RU2054040C1 - Strain of bacterium bacillus coagulans - a producer of site-specific endonuclease which recognizes and splits the nucleotide sequence 5′-ctcttc-3′ - Google Patents

Abstract

FIELD: microbiology, biotechnology. SUBSTANCE: method involves the detection of strain-producer providing the preparing thermostable site-specific endonuclease which recognizes and splits the nucleotide sequence 5′-CTCTTC-3′. The new strain Bacillus coagulans ВКПМ B-6309 (116) is proposed - a producer of restriction endonuclease Bco116 I isolated from thermal sources as a result of restriction endonuclease search. The yield of enzyme is 8000 U/g wet biomass, specific activity of the end enzyme is 10000 U/ml. Restrictase Bco116 I is an isoschizomer of restrictase Bco632 I and can replace the latter in all genetic engineering studies. EFFECT: detection of new strain-producer of enzyme, high 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'-STSTTS-3 'nucleotide sequence.

 A restriction enzyme of this specificity can be used to study the primary structure of DNA, the physical carotting 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 and 5'TCCGGA-3 ', respectively) [1]
The disadvantage of this strain is that in one biomass there are 2 restriction enzymes. This complicates the purification procedure, and not one of the reductases is able to recognize and cleave the 5'STSTTS-3 'nucleotide sequence. The cultural and biotechnological properties of the strain have not been published.

Known strain Enterobacter aerogenes producing restriction enzyme Ear I (recognition site 5'STSTTS-3 ') [2]
The disadvantage of this strain is the stability of the restriction endonuclease. Data on biotechnological indicators of the strain are not published.

The closest to the claimed strain of the prototype is a strain Kl uyvera species 632, producing the restriction enzyme Ksp 632I [3]
The disadvantages of this strain is that the restriction enzyme produced by it is thermostable. The yield of restriction enzyme does not exceed 500 u / g of crude biomass. For the destruction of biomass requires special equipment. The strains of this species belong to enterobacteria, some serotypes of which are found in cases of massive foodborne infections, infections of the urinary tract, gall bladder, middle ear and meninges.

 An object of the invention is to obtain a strain that produces thermostable restrictase, recognizing and cleaving the nucleotide sequence of 5'STSTTS-3 ', with a high yield and activity of the enzyme.

 The goal is achieved by the identification and use of a strain of Bacillus coagulans 116 producer site-specific restriction enzyme Bco 116 I.

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

 The obtained strain of Bacillus coagulans 116 was deposited in VKPM All-Russian Research Institute of Genetics under registration number B-6309, and the restriction enzyme produced by it was named Bco 116 I according to the generally accepted nomenclature.

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

 Morphological signs.

 The cells are rod-shaped, straight 0.5-0.8 x x 2-12 μl, 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 round, non-pigmented columns, 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%; grows slightly on 0.002% sodium azide and on the Saburo medium; no growth was observed in anaerobic agar if tryptone was not added to the medium; indolene negative.

For the cultivation of Bacillus coagulans B-6309, a medium of the following composition (g / l) is used: peptone 10, yeast extract 5, glucose 5, and the rest distilled water. Cultivation was carried out at 60 ° ^C and intensive aeration to achieve a growth phase retardation.

 The yield of the target enzyme is 8000 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 presence of a single thermostable restriction enzyme;
high strain productivity;
high enzyme activity;
the ability to destroy biomass by ultrasonic disintegration.

 Since the proposed strain was obtained for the first time and has never been used to isolate a restriction enzyme having a recognition site 5'-CTSTTS-3 ', we can conclude that the proposed strain meets the criteria of the invention of "novelty" and "inventive step".

 The electrophoregram of the products of hydrolysis of DNA of phage lambda by the enzyme Bco 116 I, Bco 116 I and Ksp 632 I (Boehringer Mannheim) Ksp 632 I phage lambda represents the identity of the splitting bands in parallel and joint hydrolysis confirms their isoshisomerism.

 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-hour incubation, the grown cells are subcultured with a fresh nutrient medium consisting of (g / l): peptone 10, yeast extract (Difco) 5, glucose 5, and the rest is distilled water. 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 on paths 1–3 suggests that these enzymes recognize and cleave the same nucleotide sequence 5′-STSTTS-3 ′.

The yield of Bco 116 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 Nam 1 ug Lambda phage for 1 hour at ⁶⁰ ° C. Yield is 8.000 enzyme units / 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;
the ability to destroy biomass by ultrasonic disintegration.

 Since the restriction enzyme Bco 116 I has a recognition site of 5'-CTSTTS-3 'identical to the recognition site of the restriction enzyme Ksp 632 I, it can replace the prototype in all genetic engineering activities.

Claims (1)

 The bacterial strain Bacillus coagulans VKPM-B-6309 is a producer of a site-specific endonuclease capable of recognizing and cleaving the 5′-STSTTS-3 ′ nucleotide sequence.