SU1631080A1 - Strain of bacteria bacilius megaterium - producer of restrictase bme 361 i - Google Patents

Abstract

This invention relates to microbiology and genetic engineering. The aim of the invention is to obtain a new strain of Bacillus megaterium VKPM B-4723 (361), which synthesizes restrictase Bte 361 1c with an increased yield. The strain was isolated from soil as a result of searching for restriction enzyme producers among soil bacteria, undemanding to nutrient media, not human pathogenic, allows increasing the yield of Bte 361 I restriction, which recognizes and cleaves nucleotidone GGCC sequence, 10 times per 1 liter of culture. Ytamm provides an increased restriction enzyme yield of 100,000 units / g of biomass. For cultivation of B. megaterium 361, a nutrient medium of the following composition is used, g / l of distilled water: peptone 10; yeast extract 5, NaCl 10. Cultivation is carried out at a temperature of 30 ° C with good aeration for 24 hours. Table 1. a 8

Description

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

The purpose of the invention is to obtain a new strain of Bacillus megaterium VKPM B-4723 (361) that is nutrient poor and synthesizes a restriction enzyme capable of recognizing and cleaving the GGCC nucleotide sequence in high yield.

The strain Bacillus megaterium 361, isolated from the soil, as a result of a search for the restriction enzyme-producing strains among soil bacteria.

The strain B. megaterium 361 is characterized by the following features.

Morphological features: the cells are immobile, gram-positive, rod-shaped, connected in long strands, in the old culture, bloated irregular shapes, rod-shaped cells with spindle-shaped ends are possible. The cell size is 1.2-1.4x2.8-10.0 microns. The spores do not exceed the diameter of the cell, elliptical, located non-phase-like (central, extracentral, terminal).

Cultural and physiological signs.

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Respiratory metabolism. Reactions to catapase, amylase, gelatinase, phenylalanine dehydrolase and Voges-Proscaper are positive. The strain reduces nitrates, does not precipitate indole, grows on medium with the addition of 7% NaCl, grows acid and gas on glucose, grows at pH 5.7, parasporal bodies are not detected, uncolored globules are observed. The maximum growth temperature of the strain is 35-40 С, the minimum is 3-20 ° С, the growth at 50 С is absent. The test for lecithinase, tyrazinase is negative, it does not grow at 0.001% lysozyme.

The strain is stored in 15% glycerol in L-broth at minus 60 ° C and in a lyophilized state.

On the RPA (fish agar) forms opaque, whitish colonies, shining, rough over time, | smooth edge. At the RPB fish food (broth forms a film, turbidity, sediment.

Produced by the proposed restriction strain is characterized by the following properties.

Recognizes and specifically cleaves the GGCC nucleotide sequence; the optimal pH value for the action of restriction enzyme 7., 5-9.0; optimal operating temperature of 37 ° C; for restriction enzyme activity, Mg ions with an optimal concentration of 5-10 mM are required; The optimal concentration of NaCl is 0-50 mM.

For the cultivation of B.megaterium 361, a nutrient medium of the following composition is used, g / l of distilled water:

ten

5 10

Cultivation was carried out at 30 ° C with good aeration for 24 hours.

The biomass yield is 10.0 g per liter of medium.

The yield of restrictase is 1 ppm act. per 1 g of biomass.

The advantages of the proposed strain of Bacillus mega te-rium 361 compared with the known are shown in the table. ,

As can be seen from the table B.megaterium. Less-demanding to the composition of the feeder-. medium yield, the biomass yield of this strain from 1 liter of the medium is at least 5 times greater than the biomass yield of B. sphaericus. The output of restrictase with 1 l cult 0

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0

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0

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0

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d

B.megaterium fish are 10 times higher than from 1 liter of B. sphaericus culture.,

Example. B.megaterium 361 cells, stored in 15% glycerol in L-broth at minus, are thawed and sown for recovery in L-broth, incubated for 24 hours at 30 ° C. The obtained liquid culture was sown on RPA plates to obtain individual colonies, and incubated for 24 hours at 30 ° C. The culture grown on solid medium is checked for purity and inoculated with 50 ml of L-broth to obtain inoculum, grown for 18 hours at 30 ° C. The resulting inoculum (OD 3.6) is inoculated with 250 ml of LB with 1% glucose, grown 24 hours (OP 3.8). After cooling, the cells are harvested by centrifugation. Biomass yield 10 g wet weight with 1 liter of medium.

0.5 g of cells are suspended in 10 ml of 0.1 M Tris-HCl buffer, pH 8.0, containing 10 mM 2-mercaptoethanol, 0.1% Triton X-100, 1 mg / ml lysozyme, for 20 minutes at 0 ° C. Then the suspension is voiced on an ultrasonic disintegrator. Cell debris is removed by centrifugation at 18,000 rpm for 40 minutes at Ј ° C and the supernatant containing 500,000 units. Act. restrictase was applied to the column (1x5 cm) with phosphocellulose P11 equilibrated with buffer A (10 mM potassium phosphate, pH 7.4, 10 mM 2-mercaptoethanol). Then the column is washed with 5 ml of buffer A and the sorbed proteins are eluted with a NaCl concentration gradient from 0 to 1 M in buffer A, the total gradient volume is 20 ml. Collect fractions of 1 ml. The fractions containing the restriction enzyme activity (usually eluted at 0.5-0.75 M NaCl) are combined, dialyzed for 3 hours against 200 ml of buffer A. The dialyzed fraction is applied to the column (1 ml) with hydroxylapatite, equilibrated buffer A, washed with 2 ml of buffer A. Sorbed proteins were eluted with a potassium phosphate concentration gradient from 0.01 to 0.8 M. The total volume of the gradient is 10 ml, fractions of 0.5 ml are collected, each fraction is analyzed for restriction enzyme content. The fractions containing the enzyme, usually eluted at a salt concentration of 0.4-0.78 M, are collected and dialyzed against buffer A containing 50% glycerol. The resulting enzyme preparation

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(1 ml) has a concentration of 500,000 units. act./ml. Per unit of activity, take the minimum amount of enzyme necessary for complete growth over a period of

Chipping 1 μg of phage DNA for 1 hour at 37 ° C. The enzyme preparation is stored at minus 20 ° C. From 0.5 g, 500,000 units of act. restrictase Wte 361 I.

To determine the sequence of nucleotides recognized by the restriction enzyme Bte 361 I, the DNA of phage ft are digested with Bte 361.1 BspR, Hae III separately, and also co-hydrolyzed with these restrictases. The complete coincidence of the fragments obtained by hydrolysis with restrictase Bte 361 I and BspR I, Hae III separately and together indicates that the restriction enzyme Bte 361 I is an inhibitor of the restriction enzyme BspR I and Nahe III, recognizes and splits the nucleotide sequence GGCC.

The resulting strain also has the following advantages as compared to the known one: the strain is not pathogenic for humans; not demanding on nutrient media, grows well, providing a high culture yield from 1 liter of medium, 5 times larger than the known strain; provides an increased yield of the target product, amounting to 1,000,000 units of act. / ml of biomass. (2 times more than the known). I

Since restriction enzyme Bte 361 I has a GGCC site that is identical to the recognition site BspR I and Nae III, it can also replace the enzyme in all genetic engineering works.

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Claims (1)

2 Formula of invention The bacterial strain Bacillus megate- rium VKPM B-4723 - producing restrictase Bte 361 I. Dependence of biomass yield (g / l) and restriction enzymes (unit of act / l) of B.megaterium and B.sphaericus on the composition of the nutrient medium