Showing papers on "Immobilized enzyme published in 1986"

Studies on the thermal inactivation of immobilized enzymes

Abstract: The thermal inactivation of a great number of immobilized enzymes shows a biphasic kinetics, which distinctly differs from the first-order inactivation kinetics of the corresponding soluble enzymes. As shown for alpha-amylase, chymotrypsin, and trypsin covalently bound to silica, polystyrene, or polyacrylamide, the dependence of the remaining activities on the heating time can be well described by the sum of two exponential terms. To interpret this mathematical model function, the catalytic properties of immobilized enzymes (number of active sites in silica-bound trypsin, K(M) and E(a) values in silica-bound alpha-amylase and chymotrypsin) at different stages of inactivation and the influence of various factors (coupling conditions, addition of denaturants or stabilizers, etc.) on the thermal inactivation of silica-bound alpha-amylase were studied. Furthermore, conformational alterations in the thermal denaturation of spin-labeled soluble and silica-bound beta-amylase were compared by electron spin resonance (ESR) studies. The results suggest that the biphasic inactivation kinetics reflects two different pathways according to which catalytically identical enzyme molecules are predominantly inactivated.

Electrochemical concentration detector device

Abstract: Electrochemical biosensor systems or apparatus, components thereof, and methods for quantitatively measuring or assaying concentrations of selective substrates or metabolites in aqueous media as a function of electrochemical response to hydrogen peroxide concentration, using immobilized enzyme electrode means selective for the substrate or metabolite being measured.

New Immobilized Enzyme Membranes for Tailor-Made Biosensors

Abstract: Commercially available polyamide preactivated membranes for immunodiagnostic use were found suitable for the preparation of immobilized enzyme membranes adaptable to biosensors. Membranes with immobilized glucose oxidese as model enzyme, tested with a microprocessor-based device involving an enzymatic electrode, gave excellent results. The extremely simple and fast procedure allows anyone to prepare such bioactive membranes easily, possibly from his own enzyme preparation and within a few minutes set up a specific probe from commercially available sensors.

Immobilization of Enzyme into Poly(vinyl alcohol) Membrane

Abstract: Glucoamylase, invertase, and cellulase were entrapped within poly(vinyl alcohol) (PVA) membrane cross-linked by means of irradiation of ultraviolet light. The conditions for immobilization of glucoamylase were examined with respect to enzyme concentration in PVA, sensitizer (sodium benzoate) concentration in PVA, irradiation time, and membrane thickness. Various characteristics of immobilized glucoamylase were evaluated. Among them, the pH activity curve for the immobilized enzyme was superior to that for the native one, and thermal stability was improved by immobilization with bovine albumin. The apparent Km was larger for immobilized glucoamylase than for the native one, while Vmax was smaller for the immobilized enzyme. Also, the apparent Km appeared to be affected by the molecular size of the substrate. Further, immobilized invertase and cellulase showed good stabilities in repeating usage. 9 references.

Glutathione conjugates. Immobilized enzyme synthesis and characterization by fast atom bombardment mass spectrometry.

Abstract: Glutathione transferase activity was shown to be present in an immobilized preparation of microsomal protein. Chlorodinitrobenzene, ethacrynic acid, captopril, styrene oxide, and iminocyclophosphamide were found to be substrates, each providing a different kind of electrophilic functional group for conjugation. The glutathione conjugates were characterized by thin layer chromatography (visualized by reaction with ninhydrin) and by high pressure liquid chromatography. A variety of conditions was evaluated for analysis of these glutathiones by fast atom bombardment mass spectrometry.

Contact lens cleaning and disinfection.

Abstract: A method of treating a contact lens by means of hydrogen peroxide, which method comprises conducting the hydrogen peroxide treatment of the lens in the presence of an immobilized enzyme which catalyses the decomposition of hydrogen peroxide. The invention also provides apparatus and a kit for treating contact lens. Preferably the enzyme is catalase, which may be immobilized by adsorption, by absorption, by covalent binding or by antibody complexing.

Continuous production of galacto-oligosaccharides from lactose using immobilized β-galactosidase from Bacillus circulans

Abstract: β-Galactosidase-2 (β-d-galactoside galactohydrolase, EC 3.2.1.23) from Bacillus circulans was purified using hydroxyapatite gel chromatography and immobilized onto Duolite ES-762 (phenolformaldehyde resin) and Merckogel (controlled pore silica gel) for continuous production of galacto-oligosaccharides using lactose as the substrate. The maximum amount of ologosaccharides produced by the immobilized enzyme was 35–40% of the total sugar during hydrolysis of 4.56% lactose. Partially purified β-galactosidase from B. circulans was also immobilized onto various supports for the same purpose. The stability of the immobilized β-galactosidase-2 or partially purified enzyme during a continuous reaction depended on their supports and specific activity. Of the supports tested, Merckogel was best for operational stability. With this support, the enzyme was quite stable with specific activity up to 15 units/g of wet gel; it was reversibly inactivated with more.

Kinetics of enzymatic synthesis of peptides in aqueous/organic biphasic systems. Thermolysin-catalyzed synthesis of N-(benzyloxycarbonyl)-L-phenylalanyl-L-phenylalanine methyl ester.

Abstract: We studied kinetics of thermolysin-catalyzed peptide synthesis in an aqueous/organic biphasic system theoretically and experimentally. As a model reaction producing a condensation product having no dissociating groups, we used the synthesis of N-(benzyloxycarbonyl)-l-phenylalanine methyl ester (Z-Phe2 OMe) from N-(benzyloxycarbonyl)-l-phenylalanine (Z-Phe) and l-phenylalanine methyl ester (PheOMe). Usually, ethyl acetate was used as the organic solvent. First we studied the kinetics of the synthesis of Z-Phe2OMe in a buffer solution saturated with ethyl acetate. Then, factors that may affect the kinetics in the biphasic system were examined. The course of Z-Phe2OMe synthesis in the biphasic system was explained by the rate equations obtained, using the partitions of substrate and product and non-enzymatic decomposition of PheOMe. In the biphasic reaction syste, the rate of synthesis was lower for a wide range of pH due to the unfavorable partition of PheOMe in the aqueous phase, but yields were higher than in the buffer solution. The effects of the organic solvents on the rate of synthesis could also be explained by variations in the partition coefficient of PheOMe. Finally, we gave a way to predict the aqueous-phase pH change caused by partitioning of the substrate. The significance of the pH change was shown in connection with the reaction using the immobilized enzyme in an organic solvent.

Continuous hydrolysis of tallow with immobilized lipase in a microporous membrane

Abstract: Thermostable lipase from Thermomyces lanuginosus was immobilized in untreated microporous membranes. Melted tallow pumped through the membrane did not wash the enzyme out. From 0.4 to 0.9% of the soluble activity remained after immobilization with half-lives of 1-2 months or more at 50 degrees C. Membranes can be acid/base washed and reloaded with enzyme with no adverse effects. Buffer was required for a long half-life, and recycling the buffer improved the mass transfer of glycerol out of the immobilized lipase reactor. Immobilized activity was unaffected when the pH of the aqueous product changed from 5.5-6.5. 30 references.

Immobilized enzyme membrane for a semiconductor sensor

Abstract: A membrane containing an immobilized enzyme for a semiconductor sensor is prepared containing a water soluble photosensitive resin including a high molecular weight polyvinyl pyrrolidone crosslinked to 2, 5-bis (4'-azide-2'-sulfobenzal) cyclopentanone sodium salt, and an enzyme. Glutaraldehyde and bovine serum albumin, polyamino acid or polyamino amino acid copolymer may also be present to provide chemical crosslinking. The enzyme may be glucose oxidase, urease or lipase. The membrane can be directly formed on ion-sensitive protions of a pH-ion sensitive field effect transistor to form a semiconductor sensor by coating an aqueous solution of the resin and enzyme on the ion-sensitive portion, drying and irradiating with light such as ultraviolet light to provide photo crosslinking.

Enzyme technology and gas phase catalysis: Alcohol dehydrogenase example

Abstract: systems. However, little attention has been given to the use of gas-liquid systems for production of biomolecules. One of the most important potential uses of enzymes is synthesis or modification of high added value molecules. These applications necessitate the preparation of enzymes bound to a matrix or in some way retained in a reactor to permit their reuse. The use of alcohol dehydrogenases for alcohol or aldehyde production has receive attention but this synthetic strategy has serious limitations due to the operational instability of enzymes and cofactors (NAD or NADH), insolubility of most of the substrates and products in water which necessitates work in emulsions or organic solvents, product inhibition of the enzyme and the lability of some substrates and products in aqueous solutions. In the present study, we have elaborated a methodology for using immobilized enzymes with substrates and products in a gaseous form. We have used batches or column reactors with ADH and NAD (or NADH) coimmobilized into albumin-glutaraldehyde porous particles . In the reactors, gaseous substrates flow through the solid support, react with the enzyme and then gaseous products leave the reactor. The feasibility of the system has been tested in batches for the reduction of different aldehydes, and to improve the yield of regenerated cofactor we have made studies with a fixed bed reactor catalyzing the oxidation of butanol and the reduction of acetaldehyde. MATERIALS AND METHODS MATERIALS : Horse liver alcohol dehydrogenase (E.C. 1.1.1.1.), nicotinamide adenine dinucleotide (NAD +) reduced form (NADH) and bovine serum albumin were purchased from Sigma Chemical Co. All other reagents were from Carlo Erba and Fluka. IMMOBILIZATION METHOD : The immobilization process was based on a previously described method (Legoy et al, 1985). 12 ml sodium phosphate buffer 20 raM, pH 6.8 containing 0.3 g bovine serum albumin, 45 mg glutaraldehyde, 30 mg enzyme and 8 mg NAD (or NADH) were frozen at -200C for two hours and then slowly thawed at 4"C. A porous particle structure was obtained. Before use, porous particles were rinsed with sodium phosphate buffer 20mM, pH 6.8. DETECTION METHOD : Alcohol and aldehyde were determined by gas chromatography using a Carbowax 20 M column, H 2 cartier gas 2 ml rain -~, detector and injector port temperature 110 C ; the temperature of the column was 65~ BATCH REACTOR : We used a 60 ml reactor, thermostated at 400C and fitted out with gas-tight

Determination of Acetylcholine and Choline in Rat Brain Tissue by Liquid Chromatography/Electrochemistry Using an Immobilized Enzyme Post Column Reactor

Abstract: Following separation by conventional LC, acetylcholine and choline are converted to hydrogen peroxide in a packed bed reactor consisting of covalently bound acetylcholinesterase and choline oxidase Hydrogen peroxide, the ultimate enzymatic product, is detected amperometrically at a platinum electrode thin-layer cell This method is simple and highly sensitive to the detection of acetylcholine and choline, with detection limits of about 100 femtomoles The immobilized enzyme columns were stable for at least 60 days and conserved precious and expensive supplies of enzyme relative to continuous addition schemes The apparatus is generally applicable to other enzymatic reactions which yield electroactive products

Immobilization of enzyme onto poly(ethylene-vinyl alcohol) membrane.

Abstract: Invertase was ionically bound to the poly(ethylene-vinyl alcohol) membrane surface modified with two aminoacetals with different molecular length, 2-dimethyl-aminoacetoaldehyde dimethylacetal (AAA) and 3-(N,N-dimethylamino-n-propanediamine) propionaldehyde dimethylacetal (APA). Immobilization conditions were determined with respect to enzyme concentration in solution, pH value, ionic strength in immobilization solution, and immobilization time. Various properties of immobilized invertase were evaluated, and thermal stability was found especially to be improved by immobilization. The apparent Michaelis constant, Km, was smaller for invertase bound by APA with longer molecular lengths than for invertase bound by AAA. We attempted to bind glucoamylase of Rhizopus delemarorigin in the same way. The amount and activity of immobilized glucoamylase were much less than those of invertase. 16 references.

Highly Enantioselective Reduction of Ethyl 2-Acyloxy-3-oxobutanoate with Immobilized Baker’s Yeast

Abstract: Reduction of ethyl 2-acetoxy-3-oxobutanoate (4a) with immobilized baker’s yeast in calcium alginate gel gave a 18 : 19 : 63 mixture of ethyl (2R,3S)- and (2S,3S)-2-acetoxy-3-hydroxybutanoates and ethyl (2S,3S)-2,3-dihydroxybutanoate each with >95% e.e. in 58% yield. A similar treatment of a 2-benzoyloxy analog of 4a afforded a 6 : 94 mixture of ethyl (2R,3S)- and (2S,3S)-2-benzoyloxy-3-hydroxybutanoates (>95% e.e.) in 70% yield. Their absolute configurations were determined by comparison with authentic (2R,3S)- and (2S,3S)-2,3-dihydroxybutanoic acids. Effects of the immobilization and the pH of culture solution on the product ratio are also discussed.