SU1409656A1 - Method of producing immobilized cholinesterase - Google Patents

Abstract

This invention relates to the field of biochemistry, biotechnology, and is intended to produce highly active and thermostable preparations of immobilized cholinesterase. These drugs. Immobilized cholinesterase can be used in devices to control the degree of air pollution, etc. The purpose of the invention is to increase the specific activity of the target product and the release of the enzyme during immobilization. The method involves binding the enzyme to the acid chloride derivative of shtochrome C-80j obtained from the carboxyl derivative of silochrome C-80 by heating the latter with thionyl chloride in a mixture: 2 ml of thionyl chloride, 0.2 ml of dimethylformamide and 15 ml of absoluted chloroform. In the process of immobilization for 1 h, 12-220 mg of protein per 1 g of carrier with the carrier bind 45% of activity, 55.50% of protein; carrier capacity 8.6 mg / g; The specific activity of the preparation of immobilized cholinesterase was 27.6 units / g of carrier. When stored at room temperature, the activity of the preparation of immobilized cholinesterase was stored for 80 days. i (L

Description

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The invention relates to biochemistry, biotechnology, and is intended for the preparation of highly active and thermostable preparations of immobilized cholinesterase, which can be used in devices for monitoring the degree of air pollution, water bodies and rivers with cholinesterase inhibitors, carbamic acid derivatives and organophosphorus compounds commonly used in rural farms as pesticides.

The purpose of the invention is to increase the activity of immobilized cholinesterase and the release of the enzyme during immobilization.

The method is carried out as follows.

Silochrome € -80 modified to a chlorine anhydride derivative (10 mol of acid chloride groups per 1 g of carrier) is used as a carrier. Cholinesterase is added to the carrier and stirred for an hour in phosphate buffer while cooling. . Then the drug is passed through with a cooled solution of 1 M NaCl and water. The method allows to increase the specific activity of the immobilized enzyme from 32.2% to 80% of the specific activity of the initial enzyme preparation.

Example 1. Activation of a carboxyl derivative of silochrome C-80.

The activation is carried out as follows: 3 g of silochrome C-80, modified to the carboxyl derivative, are suspended in a mixture of 15 ml of absolute chloroform, 2 ml of thionyl chloride and 0.2 ml of dimethylformamide. The suspension is heated to boiling and stirred with a stirrer for 5 hours. The mixture is then filtered and washed with 300 ml of absolute chloroform on filter No. 100. The filter cake is freed from the residue of chloroform using a boiling water bath and water-jet pump for 1 hour. The carrier contains 10 mol acid chloride groups for 1 g.

The dry product of silochrome C-80 acid chloride is suspended in 10 mpO, 1 M phosphate buffer (pH 7.2) and mixed with 10 MP of cholinesterase solution (3.6 mg in 1 ml of 0.1 M phosphate buffer) (12 mg protein per 1 g carrier). The mixture is stirred for 1 h at. The resulting product is washed on the filter.

Re No. 100 cooled to 200 ml of 1 M NaCl solution and 200 ml of icy distilled water. The precipitate is suspended in 10 ml of distilled water and stored as a suspension. The specific activity is 4.2 U / mg (80% of the initial). Associated with a carrier 55.5% protein.

Example 2. The dry product chloro anhydride of silochrome C-80 is suspended in 10. ml of 0.1 M phosphate buffer (pH 7.2) and mixed with 10 np of cholinesterase solution (1 mg in 1 ml of 0.1 M phosphate buffer) (3 , 3 mg of protein per 1 g of carrier). The mixture is stirred for 30 minutes at. Next, the product is treated as described in example 1.

The immobilization method described in Example 1 is preferred, since this method provides 21 units of binding. enzyme activity per 1 g of carrier, while the second method is only 2 units. on 1 g of the carrier. In addition, the product obtained by the first method is characterized by much greater stability.

Example 3. The method is carried out as in Example 1, but 220 mg of protein per 1 g of carrier (1140 units, per 1 g of carrier) are added. The activity of the drug - 267 u / g (79 mg of protein with a specific activity of 3.5 uUmg).

The activity of the immobilized preparations remains unchanged for 80 days. Loss of the enzyme during immobilization is 40.2%.

Thus, the proposed method allows to increase the specific activity (from 32.2 to 80%) and to reduce the loss of the enzyme during immobilization from 62.2% to 40.2%.

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Claims (1)

45 claims A method for producing immobilized cholinesterase, including covalent bonding of cholinesterase with a water-soluble carrier by incubating the pre-activated carrier with the enzyme in phosphate buffer while cooling, followed by laundering the target product from the nonspecific adsorbed enzyme, which was detected by an activator with chromosterase, followed by laundering of the target product from the nonspecifically adsorbed enzyme, which was absorbed by the enzyme. the target product and the release of the enzyme during immobilization, as a carrier of j1A09656 silochrome C-80, activated 1 gnositel, and incubation is carried out to acid chloride and containing 10 - ratio of 12-220 mg of protein per - 10 ml of acid chloride groups on the episode.