SU1659479A1 - Strain of bacterium bacillus licheniformis - is a producer of restrictase bli 49 i - Google Patents

Abstract

The invention relates to biotechnology and molecular biology and can be used as a tool to obtain DNA fragments: the mapping of DNA molecules and the design of vector DNA molecules. The purpose of the invention is the detection of the strain Bacillus llchenlformls VKPM B-4268 (49), which produces the restriction enzyme HI 49 I, capable of recognizing the nucleotide sequence 5 ... GGTC TS ... 3. The strain was isolated from the soil. The output of the enzyme is 1000 units / r of biomass.

Description

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The invention relates to biotechnology and molecular biology and can be used as a tool for obtaining DNA fragments, mapping DNA molecules and constructing vector DNA molecules.

The purpose of the invention is the detection of the strain Bacillus llchenlformis VKPM B-4268 (49) producing restrictase VP 49 I, capable of recognizing the nucleotide sequence 5 ... GGTCTC ... 3 on double-stranded DNA ... 3.

The strain is isolated from the soil and is characterized by the following features.

. Morphological features - straight rods, cell size 18 hour culture 1.5x0.6 microns. The cells are arranged singly or in a chain. Ellipsoid spores are located in the cell subterminal.

The cells do not swell during sporulation.

 Cultural signs — on solid, mi-peptone agar, after 18 hours of growth at 37 ° C, colonies are formed with a matte, rough, grayish-white surface that grows into the agar. Spore formation begins after 48-72 h when grown on mi-peptone agar at 37 ° C.

After growth on glucose agar, no globules are found in the protoplasm. Growth in a liquid nutrient medium is characterized by a uniform turbidity.

Physiological and biological signs are gram-positive aerobic spore-forming rods. Catalase is produced. Culture ferments glucose, arabinose, mannitol to form acid, does not use xylose. Utilizes citrate and propionate, hydrolyzes starch, reduces nitr.

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In aerobic conditions, nitrate forms a gas that produces a positive Voges-Proskatser reaction. It grows under anaerobic conditions, does not produce arginine hydrolase, and does not hydrolyze urea.

Example, Strain B. lichenlformis 49 is stored in test tubes on agar medium under liquid paraffin. For liveliness, the culture is subcultured on beveled mac-peptone agar and incubated in a thermostat at 37 ° C for 30 hours. To obtain biomass, culture is seeded into a 3 L flask containing 700 ml of medium containing, g / l: yeast extract 5; trypton 5; sodium chloride 5, and incubated in rocking conditions to the average logarithmic phase, then the cells are separated by centrifugation at 10,000 d. The crude biomass yield is 5 g / l.

0.5 g of the cells are suspended in 0.5 ml of buffer A (0.2 M NaCI, 0.01 M K-phosphate, pH 7.0, 2 mM dithiothreitol, 0.1 mM EDTA) are sonicated upon cooling with ice, -cellular the walls are precipitated at 40,000 d. The supernatant is chromatographed on a 1.5 x 30 cm column with Toyopeari HW-SS,

equilibrated in the same buffer at a rate of 20 ml / h, fraction 0.8 ml. Restriction enzyme activity is determined by the hydrolysis of phage A DNA / the reaction products are analyzed

5 gel electrophoresis of 0.8% agarose. The fractions containing the restriction enzyme are pooled, dialyzed against buffer A containing 50% glycerol and stored at -20 ° C. The output of the enzyme is 1 thousand units / g of biomass.

0 The specificity of the restriction enzyme Bli 491 was determined by its hydrolysis of various DN K with a known primary structure (DNA of phages A, M13.0X174, plasmid p BR322). It has been established by physical mapping that restriction enzyme BH 49 recognizes and hydrolyzes B.R322 DNA at points 3435 bp at coordinates, phage A DNA at points 11424, 42715 bps, DNA X174 and M13 is not hydrolyzed. Analysis of these

0 data leads to the conclusion that the restriction enzyme VI 491 recognizes the GGTCTC nucleotide sequence.

Claims (1)

Bacillus lichenlformis bacteria strain 5 VKPM B-4268- producing restrictase BII49U