SU1686000A1 - Strain of bacteria bacillus stearothermophilus - a producer of restrictase bss ti - Google Patents

Description

The invention relates to biothography and genetic engineering, in particular to the detection of a microorganism capable of synthesizing a site-specific restriction enzyme that recognizes and cleaves the nucleotide sequence CC (A / T) (A / T) GG and called Bss Tl.

The aim of the invention is to identify the producer strain Bacillus stearothermophllus, providing Bss Tl restriction enzyme with a higher yield.

The strain of bacteria B. stearothermophllus obtained as a result of the search and selection at

extreme cultivation conditions (68-70 ° C) of restriction thai producers among soil microorganisms.

The strain was deposited in the VKPM All-Union Scientific Research Institute of Genetics under the editorial number B-4893 (collection number of depositor 360), and the restriction endonuclease produced by it is called Bss Tl.

Morphological and cultural signs. Cells grown in a liquid nutrient medium are thin, with rounded ends, gram-positive motile rods, single or in short chains. The cell size is 3-4-0.5-1.0 microns.

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After 18-24 hours on a dense starch-ammonia medium, the cells form terminally located oval spores, expanding its body. On fish nutrient agar, flat pigment-free colonies with a rough edge, 2.0-2.5 mm in diameter, are easily removed by a bacteriological loop. Water-soluble pigment in the environment is not detected.

Strict aerobic. Cells in a liquid medium grow diffusely, without film formation and sediment. Temperature limits of growth 40-70 ° С. It grows in the presence of 5% sodium chloride, with 10% it does not grow. Growth is also absent in media with 0.001% lysozyme and 0.02% sodium azide. Tests for catalase and oxidase positive, for indole, hydrogen sulfide and acetoin negative. Restores nitrates, synthesizes alkaline phosphatase. Absorbs glucose and galactose to form acid. Does not ferment arabinose, lactose, sucrose, xylose, mannitol and glycerin.

The strain is sensitive to penicillin, ampicillin, monomitsinin, tetracycline, streptomycin, chloramphenicol, oxacillin, carbenicillin, gentamicin, resistant to nalidixic acid. Strain is not pathogenic.

The cultivation is carried out in a nutrient medium containing 1% bactotrytok, 0.5% yeast extract, 1% sodium chloride, pH 7.0 in flasks on a shaker with a stirring speed of 200-250 rpm at 68-70 ° C. Biomass yield 3.0-3.5 g of wet cells per 1 liter of medium. The output of the Bss TI enzyme is not less than 10,000 units. act, / g wet biomass.

The resulting BssTI restrictase has the following properties: it recognizes and specifically cleaves the nucleotide sequence CC (A / TXA / T) GG, the optimum pH value for restrictase is 7.5-8.0, the optimal temperature is 50-55 ° C, the optimal concentration of NaCl is 50 mM .

The defining differences of the proposed strain B. stearothermophlliis (360) B-4893: a high content of Bss TI restriction enzyme of 10,000 units of act. / G of wet biomass (5 times higher), the absence of the pathogenicity of the strain, the use of simple microorganisms cheap environment.

PRI me R 1. Obtaining biomass.

B. stearotnermophllus cells (360), stored under a layer of vaseline oil at 4 ° C, are seeded on stubble nutrient agar and incubated at 68-70 ° C for 18 hours. The cells are then introduced into a flask with 50 ml.

liquid nutrient medium and inoculum is grown on a rocking chair with a rotation speed of 200-250 rpm at 68-70 ° C for 18-20 h. The resulting culture at the rate of 5-10% uses 250 ml of medium containing 1% bactotrypton for seeding , 0.5% yeast extract, 1% sodium chloride. Further cultivation was carried out for 16-18 hours on a rocking chair under the same conditions. Biomass collected

by centrifuging for 30 minutes at 5000 rpm in a Mistral centrifuge.

Crude biomass yield of 3.5 g / l culture liquid.

PRI mme R 2. Isolation and purification

restrictase Bss TI.

10 g of cells are suspended in 30 mg of 0.02 M Tris-HCl buffer, pH 7.5, containing 1x10 3M / -mercaptoethanol and 0.1% Triton X-100, destroyed in an ultrasonic disintegrator. Cell debris is removed by centrifugation and the supernatant is applied to a 500 ml A-0.5 M biogel column, equilibrated with 0.02 M potassium phosphate buffer containing 1 x 10 M

/ -mercaptoethanol, 5x10 M EDTA, 0.8 M NaCI, 0.1% Triton X-100 (buffer A). The enzyme is eluted with the same buffer at a rate of 20 ml / h. Fractions of 10 ml are collected and analyzed for the presence of

enzyme. The fractions containing the recryctase activity are combined and dialyzed against 2 liters of 0.02 M potassium phosphate buffer, pH 7.5, containing M / 3-mercapto ethanol and M EDTA (buffer B),

change buffer twice. Dialysate was applied to a 50 ml column with DEAE cellulose, equilibrated with buffer B. The column was washed with buffer B and the adsorbed enzyme was eluted with 500 ml of NaCI gradient

(0,1-OM) in buffer B. The column is washed with buffer B and the adsorbed enzyme is eluted with 500 ml of NaCI gradient (0.1-OM) in buffer B. Fractions eluted at 0.40-0.55 M NaCI containing restrictase ,

combine and dialyze against 2 L of buffer B, changing it twice. The enzyme after dialysis is applied to a 20 ml phosphocellulose P-11 column, equilibrated with buffer B. The enzyme is eluted with 300 ml

NaCl gradient (0-1, OM) in buffer B. Fractions containing the maximum activity of restrictase, eluted at 0.66-0.7 M NaCl, are combined and dialyzed against buffer B containing 0.1 M NaCl and 50% glycerol ,

Per unit of activity, the minimum amount of enzyme necessary for complete cleavage of 1 µg of phage A DNA for 1 hour at 50 ° C is taken. Enzyme

the preparation is stored at -20 ° C. The output of the enzyme is 10,000 units / r of biomass, the activity is 10,000 units / ml.

To determine the sequence of nucleotides recognized by the Bss TI restriction enzyme, the DNA of the phage is restricted by the Bss TI and Sty I restriction enzymes separately, as well as the joint hydrolysis with these restrictases. The observed picture of the complete coincidence of fragments obtained during DNA hydrolysis with Bss TI and Sty I restriction enzymes separately and together indicates that the Bss TI restriction enzyme is an isomer of Sty I, recognizes and cleaves the CC sequence (A / TXA / T) GG ,.

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The resulting strain has the following advantages compared with the known one: it grows on a more affordable, cheap medium without antibiotics, provides an increased yield of BssTI restrictase.

Since the Bss TI restriction enzyme has a CC (A / TXA / T) GG recognition site, identical to the Sty I recognition site, it can replace Sty I in DNA fragmentation experiments on these sites.

Claims (1)

BACKGROUND OF THE INVENTION Bacterial strain Bacillus stearo-15 thermophllus VKPM B-4893 - producing BSSTI restrictase.