SU1652341A1 - Pseudomonas stutzeri strain producing restrictase psu i - Google Patents

Abstract

This invention relates to biotechnology and can be used in genetic engineering and molecular biology. The purpose of the invention is to obtain a strain of Pseudomonas stutzerl B-4727 (t31), which has a higher level of synthesis of a restriction enzyme that recognizes the palindromic S CGAfCGS1 sequence: 1 and is 3 GCTAGCS homogeneous homogeneous. The strain is cultivated on medium 3 with fish hydrolyzate. As a result of using the Pseudomonas stutzeri VKPM B-4727 strain, the biomass yield is 6.0-7.5 g / l of culture liquid, and the yield of restriction enzyme Psu is 16000-9000 u / g of biomass or 36000-67500 culture liquid / l. 1 tab. yo

Description

This invention relates to biotechnology and is a strain that can be used to produce restrictase Psu J,

The purpose of the invention is to obtain a strain of Pseudomonas stutzeri (131) VKPM B-4727, which has a higher level of restriction enzyme recognition, recognizes palindromic

b CCAT3 sequence -, g and ow 3 GqjAGC5

homosexually clean.

Strain Pseudomonas stutzeri (131) VKPM B-4727 isolated from the soil, followed by the selection of options that have high productivity restrictase Psu 3. The strain has the following characteristics.

Morphological signs.

The cells are single, rod-shaped, straight or curved, but not spiral, 0.5–1 x 1.5–4 µm in size, moving with the help of polar flagella. Gram-negative.

Physiological and chemical signs.

Respiratory metabolism (assimilate glucose on Hugh-Leifson medium by oxidative type), has urease and arginine dehydrolase activity.

Does not contain pigments, grows at 4-30 ° C, does not grow at 42 ° C (optimal growth temperature is 25 ° C).

Levan does not form from sucrose, does not thin the gelatin and does not hydrolyze the poly- / -oxybutyrate, hydrolyzes starch.

As the sole carbon source, it can use glucose, L-valine, acetate, citrate and does not use trehalose, 2-ketogluconate, meso-inositol, / 9

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-alanine, DL-arginine. Acid is formed upon glucose assimilation.

Hydrogen sulfide is not formed, the Voges-Proskauer reaction is negative, the methyl red test is negative.

Phenylpyruvate is not formed (the reaction to the presence of phenylalanine disaminase is negative).

Optional aerobes. Oxidazo and catalase-positive.

Cultural features.

Growth on braids is thick and solid. The stroke is wide, clearly visible. In contact with a solid medium, moisture grows as a continuous film over the entire surface. When cultures are grown in Petri dishes on solid medium, after 24 hours, semi-transparent dotted colonies measuring 0.1–0.2 mm are formed. After 38 hours, white, round, translucent, perfectly smooth, moist, uniform, convex, shiny colonies with a smooth edge are formed. The size of such colonies is 2-2.5 mm in diameter. Throughout the consistency, they are oily (like butter).

Colonies are not pigmented.

The strain Pseudomonas stutzeri 131 has a strong odor when grown on a solid or liquid medium.

With growth in the broth a copious precipitate is formed.

It grows well on a liquid nutrient medium containing 50 g / l of fish hydrolyzate and up to 1.0 l of water, pH 7.2-7.4. On such a nutrient medium, the strain grows at a temperature of 25-27 ° C, aeration 2 air volume per 1 volume of nutrient medium for 5-6 hours. When the strain is grown on a nutrient medium of this composition, the medium becomes cloudy, which indicates a strong growth of the culture. Growth is deep and film does not form on the surface. A pellet consisting of Pseudomonas stutzeri 131 cells, t gooey, when exposed to lysozyme, cell lysis quickly occurs. The amount of sediment is abundant, the color of the precipitate is light gray.

Strain phage-resistant. Not lysed in the presence of phages Ch 4A, A, MS 2, Q ft,

The strain is sensitive to ampicillin at a concentration of 25 μg / m. The strain gives a biomass yield of 6.0-7.5 g / l of culture fluid, has a restriction enzyme yield of Psu I 6000–9000 u / g of biomass or 36000–67500 u / l of culture fluid, biosynthesizes one site-specific endonuclease Psul Which is homogeneously clean.

Culture is grown on a nutrient medium of known composition at pH 7.2, temperature 25 ° C, aeration 2 volumes to 1 volume

nutrient medium to the late logarithmic growth phase. Biomass yield 6.0-7.5 l / g of culture fluid; output restrictase Psu I 3000-4500 u / r biomass

or 18000-33750 u / l of culture fluid.

Comparison of the levels of productivity of the strains that produce restriction enzymes - isoisomers of the restriction enzyme Psu 1 are presented in the table.

Example. The strain of bacteria Pseudomonas stutzeri 131 isolated from the soil. To obtain isolated colonies, take 10 g of soil (absolutely dry).

and shaken with 90 ml of water for 5 minutes. In the future, the method of limiting dilutions is used, consisting in the sequential transfer of 1 ml of suspension into a test tube with 10 ml of sterile water, well

mix and from this tube 1 ml of the suspension is transferred to the second tube, etc. A total of 8–9 dilutions are made; from each dilution, 0.1 ml of suspension is sown into Petri dishes with solid

nutrient medium containing 3.5% dry nutrient agar and water).

All colonies grown in Petri dishes are examined microscopically for the presence of biochemical traits, including the presence of Psu I restriction enzyme activity. The obtained colonies of Pseudomonas stutzeri in the activity of the enzyme Psu I are selected in order to obtain the most active variants.

The test of the productivity of the bacterial strain Pseudomonas stutzeri (131) VKPM B-4727 is carried out in comparison with the strain of Proteus vulgaris ATCC 133115 VKPM B-1804. which is synthesized similarly to restrictase and

experimentally tested in laboratory conditions at NPO Biolar.

A medium for growing a strain of the following composition is prepared: 50 g / l fish hydrolyzate and 1.0 l water.

The recovery of the Rseudomonas stutzert 131 strain was carried out on dry nutrient agar (35 g / l of dry nutrient agar and 1.0 l of water) in Petri dishes. Incubated in a heat chamber at 26 ° C for 18h.

From the surface of the agar in Petri dishes, the culture was transferred with a bacteriological loop to the shaker flasks with a nutrient medium of a given composition, pH 7.2.

Grow seed at 26 ° C for 18 h on a rocking chair.

Fermentation of the culture is carried out on a nutrient medium of this composition at pH 7.2, temperature 2b ° C, stirring 200

rpm, air supply - 2 volumes of air per 1 volume of nutrient medium.

Grown until late logarithmic growth phase. Biomass yield 6.0–7.5 g / l of culture liquid, yield of restriction enzyme Psu J 6000–9000 u / g biomass or 36000–67500 u / l culture liquid.

Thus, when using the strain Pseudomonas stutzerl (131) VKPM B-4727 compared to the known method, the yield of restriction enzyme Psul increases from 1000 to 6000-9000 units / g of biomass.

Claims (1)

Claims of the Invention Bacterial Strain Pseudomonas stutzeri VKPM B-4727 producing the restriction enzyme Psul,