SU722197A1 - Process for producing immobilized enzymes - Google Patents

Abstract

1. THE METHOD OF OBTAINING IMMOBILIZED ENZYMES by co-polymerizing acrylic monomers in an enzyme solution in the presence of glutaraldehyde and polymerization initiators, so that, in order to increase activity and stabilize; tartaric acid diisocyanate is added to the polymerization mixture in an amount of 0.25-0.5%, hexvmethylenediamine in an amount of 0.25-0.4% and sucrose in an amount of 20-30% by weight of the polymerization mixture and the process is carried out at 5 -10 ° C.2. Method according to claim 1, characterized in that glutar is used New aldehyde in the amount of 0.5-1% by weight of the polymerization mixture. 3. The method according to claim 1, characterized in that N, N, .N ', N-tetramethylethylenediamine and ammonium sulphate are used as polymerization initiators. (O ( L

Description

The invention relates to the microbiological and chemical industry, in particular to methods for producing immobilized enzymes that can be used in the preparation of biochemical reagents used in molecular biology and genetic engineering.

A known method for producing immobilized enzymes consists in polymerizing acrylic monomers in an enzyme solution in the presence of glutaraldehyde and polymerization initiators, with powdered polycarbamide in the amount of 5-10% with an amino group content of 0.3-0.7 milliequivalents per gram of substance added to the reaction mixture. and the process is carried out at room temperature SP,

However, this method does not provide a sufficiently high absolute and relative activity and stability of the obtained product (in particular, immobilized trans-N-deoxyribosyllase is obtained with an absolute activity of not more than 30 units / g and an activity preservation coefficient of not more than 0.1, and after 5 days of storage the activity of the immobilized enzyme does not exceed 20%).

The aim of the invention is to increase the activity and stability of the immobilized enzyme.

This goal is achieved by adding tartaric diisocyanate in an amount of 0.25-0.5%, hexamethylenediamine in an amount of 0.25-0.4% and sucrose in an amount of 20-30% by weight of polymerization in a known process. the mixture and the process of polymerization are carried out at 5-10 C.

In the process of implementation, glutaraldehyde is used in the amount of 0.5-1S by weight of the polymerisation mixture; and as polymerization initiators - N, M5M; - tetramethylethylennamin and acmonium sulphate.

Example 1. The following solutions were prepared for the preparation of an imglobilizable trans-N-deoxyribosylase preparation (EC 2,4,2,6); 1% solution of the enzyme in distilled water: with an activity of 8,000 e / d.Act / ml (the amount of the enzyme is taken as the amount of the enzyme) catalyzing the conversion of 1 nmol of substrate per 1 minute at 37 ° C); A 50% solution of monomers in distilled water, which contains 19 in it. including acrylic amides. and 1 wt.h, N, N-methylenebis-acruclad / 50% sucrose solution in distilled water; 50% solution of monomers in 50% sucrose with a content of 19 weight.h. acrylamide and 1 weight, h. M, N-methylenebis-ecrilam Ra-f 10% solution of hexamethylenediamine (GMMA) in distilled water; 10% solution of tartaric acid diisocyanate (DICVK) in distilled water; 10% solution of ammonium persulphate (HCA) in distilled water; 10% solution of N, N, N, N-tetramethylethylenediamine (TEMED) in distilled water.

In a cooled vessel mixed

0.3 ml of enzyme solution, 0.3 ml of 50% sucrose solution, 1.5 ml of monomer solution in 50% sucrose, 0.12 ml of 10% HMDA solution, 0.08 ml of 25% solution

glutaraldehyde (GA) (Merck, Germany), 0.13 ml of a 13% aqueous solution of DITsVK, 0.23 ml of a 10% aqueous solution of TEMED, 0.23 ml of a 10% aqueous solution of HCA. The final concentrations of the reagents in the polymerization mixture are as follows,% g monomers 25, sucrose 30, HMDA 0.4, HA. 0.7, DRAPA 0.4, NSA 0.75, TEMED 0.75, enzyme 0.1, The temperature of the polymerization mixture should not exceed + 5-10 ° C.

The resulting mixture is thoroughly altered and held for 30-60 seconds until the formation of a gel. The formed gel block is crushed

to the size of granules 0.3-0.5 ml and

its activity is determined according to the generally accepted enzyme method. Get 4 g of the drug with an activity of 270 units of act. / G ow, gel, that

accounts for 41% of the initial activity. The obtained gel - enzyme is kept at 4 ° C for 5 days in a buffer and its activity is repeatedly determined, which occurs

equal to 210 units of act. / g ow. gel., which is 78% of the initial activity of the obtained gel.

The obtained results show that this composition of the gel provides sufficient activity and stability of the drug: h “mobilized trans-. -deoxyribosylase.

Etc :; meper 2, Imm: the abundance of the enzyme gvodod t as described in example 1, and using those zhz initial solutions with changing the number of individual reagents. Table 1 shows the final concentrations of the components in the polymerization mixture to the results obtained in determining the activity of preparations of the immobilized enzyme.

Table 1

 Under these conditions, no gel is formed. The enzyme is leaching from the gel.

Example 3. An immobilized enzyme preparation, prepared as in Example 1, is used to prepare deoxyadenosine from adenine and thymidine. For this, the enzyme preparation is added to the reaction mixture containing 1.75 nmol / ml of edenine and 8.5 nmol / ml of thymidine in citrate-phosphate buffer

The data in Table 2 convincingly show the stability and reactivity of the preparations obtained.

Example 4. The immobilization of phosphodisterase is performed as in Example 1, used as

pH 6.0. The reaction is carried out with constant stirring at 37 ° C. After completing the first cycle of using the gel enzyme, it is separated from the reaction mixture on a filter, washed with buffer and used in the next cycle.

The results of 16 consecutive cycles are described in table 2.

table 2

enzyme solution, phosphodiesterase solution from snake and with an activity of 3000 units of actin / ml (the unit of activity is that amount of the enzyme that provides an increase in optical density at L 400 nm by 0.1 p. o with Ca-bis hydrolysis para-nitrophenyl phosphate per 30 min, at the initial amount of the enzyme 2.400 unit concentration of the remaining components of the polymerization mixture,%: monomers 25, sucrose 20, HMDA 25, HA 0.63, .DICVC 0.24, HCA 0.75 , TEMED 0.75. At the same time, an enzyme preparation with an activity of 220 units of a moisture gel is obtained, the total active - 1.230 units of actus, which is 51% of the initial activity of the enzyme.When stored for 10 days with the immobilized enzyme, it retains its activity. Example 5. Immunization of carcinoma is carried out as in Example 1, used as a solution enzyme nucleoside phosphotransferase solution from seaweed with lilOO unit activity / ml (unit of activity is taken as the amount of enzyme that ensures the formation of 1 µmol nucleotide from nucleoside and para-nitrophenylphosphate per 1 hour at 3 ° C in 0.05 M sodium solution phosphate pH 5, 2) acetic acid in 0.18 M potassium solution. The initial amount of the enzyme is 550 units of act., The concentration of the remaining components of the polymerization mixture,% g monomers 25, sucrose 20, HMDA 0.24, HA 0.63, DICEC O, 24, HCA 0.75, TEMED 0.75. An ATEM obtained an enzyme preparation with an activity of 14.7 units of act. / G of a wet gel, a total activity of 10 units of act, which is 20% of the initial activity of the enzyme. When stored for 14 days at 4 ° C, the immobilized enzyme retains its activity completely.

Claims (3)

1. METHOD FOR PRODUCING IMMOBILIZED ENZYMES by co-polymerizing acrylic monomers in an enzyme solution in the presence of glutaraldehyde and polymerization initiators, which is detected in order to increase the activity and stability of the target products, tartaric acid diisocyanate in the amount of 0.25 is added to the polymerization mixture -0.5%, hexvmethylenediamine in an amount of 0.25-0.4% and sucrose in an amount of 20-30% by weight of the polymerization mixture and the process is carried out at 5-10 ° C. 2. The method according to claim 1, characterized in that they use glutaraldehyde in an amount of 0.5-1% by weight of the polymerization mixture. 3. The method according to claim 1, characterized in that Ν, Ν, Ν ', N-tetramethylethylenediamine and ammonium sulphate are used as polymerization initiators. 22197