SU1678833A1 - Strain of bacteria bacillus alvei - a producer of restrictase bavi - Google Patents

Abstract

This invention relates to biotechnology and can be used in the practice of research work in molecular biology and genetic engineering. The aim of the invention is to detect a strain of Bacillus alvei, having a higher yield of Bav I restriction. The producer strain Bacillus alvei VKM B-675 is grown on a nutrient medium containing Hottinger broth, under aeration conditions, the target product is isolated from the resulting biomass using known methods . The biomass of the Bacillus alvei 675 strain contains the Bav I restriction enzyme, which recognizes and cleaves the nucleotide sequence of the CAG CTG DNA in quantities sufficient to produce a restriction enzyme on a preparative scale. For the Bav I isoschizomer, Pvu II restriction enzyme, the yield of the purified enzyme is 370 units per 1 g wet weight of biomass. The yield of the purified Bav I enzyme is 2.0-2.4x10 units. activity per 1 g of raw biomass, which is 5-6 times higher than the known Pvu II (370 units). SO with

Description

This invention relates to biotechnology and can be used in the practice of research, molecular biology and genetic engineering.

The purpose of the invention is the detection of a Bacillus alvei strain having a higher restriction enzyme yield.

The strain producing Bacillus alvei 675 is grown on a nutrient medium containing Hottinger broth, under aeration conditions, the target product is isolated from the obtained biomass by known methods.

The Bav i restriction enzyme, Pvu II restrictase isoshizomer, recognizes a portion of the nucleotide sequence of CAG 4-CTG DNA and splits bacteriophage A DNA in 15 sites, adenovirus 2 DNA in 24, SV 40 DNA in three, plasmid pBR322 in one and does not hydrolyze DNA0X 74.

The search for the producer strain was carried out using the developed express method for testing the activity of restriction enzymes in toluene extracts from microbial cells followed by electrophoresis of agarose gel products of DNA cleavage.

The strain Bacillus alvei 675 is obtained from the All-Union Collection of Microorganisms of the Academy of Sciences of the USSR where it is stored under number VKM-675 - NCIB 8212 and is not identical to the strain Bacillus alvet ATCC 6348, the producer of the known restrictase Bav I.

Characteristics of the strain.

Morphological properties. Rods 0.5-0.8 x 2-3 microns in size are located

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singly and in pairs, spores, Gram-positive (the sign is non-permanent, varies), Cells are mobile (the sign varies).

Cultural and biochemical properties. On mowed agar - a weak gentle raid. The colonies on the MPA are very small, shiny, convex, with smooth edges. On the mot-peptone broth and Hottinger broth - uniform turbidity, greyish tender film. Attitude to oxygen - aerobic. Utilization of carbohydrates - on glucose, lactose, sucrose forms an acid. Litmus milk coagulation, peptonization. Indole - does not form. Gelatin injection - slow, crater-like liquefaction. Sensitivity to antibiotics. Culture has multiple resistance to antibiotics, resistant to penicillin, streptomycin, kanamycin, polymyxin, neomycin, methicycline, nystatin, ampicillin. It is sensitive to tetracycline, chloramphenicol, erythromycin, oleandomycin, rbenicillin, lincomycin, gentamicin, oxacillin.

Optimum growing and storage conditions (medium, pH, temperature, time of reseeding). The culture grows well on solid and liquid nutrient high-grade media: mono-peptone agar, Hottinger broth, LB medium.

On Hottinger broth for 4-5 hours, the growth reaches Aeso - 1.2-1.4. Biomass yield 6-8 g / l. When grown on LB medium, biomass accumulation is lower than 4-5 g / l.

Optimum conditions for the formation of the Bav 675I restriction enzyme are cultivation on Hottinger's medium for 4-5 hours before reaching the end of the logarithmic growth phase.

On the medium, the fish extract culture grows slowly, and the activity of restrictase in biomass is much lower than during cultivation in Hottinger and LB.

The optimal growth temperature is 37 ° С, but it also grows at 40-45 ° С,

Working cultures are subcultured on Hottinger broth once every 7-10 days. The culture is stored in semi-liquid agar under liquid paraffin or in a lyophilized state in ampoules.

The culture is non-pathogenic for mice. A significant cultivation factor is aeration. Under anaerobic conditions, the yield of Bav I restriction enzyme is reduced. The incubation mixture for determining the activity of restriction enzyme Bav 675I with a volume of 25-50 microns contains 0.01 M Tris-HCI, pH 7.5, 0.01 MgCIa, 0.001 M dithiothreitol, 0.05 M NaCi, 1 μg of phage A DNA and 1- 20 μl of enzyme. Incubate at 37 ° C for 60 minutes. The products of restriction DNA digestion are determined by electrophoresis of the incubation mixture in an agarose gel with

subsequent staining of the gel with ethidium bromide. Per unit of enzyme activity, they take the minimum amount (in μl) that is necessary for complete cleavage of 1 μg of phage A DNA at

0 37 ° C for 1 h of incubation under standard incubation conditions.

The restriction enzyme isolation was carried out by fractionation and column chromatography. The resulting enzyme develops

5 activity in the presence at a concentration of 5-410 mM at a pH of 7.5-8.3 and a temperature of 30 ° C. Addition of KCI (20–30 mM) to the incubation medium stimulates the activity of the enzyme; in a higher concentration (100 mM), KCI inhibits restriction activity.

Example 1. The culture of Bacillus alvel BKM B - 675 is preliminarily adapted on the main nutrient medium (Hottin5 Hera broth) containing 250 ml of Hottinger's basic food, 10 g of NaCI, 1 g of K2HP04 and up to 1 l of water in 1 liter. An inoculum for the fermenter is prepared by transferring the adapted culture to 1 liter of fresh medium at a rate of 1:10 and

0 propagated under aeration conditions.

Cultivation of the culture is carried out after adding the inoculum to the fermenter in a ratio of 1:10 to the volume of the medium at 37 ° C under aeration conditions (1 liter of air per 1 l of nutrient medium), pH of the medium 7.6 maintained in the range of 7.4-7.6. Growing is continued until the end of the logarithmic growth phase is reached. Raw yield

0 biomass is 8 g / l of culture fluid. The content of restrictase Bav I 6 x 103 units / g biomass.

Determination of enzyme activity is carried out in gradient fractions after chromatography on DEAE-cellulose (DE-52, Whatman) extracts from cells treated with ultrasound, followed by agarose gel electrophoresis of DNA cleavage products.

0 The selection of the restriction enzyme Bav 675 I.

10 g of Bacillus alvel 675 biomass is suspended in a buffer solution and the cells are sonicated at 2-4 ° C. After

5 removal of residues of cell walls and intact, slightly, the enzyme is fractionated in a two-phase polyethylene glycol / dextran system. The restriction-rich upper phase is chromatographed in potassium phosphate buffer in a KCI gradient.

a column with DEAE-cellulose (DE-52, Whatman), then on a column with blue sepharose.

Get 2,4x104 units. restriction activity in 4.0 ml of glycerol buffer. The output of the purified preparation of the enzyme 2,4x10 units activity per 1 g wet weight of biomass. Restriction enzyme Bav 675 I stably retains activity for at least 6–9 months. The enzyme is suitable for DNA mapping and genetic engineering.

Example 2. The cultivation of the Bacillus alvel 675 strain is carried out as described in Example 1, except that LB medium containing 10 g tryptone, 5 g yeast extract, 5 g NaCI and up to 1 l is used as a nutrient medium. water.

Biomass yield 5 g / l of culture fluid. The content of restrictase in biomass 5x10 units / g of biomass. After purification of the enzyme, the yield of restriction enzyme Bav 675 I was 2.0x103 units / r of biomass. Thus, the biomass of the strain Bacillus alvel 675 contains restrictase Bav

I, recognizing and cleaving the nucleotide sequence of the DNA of CAG and CTG, in quantities sufficient to produce a restriction enzyme on a preparative scale. The content of restrictase Bav I in biomass 5 - 6x103 units. per 1 g of raw biomass. For isoschizomer Bav I - Pvu II restrictase enzyme content is lower. The yield of the purified Bav I enzyme is 2.0-2.4x103 units. activity per 1 g raw

biomass, the yield of purified restrictase Pvu II - 370 units. per 1 g of biomass. Bav I restriction enzyme does not contain impurities of nonspecific endonucleases and exonucleases.

Claims (1)

Invention Formula The bacterial strain Bacillus alvei VKM B-675 - producing restrictase Bav I,