RU1806191C - Strain of bacterium micrococcus species - a producer of restrictase msp - Google Patents

Abstract

Usage: biotechnology and genetic engineering. SUMMARY OF THE INVENTION: a bacterial strain Mlcrococcus species, stored in VKPM All-Russian Research Institute of Genetics under registration number B-5463, was obtained by a systematic search for producers of restriction endonucleases as a contaminant during cultivation. When the strain is grown on a fresh nutrient medium at 30 ° C in a temperature-controlled rocking chair until a stationary growth phase is reached, a crop of cells is obtained, from which the Msp 20 I enzyme capable of recognizing and cleaving the 5-TGGCCA-3 nucleotide sequence is isolated. A restriction enzyme of this specificity is used in genetic engineering to create recombinant molecules and to create a physical mapping of DNA molecules. 1 ill. w yo

Description

The invention relates to biotechnology and genetic engineering, in particular to the production of a restriction endonuclease producer strain that recognizes and cleaves the 5-TGGCCA-3 nucleotide sequence. An endonucleaa of this specificity can be used for cloning, physical mapping of genes. This endonuclease is a convenient model for studying the specificity of protein-nucleic acid interactions.

An object of the invention is to identify a producer strain synthesizing a restriction endonuclease capable of recognizing and cleaving the 5-TGGCCA-3 nucleotide sequence in high yield of the target enzyme.

This is achieved by obtaining the strain Mlcrococcus species VKPM B-5463 - producer of the restriction endonuclease Msp 20 I.

The new strain was isolated as a contaminant during cultivation as a result of a systematic search for producers of restriction endonucleases using the express method.

The strain is deposited and stored in the All-Union Collection of Industrial Microorganisms of the All-Russian Research Institute of Genetics under the number VKPM B-5463.

The enzyme is named Msp 20 I according to generally accepted nomenclature.

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The strain of Mlcrococcus species has the following characteristics,

Cultural and morphological characters. Cells are spherical, 0.5-0.7 microns in diameter, located singly, in pairs, tetrads, or irregular clusters. Stationary, do not form endopores. They grow well on ordinary nutrient media (SPA, MPB) at a temperature of 30 ° C. SPA colonies are opaque, yellowish, rounded, the edge is even.

Physiological and biochemical properties, Aerob, negative for oxidase, amylase, arginine dehydrolase and reaction with methyl red, does not emit hydrogen sulfide and indole, is catalase-positive, does not utilize malonate, citrate, urea, and does not restore nitrates.

The content of HC pairs in DNA is 77 mol. %

The strain is stored under vaseline oil or in 30% glycerol at -70 ° C.

The yield of restriction enzyme Msp 20 I 3000 u / g of raw biomass.

The drawing shows an electrophoregram of the products of hydrolysis of DNA of phage lambda with enzymes Bal I and Msp 20 I.

1st lane - hydrolysis of Bal I

2 lane - joint hydrolysis of Ball and Msp 20I

3rd lane - hydrolysis of Msp 20 I

Example 1. Obtaining biomass and the selection of the enzyme,

Before starting work, the cells of the producer strain are subcultured with a bacteriological loop on an agar medium. Petri dishes are placed in a thermostat at 30 ° C. After 10-12 hours of incubation, the grown cells are subcultured into flasks with a fresh nutrient medium consisting of (g / l): bactotryptone 10, yeast extract 5, sodium chloride 10 and placed in a thermostatic shaker.

It is cultured at a temperature of 30 ° C, at 200 rpm, until a stationary growth phase is reached. Cells are pelleted by centrifugation at 4 ° C. Isolation was carried out according to the method of Blckle, Plrotta, lmber 4. The enzyme yield was 3000 u / g crude biomass.

The enzyme is stored at -20 ° C in glycerol.

Example 2. Determination of the specificity and activity of the enzyme.

The specificity of the enzyme is determined by the pattern of joint and parallel hydrolysis of substrate DNA (Fig. 1). The identity of the hydrolysis patterns on paths 1-3 indicates that these enzymes recognize and cleave the same nucleotide sequence 5-TGGCCA-3.

The yield of the Msp 20 I enzyme is determined by the electrophoretic picture of hydrolysis of phage lambda DNA. The unit of activity is taken to be the minimum amount of enzyme necessary for complete cleavage of 1 µg of phage l mbda DNA for 1 h at 37 ° C. The yield of the enzyme is 3000 u / g, the activity is 5000 u / ml.

The resulting strain has the following advantages compared with the known:

has a productivity of 3 times greater than the prototype strain,

does not require long-term cultivation,

Since restriction endonuclease

Msp 20 I has a recognition site identical

Msc I site, and the enzyme yield is higher,

then the Msp 20 t can replace the prototype in

all genetic engineering work.

The enzyme isoschizomers in the USSR are still

no pores found. Therefore, the production of restriction endonuclease Msp 20 I will allow

completely refuse to purchase the enzyme

abroad.

The introduction of the strain is planned in the NIKTI biologically active substances of the NGO Vector in 1991-1992.

Currently, the restriction enzyme Msp 20 I, obtained from Mlcrococcus species, is used to carry out various research topics at the Research Institute of Molecular Biology and NIKTI of biologically active substances Vector, at the Novosibirsk Institute of Bioorganic Chemistry, Siberian Branch of the USSR Academy of Sciences.

Formula A The strain of bacteria Mlcrococcus species 8KPM B-5463 is a producer of the restriction enzyme Msp 20 I.

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