The strain of the bacterium bacillus stearothermophilus 22-producer of the restriction enzyme that recognizes and cleave the nucleotide sequence 5`-ccnnnnn^nngg-3', and possessing growth promoting activity in relation to seeds of cultivated plants

 

The invention relates to biotechnology. The strain of the bacterium Bacillus stearothermophilus 22 - producer of the restriction enzyme that recognizes and cleave the nucleotide sequence 5'-CCNNNNN^NNGG-3' and with the property that the growth-promoting activity. Bacillus stearothermophilus 22 - producer of restrictase Bst22I extracted from soil hot springs Peninsula Kamchatka in search of producers of restricts. Provides getting restrictase, recognize and cleave the nucleotide sequence 5'-CGNNNNN^NNGG-3'. The output of the enzyme is 50000 u/g raw biomass. Restriction enzyme Bst22I is soshitara restrictase BsiYI and can replace the latter in all genetic engineering works. Biomass cells of the bacterial strain has a growth promoting activity in respect of seeds of various cultivated plants. 4 Il., 3 table.

The invention relates to the search for new strains - producers of biologically active substances, which are promising for microbiological industry and genetic engineering and the receipt of a new strain, producing site-specific endonuclease, are able to recognize and cleave the nucleotide sequence 5’-CCNNNNN^NNGG-3’ and having the property to influence ProRAE the primary structure of DNA, physical mapping of various genomes and is rarely found in natural strains.

There are several producers of isolitaire this restrictase, however, in the literature there is no detailed description of these strains and their enzymes.

There are several strains [1, 2], capable of producing a restriction enzyme (site recognition of 5’-CCNNNNN^NNGG-3’).

There is no evidence of stimulating plant growth properties of the cells.

Known nutrient composition for promoting plant growth [3, 4]. They contain strains of Bacillus and their culture liquid, and micronutrients, salts, amino acids, fillers. The compositions are presented in dry or liquid form.

The disadvantages of these compositions is the complexity of manufacturing, the presence of urea, which serves as an additional source of nitrate, which is harmful to health, and the lack of used strains ability to produce restrictase.

Known strain of Bacillus polymyxa used for the production of the growth of sugar beet [5]. The drug is derived from this strain is used in conjunction with insecticidal and fungicidal preparations for seed plants.

Fungicides and insecticides are risk factors for agricultural workers, Neizvestnaya based on ribonuclease, obtained by synthesis Century iniermedius 7p [6].

The strain does not have activity for the synthesis of the enzyme restriction enzyme.

Closest to the claimed strain is a strain Century stearothermophilus, producing a restriction enzyme BsiYI [7], a prototype.

The enzyme retains activity from two to eight months of storage at -20With depending on the selection method. The output of the enzyme is 1200 IU/g of the drug.

Culture and biotechnological properties of the strain are not published.

The disadvantage of this strain is a low output restrictase and the absence of growth-promoting activity.

An object of the invention is to obtain a strain producing a restriction enzyme that recognizes and cleave the nucleotide sequence 5’-CCNNNNN^NNGG-3’, with a high yield of enzyme stability during storage and possessing growth promoting activity in relation to seeds of cultivated plants.

The technical problem is solved by identifying and exploiting producer strain site-specific restrictase Bst22 I and possessing growth promoting activity.

The proposed strain isolated from soil samples thermal Top source of Paratunka Peninsula of Kamchatka in search of new mi signs.

Morphology: cells of strain - motile Bacillus size (0.6 to 0.8)(3,0-6,0) µm, grammarically. Form ellipsoid spores (1.5 to 2.5 microns). The surface of spores smooth, spore-direct.

Physiological and biochemical properties: aerobe, temperature optimum 55-65C, growth at 42C. pH environment of 7.0 to 7.2. The intense strain ferments glucose, maltose, lactose, sucrose, xylose, arabinose, rhamnose, galactose, mannitol, poorly - Inositol, not fermented sorbitol; restores nitrates, does not emit indole, amylase-catalase-positive.

View identified by the identifier of [8] as a strain of the bacterium Bacillus stearothermophilus 22.

The resulting strain of Bacillus stearothermophilus 22 deposited in the Institute of KCM State Research Center of Virology and biotechnology “Vector” under the registration number In-894, and produced them with restriction enzyme named Bst22 I according to the conventional nomenclature.

The strain is stored in a freeze state or in a nutrient medium under a layer of paraffin oil.

To obtain a preparation of the enzyme strain grown in half-liter flasks on a temperature-controlled shaker at 250 rpm and 55C. For culturing Bacillus stearothermophilus 22 PR is about the allocation of Bst221 carried out at a temperature of 4-5C. the Cells are suspended in buffer A (10 mm potassium phosphate buffer pH 7.5, 7 mm-mercaptoethanol, 0.1 mm EDTA) at 4 ml per 1 g of biomass and destroy Paladino ultrasound on the cage (“MSE”, England), then clarify by centrifugation in a centrifuge J-21B (“Bekman, USA) with a speed of 18000 rpm for 30 minutes, the Extract was applied to a column (1.614 cm) phosphocellulose R-11 (“Whatman, England) and washed with buffer a and wash the enzyme concentration gradient of NaCl (0.0-0.8 M) with a volume of 200 ml. Fractions containing the restriction enzyme, dialist against buffer B (10 mm Tris-HCl pH 7.5, 1 mm- mercaptoethanol, 0.1 mm EDTA). Then the enzyme is applied on a column (0.89cm) DEAE - cellulose DE - 52 (“Whatman, England), washed with buffer and wash the enzyme in the gradient of NaCl from 0 to 0.8 M with a volume of 40 ml. Fractions with the enzyme cialiswhat against 50% solution of glycerol in buffer containing 0.2 M NaCl and stored at -20C.

The release of the enzyme from 10 g of cells is 50,000 units of the act. Per unit of activity (E) take the minimum amount of enzyme that within 1 h under optimal conditions completely hydrolyzes 1 µg DNA phage20 meat and proteases. For this purpose, test 18-hour incubation of the enzyme withDNA [9], test “restriction - stitching - re restriction” [10] and the test oligonucleotide substrates.

This strain is unique not only by the presence of the restriction enzyme, but also has additional properties. Cells of strain synthesize substances that can stimulate the growth of plants, in particular the germination of seeds of various cultivated plants, such as radishes, carrots, cucumbers, radish, and other Data presented in tables 1-3 and Fig.2-4.

The results presented in the tables show that after treatment of seeds of radish, cucumber and radish cell extracts of this strain germination of these plants is enhanced compared to the control at 10 and 100-fold dilution. In the example with the seeds of radish varieties “may” conduct measurements of aboveground and underground parts of the plants (table.3)

The defining difference of the strain from the strain of the prototype is:

- the high productivity of the strain in excess of this figure, the strain-prototype

- high stability of the enzyme (conservation activity within two years of storage at -20C)

- wide temperature opelousa activity against seed of cultivated plants.

Because the proposed strain obtained for the first time and has never been used, it is possible to conclude that the proposed strain criteria invention of “novelty” and “inventive step”.

The figure 1 presents electrophoregram products of hydrolysis of DNA of phage lambda enzyme Bst22I (Dor.1), Bst22I and Bsc4I (Dor.2) Bsc4I (Dor.3), (website recognition of 5’-CCNNNNN^NNGG-3’).

As seen from the figures, the identity of the bands splitting in parallel and joint hydrolysis confirms their solitario.

The figure 2 presents the seeds of radish treated cell extracts of strain Century stearothermophilus 22 (1, 3, 4, 5, 6) and without treatment (2). As can be seen from the figure, and root and leafy part of the plant seeds after treatment, cell extracts differ from control seeds treated only with distilled water. Similar results were obtained for seedlings of cucumbers and radishes, shown in Fig.3 and 4, respectively.The figure 3 presents the seeds of cucumbers treated cell extracts of strain Century stearothermophilus 22 (1, 2, 5, 6) and without treatment (3, 4).

The figure 4 presents radish seeds treated cell extracts of strain Century stearothermophilus 22 (top row) and without treatment (bottom row).

The invention illyustriruetsya work cell producer strain subcultured bacteriological loop on agar medium. Schools are placed in a thermostat at 55C. After 10 h of incubation grown cells subcultured in flasks with fresh nutrient medium containing the following components, g/l:

Peptone - 10 g/l, yeast extract 5 g/l, sodium chloride 5 g/l, distilled water to 1 l, pH 7.2.

The cultivation is carried out at 55With aeration, under stirring of 250 rpm, the Cells are harvested by centrifugation in a centrifuge J-21 at 5000 rpm and a temperature of 4-5C. Disintegration, separation and cleaning is performed by the method described above.

Example 2. Determination of the specificity and activity of the enzyme. The specificity of the enzyme is determined by the film parallel and joint hydrolysis of substrate DNA (Fig.1). The identity of paintings hydrolysis on tracks 1-3 suggests that these enzymes recognize and break down the same nucleotide sequence 5’-CCNNNNN^NNGG-3’.

The yield of the enzyme was determined by electrophoretic painting hydrolysis of DNA of phage lambda. Per unit of activity take the minimum amount of enzyme required to complete fission of 1 µg DNA of phage lambda for 1 h at 65°C. the Yield of the enzyme is 50 000 IU/g raw biomass. The enzyme is stored n the ha lambda carried out under standard conditions, but at different temperatures from 42 to 70C. the Enzyme retains its activity under these conditions, which confirms its wide temperature range.

Example 4. Determination of enzyme activity depending on time storage at -20C. determination of the activity carried out under standard conditions periodically during two years of storage.

Because restriction enzyme has a website recognition of 5’-CCNNNNN^NNGG-3’ is identical to the saiga recognition of restrictase Bsc4 I, which is also soshitara BsiY I, it can replace the prototype of all genetically engineered works.

Example 5. The biomass of cells of a strain of Bacillus stearothermophilus 22 received in L-broth, washed with saline, destroy with the help of ultrasound in standard buffer and receive a 10-year and 100-fold dilution of cell extract. For cultivation take sterile distilled water. The extract obtained is used for soaking seeds of radish for 60 and 90 min In the result set that the growth of the root system of these seeds has increased ~ 3-fold compared with control at the same time.

Example 6. The example is similar to example 5. Except that they use carrot seed. The seeds of the>iterator

1. Repin C. E., Serov, D., Puchkova L. I., D. T. A., Lebedev, L. P., Chizhikov C. E., (1993) Bioorgan. chemistry, I. 19, No. 5, S. 583-584.

2. Repin, V. E., Lebedev, L. R., Puchkova, L., Serov, G. D., Tereschenko So, Chizikov, V. E., Andreeva, I., (1995) Gene, vol. 157, pp. 321-322.

3. International application No. 0036085, With 12 N 1/12 published IMS 46, 6/01.

4. Japan's bid No. 1172310, A 01 N 63/02, published RJ IMS 5-20-90.

5. RF patent №2035507, 05 F 11/08, published BI 14, 1995.

6. USSR author's certificate No. 1642973, a 01 N 63/00, published BI 15, 1991.

7. Mok, Y. K., Clark, D. R., Kam, K. M., Shaw, P. C., (1991) Nucleic Acids Res., vol. 19, pp.2321-2323.

8. Bergey''s Manual of Systematic Bacteriology. Baltimore: Williams & Wilkins, 1986, V. 2, P. 1105-1139.

9. Maniatis, R. A., Fritsch E., Sambrook D. Methods of genetic engineering. Molecular cloning. M.: Mir, 1984. S. 479.

10. Puchkova L. I., Ushakov, I. A., Mikhailova C. K., Serov, D., Krivobokova, N., Repin C. E., (2002) Go Active. biochem. and microbiol., so 38, No. 1, S. 20-24.

Claims

The strain of the bacterium Bacillus stearothermophilus 22 (NII ECR SRC VB “VECTOR” IN-894) - producer of the restriction enzyme that recognizes and cleave the nucleotide sequence 5'-CCNNNNN^NNGG-3', and possessing growth promoting activity in relation to seeds of cultivated plants.

 

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