RU2053298C1 - Strain of bacterium escherichia coli - a producer of restriction endonuclease eco 75 ki - Google Patents

Abstract

FIELD: biotechnology. SUBSTANCE: proposed strain Escherichia coli BKM B-2007D is isolated from clinical isolates (Kiev city) and provides the high level of enzyme content - 200000 U/g crude matter of cell biomass, not less. The yield of purified enzyme is 4000 U/g cell biomass. EFFECT: increased yield of enzyme.

Description

 The invention relates to biotechnology and relates to a new strain of bacteria Escherichia coli, which can be used to obtain restriction endonuclease (restrictase), which recognizes and cleaves the nucleotide sequence of 5'-GPuGCPy! C-3 'as part of double-stranded DNA. The restriction enzyme Eco75kI produced by the proposed strain is an isoshizomer of the well-known restriction enzyme Banll (1) and can be used in gene engineering studies in experiments on the construction of recombinant DNA in vitro, in the study of the primary structure of DNA, etc.

 An article by Sugisaki H. et al. [1] describes a Banll restriction enzyme derived from a strain of Bacillus anenrinolyticus that recognizes and cleaves a portion of 5'-GPuGCPy! C-3 'DNA. Banll restriction enzyme isoschizomers, for example, HgiJll [2] Eco261 [3] Bvul [4] EcoT381 [5] isolated from various strains, including Escherichia coli, are also known. Closest to the proposed strain is a strain of Escherichia coli RFL26 producing restriction enzyme Eco261. The yield of purified Eco261 enzyme is 200 u / g crude biomass.

 The problem to which the invention is directed, is to obtain a strain producing a restriction enzyme of a given specificity with a higher yield, rapidly growing on ordinary nutrient media.

 The proposed strain Escherichia coli 75k was obtained as a result of a targeted search for strains of restriction enzyme producers among clinical isolates (Kiev) using our developed method for testing activity in extracts obtained from individual colonies grown on an agar medium, followed by electrophoresis of DNA cleavage products in agarose gel. The content of restriction enzyme Eco75kI in the proposed strain of 200,000 units / g of crude cell biomass.

 The strain is deposited in the All-Union Collection of Microorganisms at the IBPM RAS and is stored in it under the number VKM V-2007D.

 The strain Escherichia coli VKM B-2007D has the following characteristics.

 Cultural and morphological characters.

 The cells are straight, rod-shaped, with a size of 0.7-0.8 microns across and 2-3 microns, sometimes 5 microns in length, motile, gram-negative, with lateral flagella. They are located singly, in pairs or chains, not spore-bearing.

 The strain grows well on ordinary nutrient media (MPA, MPB, LB broth and LB agar). When growing on meat peptone agar or LB agar, the colonies are smooth, round, pressed, shiny, cloudy. When growing in liquid media: in the BCH-broth or LB-broth form a uniform intense turbidity.

 Physiological and biochemical characteristics.

Cells growing in a range of from 4 ^to 50 ° C, optimum growth temperature 37 ^{° C,} the optimum pH 6,8-7,4.

 As a carbon source, many carbohydrates, alcohols, organic acids, in particular alpha glucose, alpha fructose, galactose, arabinose, lactose, trehalactose, are used.

 As a source of nitrogen, both mineral salts in ammonium and nitrate forms, and in organic form in the form of peptone, amino acids, are used.

 Nitrates are reduced to nitrites.

 Gelatin is not liquefied.

 Indole does not form.

 The storage conditions of the strain.

The bacterial strain Escherichia coli VKM B-2007D is stored on plates and jambs. Reseeding on fresh media once a month. It can be stored for at least one year in 15% glycerol at -20-70 ^{° C,} in the freeze-dried state with saharozhelatinovym agar or semisolid agar LB medium under mineral oil.

PRI me R 1. Testing restriction enzymes. To search for restriction enzymes, the express method was used, which is a modification of the method of Belavin et al. [6] for this, one or two large colonies (about 5x10 ⁶ -1x10 ⁷ cells) are suspended in 20 μl of buffer A (20 mm Tris-HCl, pH 7.6, 12.5 mm EDTA, 100 mm NaCl, 10 mm 2 mercaptoethanol) containing 10 mg / ml lysozyme, and incubated at room temperature (18 ° ^C). After 30 min, an equal volume of buffer B (20 mM Tris-HCl, pH 7,6, 2% Triton X-100, 10 mM 2-mercaptoethanol) and incubated another 60 minutes at 4 ^C. The cell suspension (1 and 4 ul ) mixed with 2 μg of phage DNA ⌀ 80 vir in 40 μl of buffer C (10 mM Tris-HCl, pH 7.6, 10 mM MgCl2, 10 mM 2-mercaptoethanol, 50 mM NaCl). Hydrolysis is carried out at 37 ^about C for 10 minutes In addition, 4 μl of the cell suspension is further incubated in 40 μl of buffer C without substrate in order to verify the possible presence of extrachromosomal DNA in the solution. The reaction is stopped by extraction with an equal volume of water-saturated phenol. For electrophoretic analysis using 20 μl of hydrolyzate.

 PRI me R 2. Obtaining and purification of restrictase.

Culture of E. coli 75k cells were grown in 1 liter LB-broth (5g yeast extract, 10 g NaCl, 10 g Bacto trypton 1 liter of water) at ³⁷ ° C to a titer of 4x10 ⁹ cells / ml. Cells are pelleted by centrifugation (6000 rpm for 15 minutes). 1 g of crude biomass is suspended in 5 ml of buffer containing 10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM EDTA, 10 mM 2-mercaptoethanol. Cells are destroyed by ultrasound, the extract is obtained by centrifugation at 2 ^about C (48000g).

To determine the activity of the Eco75kI restriction enzyme, serial dilutions of the supernatant are prepared: 1/2, 1/4, 1/8, 1/16, 1/32, 1/64, 1/128 in buffer (10 mM Tris-HCl, pH 7.5 , 50 mM NaCl, 1.0 mM EDTA, 7 mM 2-mercaptoethanol). In an incubation mixture (9 μl) containing 10 mM Tris-HCl, pH 8.0, 10 mM MgCl 2, 1 mM EDTA, 1.0 mM DTT, 1 μg lambda phage DNA, add 1 μl of the appropriate dilution of the extract and incubate at 37 ^about C for 1 h. The results of hydrolysis are checked by electrophoresis in 0.8% agarose gel.

 The amount of enzyme necessary for complete specific cleavage of 1 μg of lambda phage DNA for 1 h is taken as a unit of activity.

 The level of activity of the restriction enzyme Eco75kI, determined in the cell-free extract of the strain of E. coli 75 k, is about 200,000 units. in terms of 1 g of biomass.

 The restriction enzyme Eco75kI can be purified by fractionation of the cell extract on a column with DEAE cellulose (DE-52 Whatman) followed by chromatography on P-11 phosphocellulose (Whatman), DNA agarose [7] and hydroxylapatite. The enzyme preparation is finally concentrated during dialysis against befer 50 mM NaCl, 10 mM Tris-HCl, pH 7.5; 1 mM EDTA; 7 mM 2-mercaptoethanol; 50% glycerin. The yield of purified enzyme is 4000 u / g cell biomass.

The purified enzyme is stable at ^-20 ° C for six months. At 0 ^{° C} the enzyme retains 95% of its activity for 2 weeks at ²⁵ ° C retains 90% activity for 4 days and at 65 ° ^C loses 95% activity within 15 minutes. Restriction enzyme is suitable for DNA structure analysis and molecular cloning.

 PRI me R 3. Determination of the nucleotide sequence recognized by the restriction enzyme Eco75kI, and the site of DNA hydrolysis.

 To determine the recognition sequence for restriction enzymes, the lengths of the fragments obtained experimentally by hydrolysis of phage lambda restriction enzyme DNA are compared with the lengths of the fragments calculated for EME using appropriate programs for different nucleotide sequences. Only one sequence -5'-GPuGCPyC-3 'was found, for which the calculated fragment sizes coincided with the experimental ones and were localized in the following regions: 586, 10091, 19766, 21575, 24777, 25882, 39458.

 To confirm the obtained data, mapping of recognition sites of restriction enzyme Eco75kI onto pBR322 DNA is carried out using co-hydrolysis with restriction enzymes BamHl and SalI. The restriction enzyme Eco75kI was shown to hydrolyze this DNA at positions 476 and 490.

 M13tg 130 DNA was used to determine the site of DNA hydrolysis with Eco75kI restriction enzyme. After annealing with a universal synthetic primer and polymerization reaction, the matrix was treated with an Eco75kI restriction endonuclease. A part of the sample is polymerized a second time and both of the obtained preparations are examined under the conditions proposed by Sanger using an 8% denaturing polyacrylamide gel, which made it possible to determine the site of hydrolysis.

The obtained experiments allow us to make an unambiguous conclusion that the restriction enzyme Eco75kI is a true isochisomer of Banll restriction enzyme and recognizes and cleaves the DNA nucleotide sequence:
5'-GPuGCPy! C-3 '
In addition, the obtained restriction enzyme Eco75kI is characterized by the following optimal reaction conditions: pH for the action of restrictase 7.5; action temperature 37 ^about C.

 Thus, the obtained E. coli strain VKM B-2007D, as a producer of specific Eco75kI endonuclease, provides a high level of the enzyme of at least 200,000 units / g of crude biomass of cells, which is sufficient to obtain restriction enzymes in preparative quantities. The yield of purified enzyme is 4000 u / g of biomass of cells, which is 20 times higher than the yield of the known restriction enzyme Eco261 isolated from E. coli RFL26 strain, which is the prototype of the proposed strain.

Claims (1)

 The bacterial strain Escherichia coli VKM B-2007 D-producer of restriction endonuclease Eco 75 kI.