SU1634714A1 - Method for testing restrictases sac ii and sst ii from microorganisms of the genus streptomyces - Google Patents

Abstract

The invention relates to biotechnology and molecular biology, concerns methods for testing Sac II and Sst type II restricts of Streptomyces and can be used in molecular biological and biotechnological studies. The aim of the invention is to increase the specificity of testing. The method consists in that the cells of the microorganisms are introduced into the cooled buffer and are destroyed by ultrasound. The extract of destroyed cells is heated at 50-55 ° C for 10-15 minutes. After cooling to 4-6 ° C, the extract containing Sac II or Sst II restrictase is freed from debris. The enzyme activity per 1 g of wet cells is at least 6000 units for Sac II restrictase and at least 10,000 units for Sst II restrictase. 1 tab. Yo

Description

The invention relates to biotechnology and molecular biology, relates to methods for testing restriction enzymes Sac II and Sst II in microorganisms of the genus Streptomyces and can be used in biological biological and biotechnological studies.

The purpose of the invention is to increase the specificity of testing.

The method consists in introducing microorganism cells into a cooled buffer and subjecting it to hydrodynamic destruction by ultrasound. The extract of the destroyed cells is heated at 50-55 ° C for 10-15 minutes. After cooling to 4-6 ° C, the extract is freed from debris, and the supernatant contains Sac II restrictase or its isosthisomer Sst II. The enzyme activity per 1 g of wet cells was not less than 6000 units. for Sac II restrictases and at least 10,000 units. for Sst II. The extract of destroyed cells of a microorganism producing several ecdonucleases is subjected to heat treatment at 50-55 ° C, as a result, the accuracy of determination of the target restrictase (Sac II and Sst II) is improved due to the release of the cell extract from associated thermolabile enzymes. The heat treatment of the cell extract also makes it easier to select for the desired product, and from several producers choose the most active.

The table shows the dependence of the accuracy of restriction enzymes testing on the mode of cell heat treatment.

As can be seen from the table, optimal for testing Sac II and Sst II restriction enzymes is the treatment of the cell extract with

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50-55 ° C. At a temperature below 50 ° C, the restriction pattern characteristic of two restriction endonucleases (Sac I + Sac II or Sst I f Sst II) is maintained, inactivated at 60 ° C and above.

The method is illustrated by the following examples.

Example 1. One gram of S.achromogenes ATCC 12767 biomass is suspended in 4 ml of tris buffer pH 7.5, containing 0.5 mM EDTA, 50 mM NaCI, 10 mM 2-mercaptoethanol. The suspension is treated with ultrasound on a UZDN-2T disintegrator at a resonant frequency in the range of 22 kHz for 4-6 minutes. The extract of the destroyed cells is heated for 15 minutes at 50 ° C and centrifuged for 30 minutes at 18,000 rpm. The Sac II restrictase activity in the obtained supernatant is tested by the method of hydrolysis of phage A DNA from 857 with the subsequent separation of the obtained fragments by electrophoresis in a horizontal block of gas. Per unit of enzyme activity, they take an amount that, within 1 h, completely specifically cleaves 1 µg of DNA.

The hydrolysis reaction mixture contains 10 mM Tris buffer pH 7.5.6 mM MgCl2. 6 mM W, 6 mM 2-mercaptoethanol. 1 μg of DNA I from 1857 in 40 μl of the mixture and 2-10 μl of the coarse cell extract. The separation of the obtained fragments is carried out in 0.1 M Na-borate buffer pH 8.2, containing 2 mM EDTA, in a 1% agarose gel. Ethidium-bromide stained (1 μg / l) gels are viewed in UV light.

The activity of Sac II restrictase in the roughly purified cell extract was 6000 units. in 1 g of wet biomass or 90900 units. in 1 g of dry mycelium.

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Example 2. Testing of Sst II restrictase in coarsely purified extract of S.stanford ATCC 29415 cells was carried out analogously to Example 1. Sst II restrictase activity was 10,000 units. in 1 g of wet cells or 150500 units. in 1 g of dry mycelium.

Example 3. Testing of Sst II restrictases is carried out analogously to Example 1, except that the extract of destroyed S.stanford lightweight materials is not subjected to heat treatment. Sst II restriction activity in the coarse cell extract is not detected, the total activity of the two restriction enzymes is tested due to the presence of Sstl restriction enzyme.

Example 4. Testing of Sst II restrictase is carried out analogously to Example 1, except that the cell extract is heated for 10 minutes at 55 ° C. Activity

0 enzyme is 10,000 units. in 1 g of wet cells.

Example 5. Testing of Sst II restrictase is carried out analogously to Example 1, except that the extract of the destroyed cells is heated for 12 minutes at 52 ° C. The Sst II restrictase activity in the roughly purified preparation of S.stanford cell extract is 10,500 units. in 1 g of wet cells.,

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Claims (1)

The invention of the method for testing Sac I and Sst restriction enzymes from microorganisms of the genus Streptomyces, which involves obtaining a coarse extract by sonicating cells, centrifuging to obtain a supernatant, characterized in that, in order to increase the specificity of testing, the extract of destroyed 0 cells of microorganisms are subjected to heat treatment at 50-55 ° C for 10-15 minutes five