Showing papers on "Immobilized enzyme published in 1973"

New methods for binding enzyme molecules into a water‐insoluble matrix: Properties after insolubilization

Abstract: New methods of crosslinking enzyme molecules inside a matrix with or without an inactive protein are described. Enzyme activity yields range between 30 and 80% of the activity of the untreated preparations. Even fragile enzyme systems, for instance those using mobile cofactors, can be efficiently immobilized. Increased resistance towards heat denaturation and proteolysis results.

Enzyme immobilization with a thermochemical-photochemical bifunctional agent

Abstract: Enzymes are immobilized by thermochemically attaching to a solid support material a bifunctional agent initially possessing thermochemical-photochemical functional substituents followed by photochemically activating the bifunctional agent and covalently bonding an enzyme thereto. Preferred bifunctional agents are arylazides which allow activation by visible light.

Conversion of benzylpenicillin to 6‐aminopenicillanic acid in a batch reactor and continuous feed stirred tank reactor using immobilized penicillin amidase

Abstract: A rate equation has been derived to describe the hydrolysis of benzylpenicillin to 6-aminopenicillanic acid by penicillin amidase. The integrated from of the rate equation has been shown to predict satisfactorily the progress of the reaction in a batch reactor using either soluble or immobilized penicillin amidase. The rate equation was also used to predict the performance of a continuous feed stirred tank reactor containing immobilized enzyme. There was good agreement with experimental measurements.

Collagen-enzyme complex membranes and their performance in biocatalytic modules

Abstract: Collagen was used as carrier for the immobilization of invertase, lysozyme, urease, glucose oxidase, penicillin amidase, and glucose isomerase. Immobilization was accomplished by either impregnation of a preswollen collagen membrane with enzyme solution or electrocodeposition of collagen and enzyme from a collagen dispersion containing dissolved enzyme. The collagen-enzyme complexes prepared are in membrane form. Membranous collagen-enzyme complexes were used to construct biocatalytic reactors such as the capillaric coil modular reactor. Such biocatalytic reactors were used in a recirculation system for the conversion of substrates. The biocatalytic reactors showed initial decreases of activity to stable limits which are maintained over a large number of reactor volume replacements. The stable limits correspond to approximately 35% of the initial activities for lysozyme and invertase, 25% for urease, 15% for glucose oxidase. The mechanism of complex formation between collagen and enzyme involves multiple salt linkages, hydrogen bonds, and van der Waals interactions. This protein-protein interaction which leads to stable complexes by both impregnation and electrocodeposition processes is unique among the enzyme immobilization methods currently available.

On the regulation of the activity of immobilized enzymes. Microenvironmental effects of enzyme-generated pH changes.

Abstract: Glucose oxidase, hexokinase, trypsin and urease were entrapped, either separately or together, within polyacrylamide particles, using a bead polymerization technique. The pH optima, for the immobilized enzymes in 5 mM buffer, were displaced compared to those of the enzymes free in solution. Thus the pH optimum for trypsin activity towards benzoyl-l-arginine ethyl ester shifted 1.3 pH unit (to pH 9.6) and the pH optimum for glucose oxidase towards glucose shifted 0.3 pH units to the alkaline side (to pH 6.9). Reversely, urease activity towards urea leading to consumption of protons shifted its optimum 0.4 unit to the acidic side (to pH 5.8). The effect on glucose oxidase activity of pH changes caused by activity of coentrapped trypsin or urease was studied. It was found that glucose oxidase activity in the alkaline region was stimulated during simultaneous trypsin activity, whereas at pH values below that of the optimum for glucose oxidase inhibition occurred. A reversed effect was measured during urease activity. At pH 8.6, for example, glucose oxidase activity was increased by a factor of three to 75% of its activity found at pH optimum due to trypsin activity. On the other hand, at pH 6.0, i.e. below the optimum, it increased by a factor of two to about 80% due to urease activity. Both simultaneous trypsin or urease activity gave distorted “two-peak” pH-activity profiles of glucose oxidase. The effect of protons produced by trypsin on the simultaneous activities of glucose oxidase and hexokinase, both competing for the same substrate, glucose, was studied with a gel containing all three enzymes. It was found that the amount of glucose converted per minute by each enzyme reaction was effected. In one case, at pH 8.5 in the absence of trypsin activity, about one seventh of the glucose consumption was due to hexokinase activity. On addition of benzoyl-l-arginine ethyl ester, hexokinase activity decreased almost to zero, while glucose oxidase activity was stimulated, resulting in practically all conversion being due to the latter.

ATP regeneration using immobilized carbamyl phosphokinase.

Abstract: Most of the biochemical processes now employing immobilized enzymes are of the type which require no cofactors (1,2). Large scale use of immobilized enzymes which require cofactors, such as NAD or ATP, depends in part on efficient regeneration of the cofactors. Progress in this direction has been reported for NAD reuse (3,4). The work reported here describes a method for ATP regeneration. The procedure is based on the enzyme catalyzed reaction between carbamyl phosphate and ADP. The carbamyl phosphate is easily generated in situ by reaction between potassium cyanate and potassium phosphate. The enzyme, carbamyl phosphokinase, previously described by Jones and Lipmann (5), was immobilized on porous, alkylamine glass and used continuously to convert ADP into ATP.

[3] Active enzyme centrifugation

Abstract: Publisher Summary This chapter describes active enzyme centrifugation, which is based on the principle that an enzymatic reaction occurring in the cell can be followed by some physical method during centrifugation. In many cases, enzymatic reactions may be monitored spectrophotometrically, which is convenient as well as highly accurate. This technique can be used in the centrifuge by utilizing a photoelectric scanning system. The method is not necessarily limited to reactions in which optical density changes occur in either substrates or products during catalysis. Enzymatic reactions may also be monitored if the enzymatic reaction can be coupled to a secondary reaction, which involves a change in optical density. As only the displacement of the active form of the enzyme is observed with this method, any inactive forms of the enzyme, which may be present in the preparation, are not observed. For this reason, it is not always necessary to use highly purified enzymes to obtain meaningful results. In fact, successful experiments may be done with relatively crude dialyzed tissue extracts. Active enzyme centrifugation may, thus, be applied to many enzyme systems.

Multiple immobilized enzyme reactors: determination of pyruvate and phosphoenolpyruvate concentrations using immobilized lactate dehydrogenase and pyruvate kinase.

Abstract: Lactate dehydrogenase (LDH) and pyruvate kinase (PK), immobilized on solid glass beads by diazotization, were used in packed bed reactors to analyze for both pyruvate (PYR) and phosphoenolpyruvate (PEP) through the disappearance of β-nicotinamide adenine dinucleotide (NADH) monitored spectrophotometrically at 340 nm. Packed bed reactors containing PK and/or LDH were also capable of monitoring continuously varying concentrations of adenosine-5′-diphosphate (ADP), PEP, and PYR. The immobilized enzymes (∼40 μg/g glass) retained about 4% of the activity of the soluble enzymes. Preparations of immobilized LDH and PK exhibited enhanced stability when maintained in the presence of β-mercaptoethanol and NADH or EDTA, respectively, and were shown to regain 75% of the original activity after four months storage at 4°C.

Enzymes immobilized on porous inorganic support materials

Abstract: An immobilized enzyme composite is disclosed having an enzyme adsorbed to the inner surface of a porous ceramic body consisting of agglomerated metal oxide particles having an average pore diameter of at least as large as the largest dimension of the enzyme but less than 1,000 DEG A.

Preparation of immobilized invertase

Abstract: Immobilized invertase was prepared by ionically binding the enzyme to diethylaminoacetyl cellulose (DEAA-cellulose). DEAA-cellulose-invertase complex was quite stable to electrolyte in the range of pH 5–7. Bound invertase was less active than the native enzyme, and approximately 55–70% of the enzyme activity was lost on binding. The complex was stable for 9 days' continuous inversion in a column system at 30°C, but was rather unstable at 40°C. Heat stability and the effect of temperature on the reaction rate of the complex were almost identical with those of the native enzyme.

Method of making fructose with immobilized glucose isomerase

Abstract: Method of converting glucose to fructose which comprises incubating a glucose-containing solution with an immobilized enzyme composite comprising glucose isomerase adsorbed within the pores of a porous alumina body having an average pore diameter between about 100 and 1000A

Synergistic enzymes adsorbed within porous inorganic carriers

Abstract: 1. AN IMMOBILIZED ENZYME COMPOSITE COMPRISING TWO SYNERGISTIC ENZYMES ADSORBED TO THE INTERNAL SURFACE OF A HIGH SURFACE AREA, POROUS TITANIA BODY, THE POROUS TITANIA BODY HAVING AN AVERAGE PORE DIAMETER AT LEAST AS LARGE AS THE LARGEST DIMENSION OF THE LARGER ENZYME BUT LESS THAN 1000 A., AND THE TWO SYNERGISTIC ENZYMES SELECTED FROM THE GROUP CONSISTING OF GLUCOSE OXIDASE AND CATALASE, GALACTOSE OXIDASE AND CATALASE, D-AMINO ACID OXIDASE AND CATALASE, CHYMOTRYPSIN AND TRYPSIN, AND PAPAIN AND STREPTOCOCCUS PEPTIDASE A.

Immobilized enzymes and method for preparation

Abstract: The instant invention relates to immobilized enzymes which comprise an enzyme containing-solution emulsified in immiscible liquid. Preferably the enzyme containing-solution is aqueous and will comprise from about 0.1 to about 10 weight percent enzyme. A surfactant is provided in the immiscible liquid to stabilize the emulsion, the surfactant comprising from 0.01 to 90 weight percent, preferably 0.5 to 20 weight percent of the immiscible liquid. The instant invention also relates to a general procedure for preparing immobilized enzymes by emulsifying a solution containing said enzyme in an immiscible liquid. The emulsions of the instant invention are designed to be stable at the conditions at which the immobilized enzymes will be utilized. For example, in a chemical conversion process wherein phenol is oxidized in the presence of the immobilized enzymes of the instant invention, e.g. an aqueous phenol oxidase containing solution emulsified in a hydrocarbon, e.g. cyclohexane, sufficient surfactant is added to said hydrocarbon liquid so as to provide an emulsion which does not break down during the contact with the phenol reactant.