RU2007453C1 - Strain of bacterium bacillus sphaericus - a producer of endonuclease restriction bsh 451 - Google Patents

Abstract

FIELD: biotechnology, genetic engineering. SUBSTANCE: strain of bacterium Bacillus sphaericus No. 209 is isolated from the soil by screening in the search of strains - producers of site-specific endonucleases among Bacillus species. Enzyme Bsh 451 recognizes and splits nucleotide sequence 5′-G(A/T)GC-(A/T)-C-3′. Strain grows on the simple low-cost monocomponent medium, which is prepared on the basis of fish-hydrolyzate at 30 C under effective aeration up to achievement of stationary growth phase. The yield of the end enzyme is 30000 U/g wet biomass. Specific activity of enzyme Bsh 451 is 10000 U/ml. EFFECT: enhanced yield of enzyme activity.

Description

The invention relates to biotechnology and genetic engineering, in particular to the production of a strain used to isolate a restriction endonuclease (restrictase) that recognizes and cleaves the nucleotide sequence 5 ^' -G (A / T) GC (A / T) C-3'.

 A restriction enzyme of this specificity can be used for cloning and physical mapping of substrate DNA. This restriction enzyme is a convenient model for studying the specificity of protein-nucleic acid interactions.

Closest to the claimed strain of the prototype is a strain of Bacillus brevis (VKPM B-4906) - the producer of site-specific restriction endonuclease Bbv 12 I, recognizing and cleaving the sequence 5 ^' -G (A / T) GC (A / T) C-3 ^' . The strain grows well on a domestic agar medium prepared on the basis of a 3.7% sprat hydrolyzate. Biomass is lysed by lysozyme. The main disadvantage of this strain is the insufficiently high yield of the enzyme - 6000 u / g of raw biomass.

The aim of the invention is to identify the producer strain synthesizing restriction enzyme capable of recognizing and cleaving a sequence nekleotidov 5 ^{'-G (A / T) GC} (A / T) C- ^3', with an increased yield of the desired enzyme.

 The goal is achieved by obtaining a strain of Bacillus sphaericus 45 - producer of site-specific restriction enzyme Bsh 45 I.

 The new strain was isolated from natural material (soil) as a result of a systematic search for strains producing restriction endonucleases in the genus Bacillus.

 The restriction enzyme from Bacillus sphaericus 45 is named Bsh 45 I according to generally accepted nomenclature.

The strain of Bacillus sphaericus 45 is characterized by the following features:
Cultural and morphological characteristics: on the medium of MPA and RA forms slightly convex colonies with smooth edges. The cells are rod-shaped, sizes 1.6-2.5 microns in length and 0.6-0.8 microns in width. They form endospores of a spherical shape, in a bloated sporangium of a mainly terminal or subterminal location. Aerobes.

Physiological and biochemical properties: Catalase-positive. Tyrosine not hydrolyze starch and do not form indole and H ₂ S, Voges-Proskauer negative reaction, gives growth in anaerobic conditions at 50 ^° C and alcohols Carbohydrates (glucose, arabinose, xylose, mannitol, etc.) Are not fermented.

Strain deposited under the mineral oil is carried out in philous dried state or in 30% glycerol at -70 ° ^C.

 For the deep cultivation of biomass, an environment of the following composition is used: (g / l) Fish hydrolyzate 37 Water Else.

Cultivation was conducted at 30 ^{° C} with good aeration until the stationary growth phase. The restriction enzyme yield of Bsh 45 I is 30,000 units / g of crude biomass.

The obtained restriction enzyme Bsh 45 I is characterized by the following properties:
- recognizes and cleaves the sequence of nucleotides 5 ^' -G (A / T) GC (A / T) C-3 ^' ;
- the optimal pH for the action of restriction enzyme 7.9-9.0;
- optimum temperature action of 37 ^{° C;}
- to activate restriction enzyme requires Mg ^{2+ ions} , the optimal concentration of 5-15 mm.

The defining differences of the proposed strain of Bacillus sphaericus 45 are:
- the strain contains one site-specific restriction enzyme Bsh 45 I, capable of recognizing and cleaving the nucleotide sequence of 5'-G (A / T) GC (A / T) C-3 ';
- the strain productivity is 5 times higher than the prototype strain in the target enzyme.

PRI me R. The culture is stored in jambs under liquid paraffin. Before starting work, the cells are subcultured with a bacteriological loop on a solid agar medium (3.7% SPA). Petri dishes are placed in a thermostat at 30 ^° C. After a 24-hour incubation, the grown cells are transplanted into flasks with a fresh nutrient medium consisting of 3.7% sprat hydrolyzate and placed in the thermostat overnight. The overnight culture of the strain of Bacillus sphaericus 45 is seeded in a liquid nutrient medium consisting of 3.7% sprat hydrolyzate, the rest is water. Cultured in incubator shaker at ³⁰ ° C at 200 rev / min until the stationary growth phase. Cells were pelleted by centrifugation at 4 ^C. The yield of biomass was 6 g / l culture medium. Isolation is carried out according to the method of Bickle, Pirota, Imber. The enzyme yield is 30,000 units / g of raw biomass. The specific activity of the restriction enzyme Bsh 45 I is 10,000 units / ml. The enzyme was stored at ^-20 ° C in glycerol.

 The obtained strain in comparison with the prototype is characterized by a significantly higher enzyme yield (30,000 units / g instead of 6,000 units / g), i.e. 5 times and produces only one restrictase.

 Since the restriction enzyme Bsh 45 I has a recognition site that is identical to the recognition site for the restriction enzyme Bbv 12 I (prototype), and the enzyme yield upon cultivation on identical media is 5 times higher, Bsh 45 I can replace the prototype in all genetic engineering studies. (56) Brown N. L. Mc. Clealland M., Whitehead P. R. Hgi AI: a restriction endonuclease from Herpetosiphon giganteus HP 1023. Gene. 1980, v. 9, p. 49-68.

 USSR author's certificate N 1694647, cl. C 12 N 9/14, 1990.

Claims (1)

 The bacterial strain Bacillus sphaericus GISK N 209 - producer of restriction enzyme bsh 45 I.