RU2040540C1 - Strain of bacteria bacillus coagulants being producer of side specific endonuclease which is enable to identify and to cleave series of 5′-ct(a/g)(t/c)ag-3′ nucleotides - Google Patents

Abstract

FIELD: microbiology, biotechnology. SUBSTANCE: proposed strain is isolated of thermal sources. Yield of ferment is 1000 units per 1 g of raw biomass, specific activity of desired ferment is 3000 units per 1 ml. EFFECT: recognizes strain producer.

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 nucleotide sequence 5'-CT (A / G) (T / C) AG-3 '. A restriction enzyme of this specificity can be used to study the primary structure of DNA and 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 in one biomass there are 2 restriction enzymes. This complicates the purification procedure, and not one of the restriction enzymes is able to recognize and cleave the nucleotide sequence 5'-CT (A / G) (T / C) AG-3 '. The cultural and biotechnological properties of the strain have not been published.

A known strain of Streptococcus faecalis producing the restriction enzyme Sfe I (recognition site 5'-CT (A / G) (T / C) AG-3 ') [2]
The disadvantage of this strain is the thermal lability of restriction endonuclease. Data on biotechnological indicators of the strain are not published.

Closest to the proposed is a strain of Bacteroides distasonis, producing the restriction enzyme Bdi I (recognition site 5'-CT (A / G) (T / C) AG-3 ') [3]
However, the restriction enzyme produced by this strain is thermolabile. For the cultivation of strains of this species, media of the following composition are required, g / l: casamic acids 4, yeast extract 3, polypeptone (Difco, USA) 3, glucose 10, NaCl 1,5, Tween-80 1, L-cysteine 0.4, water the rest. Cultivation lasts 48 hours and requires special equipment, since the strain is an anaerobic.

 The purpose of the invention is the obtaining of a strain that produces thermostable restriction enzyme that recognizes and cleaves the nucleotide sequence of 5'-CT (A / G) (T / C) AG-3 ', which is not fastidious to nutrient media and does not require special equipment for growing.

 This goal is achieved by the identification and use of a strain of Bacillus coagulansi 63 producer site-specific restriction enzyme Bco163 I.

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

 The resulting strain of Bacillus coagulans163 was deposited at VKPM Research Institute of Genetics under registration number B-6311, and the restriction enzyme produced by it was named Bso163 I according to the generally accepted nomenclature.

The strain of Bacillus coagulans163 is characterized by the following features:
Morphological signs.

 The cells are rod-shaped, straight, 0.5-0.8x2-12 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 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; does not grow at a NaCl concentration above 5%; weak growth at 0.002% sodium azide and on Saburo medium; weak growth was observed in anaerobic agar only with the addition of tryptose extract; indolene negative.

For the cultivation of Bacillus coagulans B-6311, a medium of the following composition is used, g / l: peptone 10, yeast extract 5, glucose 5, the rest is distilled water. Cultivation was carried out on incubator shaker or fermenter at 60 ° ^C and intensive aeration to achieve a growth phase retardation.

 The yield of the target enzyme is 1000 u / g crude biomass with a specific activity of 3000 u / ml.

The defining differences of the proposed strain from the strain of the prototype are:
the presence of thermostable restriction enzyme;
simple nutrient medium;
conventional equipment used to cultivate aerobic bacteria.

 Since the proposed strain was obtained for the first time and was never used to isolate a restriction enzyme having the recognition site 5'-CT (A / G) (T / C) AG-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 lambda phage DNA with the enzyme Boc163 I, Sfe I (5'-CT (A / G) (T / C) AG-3 ', Pst I, lambda phage DNA represents the identity of cleavage bands during parallel hydrolysis and 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. The petri dishes were placed in an incubator at 60 ^o C. After 10-12 hours of incubation the grown cells passaged in flasks of fresh growth medium consisting, g / l: Peptone 10, yeast extract (Difco) 5, 5 glucose, distilled water rest. Cultivation was carried out in a fermenter at 60 ° ^C with aeration of 1.5 volume / min, agitation 150 rev / min, until the deceleration phase growth. 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 hydrolysis of substrate DNA using fragments obtained after hydrolysis of phage lambda restriction enzyme DNA of known specificity as markers. The identity of the hydrolysis patterns suggests that these enzymes recognize and cleave the same nucleotide sequence: 5'-CT (A / G) (T / C) AG-3 '.

The yield of the Bso163 I enzyme 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 1000 U / g wet biomass, specific activity 3000 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;
simple nutrient medium;
conventional equipment used to cultivate aerobic bacteria.

 Since the Bst163 I restriction enzyme has a 5'-CT (A / G) (T / C) AG-3 'recognition site identical to the Bdi I restriction enzyme recognition site, it can replace the prototype in all genetic engineering activities.

Claims (1)

 The bacterial strain Bacillus coagulans VKPM B-6311 produces a site-specific endonuclease capable of recognizing and cleaving the 5′-CT (A / G) (T / C) AG-3 ′ nucleotide sequence.