SU1059002A1 - Method for immobilizing l-lysine decarboxylase - Google Patents

Abstract

METHOD FOR IMMOBILIZATION L-LIEINDEKARBOKSNLAZY by binding the enzyme with a water-insoluble carrier / characterized in that, in order to simplify the process, as a carrier used aminopropilsilohrom modified monobromuksusnoy acid and N, N N'-dicyclohexylcarbodiimide, taken in tymi Equivalent amounts. a Jo t90ea-j 70W CL with JsP Figure 1

Description

The invention relates to biotechnology, in particular, to methods of immobilizing the enzyme L-lysine decarboxycyl, and is intended for use in the microbiological industry and in the production of biochemical reagents. There is a known method for immobilizing L-lysine decarboxylase by depositing CO - selective electrodes with a solution of bovine serum albumin, dissolving the enzyme in it, and treating the mixture with glutaraldehyde l. However, this method of immobilization is only suitable for obtaining immobilized b-lysine decarboxyses, which is localized on the surface of a COj - selective electrode. The immobilized L-lysine decarboxylase obtained by this method cannot be used in installations of other types (except for those where CO is used as a selective electrode), which limits its scope. Closest to the proposed. Technically, it is a method of immobilizing L-lysine-decarbo silase by binding the enzyme to a water-insoluble carrier, which uses the inner surface of a glass tube silanized with vinyltriethoxysilane (using a mixture of vinyltristoxysilane, methanol and water). The immobilization is carried out by depositing on the silanized surface of the steel tube an enzyme solution containing benzoinisobutyl ether {dissolved in polypropylene glycol and an emulsion type prepolymer consisting of polyvinyl acetate, polyvinyl alcohol and hydroxymethyl acrylamide. The mixture is illuminated with ultraviolet light in a nitrogen atmosphere. The layer of immobilized enzyme is covered twice more with a mixture of benzoinisobutyl ether and prepolymer, each time it is replenished by ultraviolet light in a nitrogen atmosphere 2. The disadvantage of this method is that it is technologically complex and laborious, requires a large range of reagents, including such hard-to-reach as benzoinisobutyl ester. The aim of the invention is to simplify the process for the preparation of immobilized L-lysine decarboxylase. This goal is achieved by the method of immobilizing L-lysine decarboxyc silase by binding the enzyme to a water body with an anemomer, as a carrier using aminopropylsilochrome modified with monobromic acetic acid and N, N-dicyclohexylcarbodiimide, taken in equivalent amounts of B. the silochrome and the enzyme are bound to such a silochrome by the alkylation reaction of the nucleophilic groups of the enzyme. The proposed method is quite simple in technology and, in addition, allows to obtain an immobilized L-lysine decarboxylase with a rather high specific activity (3000-3500 mk mol / min-g). The level of activity obtained by the proposed method significantly exceeds the average level of activity of immobilized enzymes (300-500 µmol / min by the known method of obtaining immobilized enzyme with the highest activity (Figs. 1 and 2) it is advisable to carry out the process at pH 6.6-7, 3 and the weight ratio of protein / carrier is 45-55 mg / g. With the remaining values of these parameters, the process of immunization is less effective from the point of view of the level of activity achieved.Figure 1 shows the dependence of the activity of immobilized L -lizin decarboxylase on immobilization pH; Fig. 2 shows the dependence of the activity of immobilized L-lysine decarboxylase on the initial weight ratio of enzyme / carrier at immobilization pH of 7.2, Example 1. Method of immobilizing L-lysine decarboxylase. in groups, 10 g of aminopropylsilochrome CX-2.5 are suspended in 4-0 ml of dioxane containing 1.12 g of monobromoacetic acid, and a solution of an equivalent amount (1.16) is added. d) N, N-dicyclohexylcarbodiimide in 10 ml of dioxane. The mixture is stirred in a shaker at room temperature for 2 hours, after which the carrier is nicknamed by decanting with water to remove the reaction by-product, dicyclohexylurea. . Binding of L-lysine decarboxylase with a carrier, To 10 g of modified silochrome, add 100 ml of p. Escherichia L-lysine decarboxylase solution in 0.05. M phosphate buffer pH 7.2 and mix on a rocking chair for 10-15 hours. Immobilized enzyme is filtered and washed on the filter 0.05 M phosphate buffer pH 5.7, containing 510 M pyridoxyl phosphate, 10 M Trilon B and 0.01% mercaptoethanol. The resulting preparation is stored in a refrigerator (+ 4 ° C). Activity immobilized enzyme 3400 micromol / min-g, the output in the process of immobilization 55%.

The use of the proposed method for obtaining immobilized L-lysine decarboxylase provides the following advantages in comparison with the known method: the proposed method is technologically simpler, since it does not require such hard-to-scale operations as silanization / glass surface, a thin layer of reagent mixture deposited on the glass surface eliminates the need to work in the atmosphere nitrogen. To implement the method, a large assortment of reagents is not required, including such a hard-to-reach and expensive reagent such as benzo-e-butyl ether. The proposed carrier and reagents for its activation are cheap and are produced by domestic industry. The remaining reagents, namely L-lysine decarboxylase, pyridoxal phosphate, Trilon B, and mercaptoethanol, are used in any method of immobilization, since they are necessary for the manifestation and stabilization of enzyme activity.

Immobilized L-lysine decarboxylase is used to determine the concentration of L-lysine in its microbiological production, as well as for the analysis of L-lysine in food and

0 feed raw materials and products, in research works and other areas. The high activity of the immobilized enzyme allows for the complete decarboxylation of the substrate to be achieved over several

5 minutes. This significantly speeds up and simplifies the determination of the concentration of L-lysine, in particular, makes it possible to eliminate the temperature control, which is necessary when using little active immobilized preparations operating in the kinetic mode ....

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fOO 80504529Yu 20 30 0, 50

Perpent carrier, g / g Figure 2

Claims (1)

METHOD FOR IMMOBILIZING L-LYSINDECARBOXCLOSE by binding the enzyme to a water-insoluble carrier; distinguishing S.I. in that, in order to simplify the process, aminopropylsilochrome modified with monobromoacetic acid and Ν, Ν'-dicyclohexylcarbodiimide, taken in equivalent amounts, are used as a carrier.