SU922142A1 - Method for immobilizing cells for microorganisms with beta-tyrosinase activity - Google Patents

Description

(54) METHOD FOR IMMOBILIZATION OF MICROORGANISM CELLS WITH |) -TYROSYNASE ACTIVITY

I

This invention relates to organic chemistry, and specifically to methods for producing immobilized microorganism cells; mov with fb-tyrosinase (EC 4.1.99.2) activity.

The use of immobilized microbial cells for the production of various substances has recently been greatly expanded. Immobilization of cells allows more efficient use of the catalytic properties of microorganisms, since the costs of isolation and purification of enzymes are eliminated. Immobilized cells with 1-tyrosinase activity can be used for enzymatic synthesis, Li-tyrosine, an amino acid, which is difficult and expensive to produce by other means.

There is only one method of immobilization of cells of microorganisms with | tyrosinase activity by incorporation into a gel.

According to this method, the immobilization of cells Erwimq Vierb uOBc | The ATCC strain 21434 is carried out by incorporation into a gel, formed by mixing water-soluble polymers of polyvinyl alcohol, gelatin, carboxymethylcellulose and tetraalkoxysylane with the addition of acid. The gel is dried in a frozen state for several days. All operations are covodt at a temperature of no less than 5 ° C. The specific activity of the obtained preparation is

mole

l

7-10

. Further

cmg of cell protein according to the stability of the preparation; in the description of the invention, there is no Cl.

The disadvantages of the method are not high enough jb tyrosine, the specific activity of the preparation, the complexity and duration of the immobilization process due to the implementation of all operations at low temperature, the use of scarce and expensive polymers, including food raw materials. The purpose of the invention is to increase the noise immunity of the drug, simplifying and cheapening the process. This goal is achieved in that according to the method of immobilizing microbial cells with (L-tyrosinase activity by incorporating them into the gel, immobilization is carried out in the presence of ammonium acetate or pyruvate sodium or I tyrosine, while polyacrylamide gel is used as gel, from microorganisms, the strain Citrobcioter reund N 62 is used. Immobilization of cells is carried out in phosphate buffer pH 7-8 with the addition of ammonium acetate, or in acetate buffer at pH 8. The buffer is mixed with suspension of cells, solutions of acripamide megylenbisacrylamite, N, S, N4 N of tetramethylenedimine and ammonium persulphate. The reaction vessel is immersed in an ice bath. Polymerization is completed in a minute. includes 89% of the cells. When immobilized, 65% of the initial activity is retained. The specific catalytic activity of the immobilized cells is (depending on the batch) 3-Oj4-10 mol / s mg protein. The preparation is stored in a buffer at pH 8 and +4, after a month of storage, the immobilized cells retain 6% of the activity of the original while the nodular cells completely lose the I-tyrazinase activity. Immobilized cells can be repeatedly used for. tyrosine synthesis. At the same time, after the first use, 95% of the activity is retained, after the second - 80%, after tert its use - 50%. EXAMPLE 1. To O, 4 ml of 0.01 M phosphate buffer pH 7.0, add 1 mM ammonium acetate (adjusted to pH 8 with a concentrated ammonium solvent) 0.2 ml of a suspension of Citr batter freoncliV 62, containing 53 mg of protein with a specific (Js-tyrosinase catalytic activity, determined by the standard method for the synthesis of tyrosis on 4.6 "mol / s-mg protein, 1 ml of 5p% acryl amide solution with the addition of methylene bisacrylamide in a ratio of 19: lj 0.3 ml of a 109 & solution of N, N, N-tetramethylethylenediamine and 0.3 ml of a 10% solution of ammonium persulfate. All solutions are prepared in 0.01 M fo. fat buffer pH 7.0. The mixture is rapidly stirred in a Petri dish placed in ice. Polymerization begins after 30 seconds and ends after 1 minute. A block of polyacrylamide gel with cells included in it is rubbed through a 1 × 1 mm polyethylene screen. Gel particles 0.05 M ammonium acetate buffer pH 8.0 is washed free of free cells. The gel contains the number of cells corresponding to 47.7 mg of protein, i.e. 90%. The specific catalytic activity for the synthesis of {i tyrosine immobilized cells is 3 mol / s, mg protein, which is 65% of the original. The drug is stored in a buffer at pH 8 and + 4 ° C. After a month of storage, the activity of the immobilized cells is 6 O% of the original. When immobilized without ammonium acetate, only 20% P-tyrosinase activity is retained. PRI mme R 2. The method is carried out according to Example 1, but all solutions are prepared in 0.05 M ammonium acetate buffer pH 8.0. The results are similar to those obtained in Example 1. PRI me R 3. The method is carried out according to Example 1. Another batch of cells with initial JS tyrosinase activity equal to 1.2510 mol / s, mg protein, is used. To the mixture for the polymerization of acrylic monomers, instead of b, 5 M ammonium acetate is added. nor 0.5 M sodium pyruvate to a final concentration of 0.2 M. C. Immunized C.ire.UT cells are obtained (3ii 62 with a specific activity of 0.74-10 mol / cmg of protein, i.e. with 59% of free cells remaining active and 9O% cell incorporation into polyacrylamide Example 4: The method is carried out according to the predler 1. The same batch of cells is used. 4 geips1m as in example 3. To the mixture for the polymerization of acrylic monomers add 2 , .25 mg 1b-tyrosine and made up to a total volume of 5 ml with phosphate buffer. 5 cells obtained immobilized by inclusion in polyacrylamide 1 gel are obtained. C. reur (3ii 62 with a specific L-tyrosinase activity of 0.5 10 mol / s M MI protein, i.e., equal to 40% of the initial 93% inclusion of the cells in the chloracrylamide gel.

Claims (1)

1. US patent No. 4148689, CL. 195-65, published. 1979