detergent. This preparation almost completely biospecifically adsorbed to the same ATP-resin and was not eluted with 5 mM cyclic AMP, pyrophosphate, or GTP. If the GTP-washed immobilized enzyme was subsequently desorbed with ATP, then expected Gpp(NH)p (5'-guanylyliminodiphosphonate) sensitivity persisted. A preliminary purification scheme that resulted in an approximate 5000-fold increase in specific activity is presented. These observations indicate that a membrane-bound enzyme may appear to be intrinsically hydrophobic only by virtue of aggregation with other hydrophobic constituents and that prior separation of hydrophobic chromatography may permit such proteins to be fractionated subsequently by methods conventionally applied to hydrophilic proteins.

Some factors affecting the stability of glucoamylase immobilized on hornblende and on other inorganic supports

Abstract: Glucoamylase from four different companies was studied: three had similar stability (half-life at 50°C about 140 hr); the fourth was less stable (half-life at 50°C about 20 hr). The immobilized enzymes were all less stable than their soluble counterparts: immobilized enzyme stability depended on the soluble enzyme used, the support, and method of immobilization. Thus enzyme bound to Enzacryl-TIO was less stable than enzyme bound to hornblende (metal-link method); this, in turn, was less stable than enzyme bound to hornblende by a silane–glutaraldehyde process. Bound enzyme stability was also improved by the presence of substrate or product (starch maltose or glucose). After 110 hr at 50°C in the presence of maltose (10% (w/v)) one preparation (a more stable soluble enzyme boul1d to hornblende by a silane–glutaraldehyde process) retained over 95% of its activity: activity loss was too low to permit the estimation of a half-life.

Immobilized-enzyme nylon-tube reactor for routine determination of uric acid in serum.

Abstract: We report here the preparation of an immobilized-enzyme nylon-tube reactor containing uricase (EC 1.7.3.3) and the assembly of a flow-through system (Technicon AutoAnalyzer II) for the routine determination of uric acid in serum. Results of these uric acid analyses by use of immobilized uricase, in conjunction with peroxidase (EC 1.11.1.7) and aminophenazone-dichlorophenol in solution, are compared with those obtained with the same enzyme in solution by use of the uricase-PAP (peroxidase, 4-aminophenazone, dichlorophenol) method. Clinical trials carried out routinely with the uricase reactor give reliable and reproducible results with high precision at an appreciably lower cost. The reactors are stable to continued or intermittent use for at least three months or for 4000 tests.

Flow kinetics of lactate dehydrogenase chemically attached to nylon tubing.

Abstract: Rabbit muscle lactate dehydrogenase (EC 1.1.1.27) was attached covalently to the inner surface of nylon tubing; a modified technique, involving benzidine and glutaraldehyde, was used, and the resulting immobilized enzyme showed no loss of activity over a period of several months. An experimental study was made of the flow kinetics for the reaction between pyruvate and reduced nicotinamide adenine dinucleotide in two limiting cases, one substrate in excess and the concentration of the other one varied. A range of flow rates and temperatures was covered. The results were analyzed in various ways on the basis of the Kobayashi--Laidler treatment of flow systems. It was concluded that the kinetics are largely diffusion-controlled, especially at the lower substrate concentrations and flow rates. The values of the apparent Michaelis constants vary with flow rate vf, being linear in vf-1/3, and the values extrapolated to infinite flow rate (vf-1/3 = 0) approach the values for the enzyme in free solution. Analysis of the rates led to activation energies for the diffusion of the two substrates.

Practical Process Conditions for the Use of Immobilized Glucose Isomerase

Abstract: The present world-wide rush into the production of high fructose corn syrups has increased the demand for the enzym glucose isomerase for the isomerization process. Economics have forced producers of these sweeteners to use the immobilized enzyme in a continuous process. The most suitable way of performing continuous isomerization turned out to be a process in a fixed-bed column. However, a downflow mode of the substrate through the enzyme bed required special properties of the beads, especially when using very high enzyme beds. Practical process conditions are described for the use of immobilized Maxazyme glucose isomerase in a continuous fixed-bed column operation procedure, e. g. pH, temperature, concentration of activators, quality of the substrate and enzyme bed dimensions. Special attention is paid to compressibility of the enzyme particles and the characteristical pressure drop of this product. Improvements are described for the production of this immobilized enzyme, which make it possible to use Maxazyme Gl-Immob in columns up to 5 m bed height. Praktische Prozesbedingungen bei der Verwendung von immobilisierter Glucoseisomerase. Der heutige weltweite Trend zur Herstellung hochfructosehaltiger Glucosesirupe hat die Nachfrage nach dem Enzym Glucoseisomerase fur das Isomerisierungsverfahren erhoht. Aus wirtschaftlichen Grunden sind die Hersteller dieses Susmittels gezwungen, immobilisierte Glucoseisomerase einzusetzen. Dabei ist es am einfachsten, das immobilisierte Enzym in einer Saule mit einem Festbett zu verwenden. Der absteigende Lauf durch das Enzymbett erfordert jedoch spezielle Eigenschaften der Enzymkorper, insbesondere bei Verwendung sehr hoher Enzymschichten. Die praktischen Prozesbedingungen bei der Verwendung von immobilisiertem Maxazyme in einem kontinuierlichen Festbettverfahren, insbesondere pH, Temperatur, Konzentration des Aktivators, Substratqualitat und Abmessungen des Enzymbettes, werden erlautert. Besondere Aufmerksamkeit wird der Zusammenpressung der Enzymkorper und dem kennzeichnenden Druckabfall des Produktes gewidmet. Es werden Verbesserungen hinsichtlich des Enzymproduktionsverfahrens erlautert, welche es ermoglichen, immobilisiertes Maxazyme GI-Immob in Saulen bis zu 5 m Betthohe einzusetzen.

Enzymatic conversion process

Abstract: Chemical enzymatic conversions are conducted by contacting an aqueous solution of the substrate with a granular immobilized enzyme, the substrate solution being passed through several series-connected, separate fluidized beds of the granular enzyme, while the enzyme particles are passed from one fluidized bed to the next countercurrently and against the direction of flow of the substrate solution. Multi-compartment reactor columns are also disclosed.

HYDROLYSIS OF LACTOSE IN ACID WHEY USING β‐GALACTOSIDASE ADSORBED TO A PHENOL FORMALDEHYDE RESIN

Abstract: The feasibility of using immobilized enzymes for the commercial hydrolysis of lactose in acid whey was investigated. The areas of enzyme kinetics, enzyme adsorption, mass transfer, reactor design and economics were considered. All reaction studies were carried out at 40°C, pH 4. Adsorption of β-galactosidase into a porous phenol formaldehyde resin (Duolite ES-762) followed an S-shaped isotherm at low concentrations and adsorption increased with temperature at 0, 24 and 26°C. Up to 0.6g of Wallerstein enzyme preparation (4043 activity units) were adsorbed to 1g of dry resin. Adsorption had little effect on enzyme activity. An integral reactor of immobilized β-galactosidase retained nearly full activity for over 120 days of continual hydrolysis of lactose in acid whey and pure lactose solutions. Throughout the long term study, mold was effectively inhibited by the use of 0.1% w/v of potassium sorbate and CO2. Based on 120-day catalyst life and the integral reactor data for Duolite ES-762 loaded with 0.23g enzyme/g dry resin, the catalyst cost for the hydrolysis of 1 lb of lactose is 1.65 cents at 40% conversion using 50 mesh resin.

Preparation of water-insoluble enzyme compositions

Abstract: Enzymes are covalently bonded to porous inorganic supports by a process wherein maximum activity of enzyme in the water-insoluble enzyme composition is achieved with the lowest possible amount of enzyme. The process involves selecting an inorganic carrier having a most frequent pore-diameter that results in highest enzymatic activity possible per weight unit of the insoluble enzyme composition at the given enzyme concentration within the insoluble enzyme composition, and contacting the selected inorganic carrier with an enzyme solution containing an amount of enzyme which is sufficiently low so that the enzyme in the insoluble enzyme composition has a specific activity which is substantially the same as that of the enzyme in solution in the free state.