RU1838409C - Immobilized adenylate cyclase of plague microbe - Google Patents

Abstract

Usage: biochemistry and biotechnology refers to the preparation of matrices with covalently bound proteins. SUMMARY OF THE INVENTION: An immobilized plague microbe adenylate cyclase is prepared by covalently linking adenylate cyclase to periodate-oxidized CL 6B sepharose. The activity of the immobilized enzyme in relation to the native 95%. Activity is maintained for 6 months.

Description

The invention relates to biochemistry and biotechnology, in particular to methods for producing matrices with covalently bound proteins.

The purpose of the invention is the immobilization of plague microbe adenylate cyclase.

. The goal is achieved by the fact that plague adenylate cyclase is covalently linked to plague microbe adenylate cyclase on a periodate oxidized by CL 6B. Unlike the prototype, the enzyme accession solution does not contain phosphate ions that inactivate adenylate cyclase.

Example 1. 1.5 g of Sepharose CL 6B, washed with 10 times the volume of water, is placed in a plastic bottle with a volume of 15-20 ml. 3 ml of a 0.07 M sodium periodate solution was added and the reaction mixture was slowly stirred for 45-60 minutes at room temperature. 0.5 ml of 2M ethylene glycol was added and stirring was continued for another 30 minutes. The gel is filtered on a porous glass filter,

washed with water and then 0.01 M sodium carbonate buffer (pH 9.5).

An activated matrix is added to 5 ml of a plague microbe adenylate cyclase solution dialyzed against 0.01 M carbonate buffer (pH 8.5) (0.5 mg / ml adenylate cyclase with an activity of 2 nmol / mg) and the pH of the suspension adjusted to 9.5 with 2 M sodium hydroxide. The reaction is continued for 24 hours, the reaction vessel is slowly rotated at 8-10 ° C.

To reduce the azomethines and the remaining aldehyde groups, 3 ml of a freshly prepared solution of sodium borohydride at a concentration of 1 mg / ml in the same buffer was added. The reaction is continued with slow stirring for 1 hour in a cold room. The gel is washed with 0.01 M carbonate buffer pH 8.5, then 0.01 Mtris-HCI pH 7.6. The activity associated with the adenylate cyclase matrix is determined by the method of (Tovey K.S., Whelan J.A. M. A simple direct assay for cyclic AMP in plasma and other biological sampe using an improved

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ompletive protelin binding technique. Clin. hlm. Acta, 1974, v. 56, P.221-224). Ohm is 2.5 u / g. The unit of denylate cyclase activity is taken as the number of cAMP anomeles synthesized by one g of the enzyme in 1 minute at 37 ° C. The number of adenylate cyclases immobilized per gram of carrier is 1.3 mg, which is 80% of the amount added for binding. The immobilized enzyme retains 95% activity in relation to the native state. Immobilized adenylate cyclase fully retained its activity for 6 months (observation period).

Example 2. All operations were carried out as in example 1, but the concentration of adenylate cyclase was 0.6 mg / ml, incubation 12 hours, temperature 22 ° C. Further operations are as in Example 1. The amount of adenylyltase-clase immobilized per 1 g of carrier is 1.7 mg, which corresponds to 85% of that added for binding. The activity associated with the adenylate cyclase matrix corresponds to 3.2 u / g.

Example 3. All operations are carried out as in Example 1, but adenylate cyclase is dialyzed against 0.01 M carbonate buffer, pH 9.5. The concentration of the enzyme is 0.5 mg / ml, the incubation time is 10 hours, and the temperature is 28 ° C. The amount of adenylate cyclase immobilized per gram of carrier is 1.4 mg, which is 82% of that added for binding. The activity associated with the adenylate cyclase matrix is 2.7 u / g.

As can be seen from the above examples, the obtained immobilized plague microbe adenylate cyclase is a highly active and stable product with great biotechnological and biochemical significance. Since adenylate cyclase is an enzyme for the synthesis of cyclic adenosine monophosphate, a biologically active substance used in biology and medicine,

the immobilized form of the enzyme can be used in the biotechnological process for the preparation of cAMP, due to the easy extractable nucleotide ™ from the reaction mixture and the possibility of reuse of the matrix. In addition, the associated adenylate cyclosis is characterized by the stability of its structure, which makes it possible

transport over long distances without involving low temperatures and other conditions commonly used for enzymes.

Covalent binding of adenylate cyclase can play a role in studying the mechanism of its action, because allows you to quickly change and control the environment of the enzyme. If we take into account that most of the proteins of the cell, including adenylate cyclase, are activated by the addition or cleavage of inorganic phosphate, then, having a protein attached to the matrix, sequentially introducing or removing the studied substances from the reaction system, it is possible to determine exactly which ion catalyzes its activity and which cell enzymes are involved.

Formula-and on acquisition Immobilized plague microbe adenylate cyclase general formula

n and

P-C-N-E-. Mgde P is the residue of the modified by periodic oxidation of sepharose CL 6B,

H - N-E residue of the adenylate cyclase molecule after reduction with sodium borohydride,

capable of synthesizing cyclic adenosine monophosphate.