SU1659480A1 - Strain of bacterium klebsiella pneumoniae - a producer of restrictase kpn 378 1 - Google Patents

Abstract

This invention relates to biotechnology and genetic engineering. The aim of the invention is the detection of the Klebslella pneumoniae strain VKPM B-4894 (378), which is undemanding to nutrient media, providing a higher yield of restrictase, capable of recognizing and cleaving the sequence of nucleotides CCGCGG. The strain obtained as a result of searching for producers of restriction enzymes, isolated from air, grows on a simple cheap nutrient medium of domestic production, provides an increased yield of restrictase with a simpler, more economical method for isolating the enzyme compared to the known one. From 1 g of wet biomass release 40000 units of the act. enzyme with an activity of 30,000 units of act / ml

Description

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This invention relates to biotechnology and genetic engineering, in particular to the production of a restriction enzyme capable of recognizing and cleaving the nucleotide sequence CCGCGG.

The aim of the invention is the detection of the Klebsiella pneumoniae VKPM B-4894 (378) strain, which is undemanding to nutrient media and provides a higher yield of restrictase capable of recognizing and cleaving the nucleotide sequence CCGCGG.

The strain Klebslella pneumoniae 378 isolated from the air as a result of the search for strains - producers of restriction enzymes.

The strain Klebsiella pneumoniae 378 is characterized by the following features.

Morphological features The cells in the form of shortened rods are often oval in shape, the fixed spores do not form gram-negative, as a rule they have a capsule, the length and thickness of the cells vary from 0.5 to 7 µm and from 0.3 to 1.5 µm, respectively, the cells are located single, in pairs, rarely in chains

Culture signs. On dense nutrient media it forms, as a rule, whitish, convex, luminous colonies, opaque, there are colonies different in morphology from the main type - small dryish, large transparent, differing from each other in texture of the colony.

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The optimal growth temperature of the strain is 30-37 ° C, the limit is from 12 to 43 ° C, the optimum pH is 7.2.

Physiological and biochemical signs. The strain Klebsiella pneumoniae 378 is distinguished by its high biochemical activity: it assimilates glucose, lactose, mannitol, sucrose, dulcite, rhamnose, xylose, maltose, sorbitol, arabinose, raffinose, glycerin, starch; restores nitrates, positive for catalase, lysine decarboxy-force, in the reaction of Voges-Proskauer; absorbs malonat, citrate; does not release indole, hydrogen sulfide; negative for urease, ornithine decarboxylase, arginine dehydrolase, in reaction with methyl red, for oxidase, does not liquefy gelatin. The ratio G + C 55.4 mol.%.

The strain Klebsiella pneumoniae 378 is resistant to penicillin, erythromycin, oleandomycin, oxacillin. ristomycin, sulfathiazole, mencomycin, is sensitive to tetracycline, streptomycin, monomitsin, neomycin, levomycetin, kanamycin, polymyxin, ampicillin, gentamicin, rifampicin, is slightly sensitive to carbenicillin.

The strain is stored in a liquid nutrient medium with the addition of glycerin up to 15% at minus 60 ° C.

Produced by the proposed restriction enzyme strain, CrP 378 1 is characterized by the following properties: it recognizes and specifically cleaves the sequence of nucleotides CCGCGG; the optimum pH for restriction enzyme action is 7.5-8.5; MD2 + ions are required for the activity of restriction enzymes, the optimal concentration is 10 mM, the optimal concentration of NaCl in the reaction mixture is 50-70 mM; the optimum temperature is 37 ° C.

To maintain the Klebsiella P378 strain, fish nutrient agar (RPA) is used.

For cultivation and fermentation of K. pneumoniae 378, nutrient medium of the following composition is used, g / l distilled water:

Peptone10

Yeast extract5

NaCI5

Glucose is added before seeding to 1%.

The cultivation is carried out at 30 ° C with good aeration (200 rpm) on a rocking chair for 15-16 hours.

Biomass yield: 8-12 g / l of culture fluid.

The output of restrictase Krp 3781: 40000 units of act / g of biomass.

Example. Cultivating the strain and obtaining biomass.

Klebsiella pneumoniae 378 cells are thawed at 37 ° C and seeded into liquid nutrient medium containing peptone (10 g), extract (5 g), NaCI (5 g) and distilled water to 1 l. Glucose is added before seeding to 1%. After 24 h incubation in a thermostat at 30 ° C

The 0 strain is plated on solid medium with RPA to obtain individual colonies. Grown under the same conditions. Grown cells are looped from agar to flasks with 50 ml of liquid nutrient medium to obtain

5 inokul ta. Incubate on a rocking chair (200- 220 rpm) for 24 hours at 30 ° C.

The resulting inoculum is seeded with a volume of medium (250 ml of medium flasks), intended for fermentation, from

0 calculate 1.0 ml of inoculum (stationary culture) per 100 ml of medium. Incubate under the same conditions for 20 hours. After cooling, cells are harvested by centrifugation at 5000 rpm. Biomass yield 9 g

5 wet cells per 1 liter of medium.

Isolation and purification of restrictase. 4 g of cells are suspended in 10 ml of buffer A containing 10 mM Tris-HCl, pH 7.6, 10 mM 2-mercaptoethanol, 0.1 mM EDTA, and

0 destroy the ultrasonic disintegrator. Cell fragments were removed by centrifugation (18,000 rpm, 40 minutes at 2 ° C). The supernatant is collected, 2 M KCl is added to it to a final concentration of 0.1 M and 10% polyethyleneimine to a final concentration of 0.1%. The mixture is thoroughly mixed and kept in ice for 30 minutes, then centrifuged (6000 rpm, 10 minutes at 2 ° C). The supernatant is removed and ammonium sulfate is added to 70% saturation (with stirring on a magnetic stirrer). The mixture was kept in ice for 30 minutes, then centrifuged.

5 (6000 rpm, 10 min at 2 ° C). The supernatant is discarded, the precipitate is dissolved in 10 ml of buffer A and dialyzed against 300 ml of buffer A for 2 hours. Next, the protein solution is applied to a column (2x20 cm) with DEAE-cellulose (DE-52) and washed with buffer A to disappearance of the UV absorbing material in the eluate. The adsorbed material is eluted with 400 ml of a linear gradient of CS (0-0.8 M) in buffer A. Aliquots from each

Five fractions (2 μl each) are analyzed for restriction content, incubated for 30 min at 37 ° С with 1 μg of phage A DNA, and analyzed by agarose gel electrophoresis. The fractions containing the enzyme are combined (total volume 30 ml) and applied to the column (1, cm)

with hydroxylapatite. The column is washed with buffer B containing 10 mM potassium phosphate, pH 7.6, 10 mM 2-mercaptoethanol and 0.1 mM EDTA until the UV absorbing material disappears from the eluate. The adsorbed material is eluted with 150 ml of a linear gradient of potassium phosphate, pH 7.6 (0.01-0.6 M) in buffer B. Aliquots from each fraction (2 μl) are analyzed for restriction enzyme content. The fractions containing CrP 378 1 restriction enzyme are combined (total volume 15 ml) and dialyzed for 16 hours against 150 ml of buffer A containing 50% glycerol and stored at -20 ° C.

The yield of 40000 units of act. per 1 g of biomass. The resulting preparation has a concentration of 30000 units of act. / Ml.

The enzyme activity is determined in 20 µl of the reaction mixture containing 10 mM Tris-HCl, pH 8.0,10 mM MgCl, 50 mM NaCl, 10 mM 2-mercaptoethanol and 1 μg phage A DNA at 37 ° C. Per unit of activity, the minimum amount of enzyme necessary for the complete specific cleavage of 1 µg of phage I DNA per 1 h at 37 ° C is taken.

The resulting enzyme preparation is free from significant impurities of nonspecific nucleases and phosphate. Incubation 120 units. Act. enzyme with 1 µg of phage I DNA for 2 hours at 37 ° C. A standard set of DNA fragments was observed. With

incubation 5 units of act. the enzyme with 1 µg of phage I DNA for 1 h at 37 ° C, the DNA fragments are stitched with DNA ligase and re-hydrolyzed with restriction enzyme Krp 378 1.

To determine the nucleotide sequence recognized by the restriction enzyme CRP 378 1, the DNA of the phage YR restriction enzyme CRP 378 1 and Sst II is hydrolyzed, as well as co-hydrolysis with these restrictases.

The observed pattern of complete coincidence of the fragments obtained during DNA hydrolysis with Krp 378 1 and Sst II restrictases separately and together indicates that the Krp 378 1 restriction enzyme is an Sst II restriction enzyme, it recognizes and splits the nucleotide sequence CCGCGG.

The resulting strain, in comparison with the known, provides an increased yield of the target enzyme, which is 40000 ac./g biomass.

To cultivate the proposed strain, a simple medium of the available components is used.

Since restriction enzyme CRP 378 1 has a recognition site CCGCGG. identical to the Sst II recognition site and known, it can replace these restrictases in all genetic engineering works.

Claims (1)

Claim Formula Bacteria Strain Klebsiella pneumonlae VKPM B-4894 - producer of restriction enzymes Krp 3781.