SU1645293A1 - Strain bacteria streptococcus faecalis - is a producer of restrictase sfa n - Google Patents

Description

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The invention relates to biotechnology and genetic engineering, in particular, to the detection of a microorganism capable of synthesizing site-specific restriction enzyme and cleaving the sequence of nucleotides GCATC

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The aim of the invention is the detection of a strain of Streptococcus faecal is VCLO1 B-4426 (555), which produces an restriction enzyme Sfa N 1 with an increased yield

The strain Streptococcus faecal is 555 B-4426 - producing SfaN 1 restrtase, which recognizes and uncouples the GCATC nucleotide sequence, was isolated as a result of selection work with museum cultures.

The strain is characterized by the following properties.

Morphological. The cells are in the form of slightly oval small gram-positive fixed cocci located singly, in pairs or in. form of short chains Small, up to 1 mm in diameter, slightly convex, round with a smooth edge, shiny, semi-transparent pasty consistency of colonies are formed on a dense nutrient medium. Growth in nutrient broth leads to a uniform turbidity.

Physiological and biochemical. On the first day it grows at a temperature of 45 ° C, on the second at 10 ° C, It grows at pH 9.6 and in milk in the presence of 0.1% methylene blue, causing its reduction. It grows on nutrient agar in the presence of potassium tellurite, which at the same time is regenerated.

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fermentation of gasification of melibiose, mannose, galactose, glucose, lactose, sucrose, maltose, arabinose, mannitol. Does not ferment ramnoe, raffinose, trehalose, starch, inositol, dulcito Ferments in aerobic and does not ferment in anaerobic conditions glycerin. The test with malonate is negative. Indole, hydrogen sulfide does not form negative reactions with methyl red and to acetone, oxidase, catalase and urease “Nitrates do not reduce. Gelatin does not hydrolyze, does not possess lysine and ornithine decarboxylase, the test for arginine dihydrolase is positive „

Attitudes towards antibiotics, cells are resistant to penicillin, ampicillin monomitsin, antsha, aksy, and oxycylline, ristomitschgu, sulphathiazole, antifc, and methicillin are sensitive to streptomycin, neomycin, oleandromycin, erythromycin, and erythromycin, and necrosis.

Cytamma is stored in a freeze-dried state and on a semi-liquid nutrient medium under a layer of vaseline oil.

For cell culture, a nutrient medium of the following composition is used, g / l: casein hydrolyzate 20, glucose 10, yeast extract 5, sodium chloride 5, pH 7.2.

The cultivation is carried out at a temperature of 30 ° C with good aeration until the late logarithmic growth phase. The yield of wet cells is 2.4-3.0 g / l nutrient medium

The enzyme yield is 2000 units of act. / G of raw biomass, which is more than 3 times higher than the known

The resulting restriction enzyme Sfa N 1 is characterized by the following properties.

recognizes and specifically cleaves the nucleotide sequence GCATC

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optimal value. pH for action of restriction enzyme 7.2;

optimal operating temperature 37 ° Cj

optimal concentration of ions Nad - 150 иН, MgCl - 5-15 shM „

The enzyme is stable for at least 6 months. at a temperature of -20 ° C, it is free from impurities of non-specific ecoeos and endonucleases, phosphatases.

The invention is illustrated by the following examples.

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Example 1 "Cells are grown in a tube on beveled nutrient agar with the addition of 5 g / l yeast extract at 30 ° C for 16-18 h. This volume of inoculum is evenly distributed in 4 flasks with a volume of 200 ml of the same nutrient). environment. Cultivation in a liquid nutrient medium was carried out on a thermostatted rocking chair. The resulting culture liquid was centrifuged at 5 thousand rpm for 30 min. 0 The yield of wet cells was 2.4-3.0 g / l of nutrient medium.

Example 2. TO g of Streptococcus faecalis 555 biomass is suspended in 30 ml of 0.02 P Tris-HCl buffer, pH 7.5, containing M / 3-mercaptoethanol and 0.1% Triton X-100, and destroyed by ultrasonic disintegrator . Cell debris is removed by centrifugation. A supernatant is applied to a 500-ml Biogel A 0.5t column (Bio-Rad) with a volume of 500 ml, equilibrated with 0.02 M potassium phosphate buffer containing AND ft-mercaptoethanol, 5-10-4 M EDTA , 0.8 And NaCl and 0.1% Triton X-SO (buffer A). The enzyme is eluted with the same buffer at a rate of 20 ml / h0. Fractions of 10 ml are collected and analyzed for enzyme activity. The enzyme is dialyzed against 2 liters. , 02 M potassium phosphate buffer, pH 7.5 containing M-mercaptoethanol and 5 (buffer B), twice the dialysate buffer is applied to a 30 ml column with ben zil-DEAE cellulose (Sigma, CL1A), equilibrated with buffer B0 The column is washed with buffer B, the adsorbed enzyme is eluted with 300 ml of gradient Nad (0.0-1.0 M) in buffer B. Fractions eluted at 0.45-0.55 And Nad containing restriction enzyme is pooled and dialyzed against 2 liters of buffer B, changed twice, after the dialysis enzyme is applied to a 10 ml omanpocellulose column (equilibrated with buffer B) and washed with a column The same buffer elution of the enzyme was carried out with 200 ml of Nad gradient (0.0–1.0 M) in buffer В “The fractions containing retrictase eluted at 0.43–0.55 M iiaCl, dynes dissolved and dialyzed against 2 liters of buffer B0 then carried rehro- matografiyu enzyme per se fosfotsellyulo- P-11 2 ml volume to concentrate the enzyme ,, Elution was carried out with 40 ml

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NaCl gradient (0.0-1.0 M) “Fractions The obtained strain has the following eluted at 0.5–0.55 M NaCl, the volume advantages compared with

Dinut and dialyze against buffer B, known:

containing 0.1 M NaCl and 50% -5 - provides povypenky yield

glycerol restrictase Sfa N 1;

The enzyme preparation is stored when it grows on more affordable 20VS. Enzyme yield 2000 units „act” / nutrient media of domestic

biomass, activity of 20,000 units „/ ml0production.

Per unit of activity take 10

Claims (1)

minimum amount of enzyme non-formula inventions required for complete cleavage Utahim bacteria Streptococcus 1 µg JF phage fl C1 857 during faecalis VKPM D-4426 - producer hour at a temperature of 37 ° C „restrictase Sfa N 1.