RU2037522C1 - Strain of bacterium bacillus species - a producer of restriction endonuclease recognizing and cleaving nucleotide sequence 5'-(a/g)-ccgg-(t/c)-3' - Google Patents

Abstract

FIELD: biotechnology. SUBSTANCE: detection of strain-producer Bacillus species providing preparing thermostable restriction endonuclease which can recognize and cleave nucleotide sequence 5′-(A/G)-CCGG-(T/C)-3′ with high yield and activity of enzyme. Strain Bacillus species ВКПМ-B5941 is isolated from thermal sources after screening of site-specific endonuclease restriction (restrictases). The yield of enzyme is 4000 U/g wet biomass, specific activity of the end enzyme is 10000 U/ml. Restrictase Bse 118I is an isoschizomer of restrictase Cfr 10I and can substitute the latter in all genetic-engineering studies. EFFECT: increased yield of enzyme.

Description

 The invention relates to the microbiological industry and genetic engineering and relates to the production of a new strain producing a site-specific endonuclease capable of recognizing and cleaving the 5 '- (A / G) CCGG (T / C) 3' nucleotide sequence.

 A restriction enzyme of this specificity can be used to study the primary structure of DNA, the physical mapping of various genomes. This endonuclease is a convenient model for studying the specificity of protein-nuclein interactions.

A known strain of Bacillus species M. producing restriction enzymes BspM I and BspM II (recognition sites 5 'ACCTGC 3' and 5 'TCCGGA 3', respectively) [1]
The disadvantage of this strain is that there are two restriction enzymes in one biomass. This complicates the purification procedure, and not one of the restriction enzymes is able to recognize and cleave the nucleotide sequence 5 '(A / G) CCGG (T / C) -3'. The cultural and biotechnological properties of the strain have not been published.

A known strain of Bacillus stearothermophilus producing the restriction enzyme BsrF I (recognition site 5 '(A / G) CCGG (T / C) 3') [2]
The disadvantage of this strain is the low yield of restriction endonuclease. Data on biotechnological indicators of the strain are not published.

The closest to the claimed strain of the prototype is a strain of Citrobacter freundii, producing the restriction enzyme Cfr10 I [3]
The disadvantage of this strain is that the restriction enzyme produced by it is thermolabile. The yield of restrictase does not exceed 300 u / g of raw biomass. Strains of this species belong to enterobacteria, some serotypes are found in cases of massive food poisoning, infections of the urinary tract, gall bladder, middle ear and meninges.

 An object of the invention is to obtain a strain that produces thermostable restriction enzyme that recognizes and cleaves the nucleotide sequence of 5 '- (A / G) CCGG (T / C) 3', with a high yield and activity of the enzyme.

 The goal is achieved by identifying and using a strain of Bacillus specis118 producer site-specific restriction enzyme Bse118 l.

 The proposed strain isolated from thermal sources as a result of a search for restriction enzyme producers.

 The obtained strain of Bacillus species118 was deposited in VKPM All-Russian Research Institute of Genetics under registration number B-5941, and the restriction enzyme produced by it was named Bse118 l according to the generally accepted nomenclature.

 The strain of Bacillus species118 is characterized by the following features.

 Morphological signs.

 The cells are rod-shaped, straight, 0.5-0.8x2-12 microns, motile. They form oval endospores located centrally and terminally, swollen relative to the size of the vegetative cell. Gram stain is gram-positive.

 Cultural signs.

 The strain is undemanding to growth factors, grows well on ordinary nutrient media (SPA, MPA, MPB). On agarized media forms small, round, unpigmented colonies, shiny, with a smooth edge.

Optimum growth temperature 60 ° ^C, pH 7.0-7.2.

Reseeding culture was maintained on solid media, is stored in a glycerol solution at ^-70 ° C or freeze-dried state.

 Physiological signs.

 Does not reduce nitrates, negative in reaction with methyl red and Voges-Proskauer; does not hydrolyze starch, casein, gelatin; catalase-positive, grows at a NaCl concentration of up to 5% 0.002% sodium azide, on Saburo's medium; no growth was observed in anaerobic agar; resistant to streptomycin.

For the cultivation of Bacillus species B-5941, a medium of the following composition is used, g / l: peptone 10, yeast extract 5; glucose 5; distilled water the rest. Cultivation was carried out at 60 ° ^C and intensive aeration to achieve a growth phase retardation.

 The yield of the target enzyme is 4000 u / g crude biomass with a specific activity of 10,000 u / ml.

The defining differences of the proposed strain from the strain of the prototype are the following:
the presence of thermostable restriction enzyme;
high strain productivity;
high enzyme activity.

 Since the proposed strain was obtained for the first time and was never used to isolate a restriction enzyme having a recognition site 5 '- (A / G) CCGG (T / C) -3', we can conclude that the proposed strain meets the criteria of the invention of "Novelty" and "Inventive level".

 The electrophoregram of the products of hydrolysis of lambda phage DNA with the enzyme Bst118 I, Cfr10 I and Bse118 I, Cfr10 I represents the identity of the cleavage bands during parallel and joint hydrolysis, confirms their isoschisomerism.

 PRI me R 1. Obtaining biomass and isolation 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 60 ^° C. After a 10-12-hour incubation, the grown cells are transplanted into flasks with a fresh nutrient medium consisting of, g / l: peptone 10; yeast extract (Difco) 5, glucose 5; distilled water the rest. The cultivation is carried out at 60 ^about With aeration of 1.5 volume / min, with stirring 150 rpm, until the phase of growth retardation. Cells were collected by centrifugation at 1500 g and ⁴ ° C. disintegration, separation and purification were performed according to the procedure of Bickle, Pirotta, Imber.

 PRI me R 2. Determination of the specificity and activity of the enzyme.

 The specificity of the enzyme is determined by the pattern of parallel and joint hydrolysis of substrate DNA. The identity of the hydrolysis patterns in lanes 1-3 indicates that these enzymes recognize and cleave the same nucleotide sequence 5 '- (A / G) CCGG (T / C) -3'.

The yield of Bse118 I is determined by the electrophoretic picture of hydrolysis of lambda phage DNA. One unit of activity is defined minimum quantity of enzyme required for complete digestion 1 ug Lambda phage DNA for 1 h at 60 ^C. The yield of the enzyme is 4000 U / g wet biomass, specific activity 10,000 U / ml.

The enzyme was stored at ^-20 ° C in glycerol.

Thus, the obtained strain having the following advantages compared with the known:
the presence of a single thermostable restriction enzyme;
high strain productivity;
high enzyme activity.

 Since the restriction enzyme Bse118 I has a recognition site 5 '- (A / G) CCGG (T / C) -3' identical to the recognition site for the restriction enzyme Cfr10 I, it can replace the prototype in all genetic engineering activities.

Claims (1)

 The bacterial strain Bacillus species VKPM B-5941 is a producer of a restriction endonuclease capable of recognizing and cleaving the 5 ′ (A / G) CC GG (T / C) 3 ′ nucleotide sequence.