SU1761803A1 - Strain of bacteria bacillus alvei - producer of restriction endonuclease bav bii - Google Patents

Abstract

Usage: biotechnologists and in molecular genetic studies and genetically engineered studies. SUMMARY OF THE INVENTION: Biomass B alvei BKM B-674 is grown under aerobic conditions in a nutrient medium containing sources of carbon, nitrogen, and mineral salts until the late logarithmic growth phase, the target product is isolated from the obtained biomass by known methods. The biomass of the bacterial strain B. alvei BKM B-674 contains the new Bav B II restriction endonuclease that recognizes and cleaves the nucleotide DNA sequence 5 ... G CNCC ... 3, in quantities sufficient to produce restrictase on the preparative scale: the content of Bav restriction enzyme BII in biomass 3-4 x 10 u / g wet microbial mass. Asu I restriction enzyme, Bav BII restriction endonuclease isoshizomer, the content of the enzyme in the biomass and the yield of the purified preparation of the enzyme are not indicated. The yield of the purified Bav BII preparation is 8-12 x 103 units per 1 g of raw biomass. Bav BII restriction enzymes do not contain impurities of phosphatases, nonspecific endonucleases and exonucleases. V fe

Description

The invention relates to the field of biotechnology and can be used in molecular genetic work and genetic engineering research.

Class II restrictases (synonyms: restriction endonucleases, site-specific endonucleases), which break down phosphodiester DNA bonds by specific nucleotide sequences (sites), are an indispensable research tool in molecular genetic and molecular practice. genetic engineering works are a unique object for studying the nature and mechanisms of protein – nucleus interactions. These circumstances explain the great interest

researchers in the field of biochemistry, molecular biology, microbiology, virology to identify producers of new restricts, to develop methods for their purification, to study the properties used in molecular genetic experiments.

Restriction enzymes were isolated and partially or fully characterized from more than 1,100 microorganisms of various taxa / 1 /, One of the restriction enzymes Asul was isolated from the strain Anabaena subcylindrica CCAP 1403 / 4B 111.

The restriction endonuclease Asul cleaves the DNA of phage I in 74 sites, the DNA of adenovirus Ad 2 in 164, the DNA of monkey virus SV 40 in II, the DNA of phage $ X174 in 2 and the plasmid

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pBR322 in 15, the enzyme hydrates the region of the nucleotide DNA sequence 5 ... GAGNCC ... 3

The content of the Asul restriction enzyme in the biomass of Anabaena subcyllndrica CCAP 1403 / 4B and the yield of the enzyme purified from impurities of non-specific nucleases are not given.

The aim of the invention is to obtain a strain of bacteria producing with high yield a new restriction enzyme Bav BII, recognizing and cleaving the nucleotide sequence 5 ... G CNCC ... 3.

The task is achieved by the fact that the biomass of B.alvei BKM B-674 is grown under aerobic conditions in a nutrient medium containing sources of carbon, nitrogen and mineral salts until the late logarithmic growth phase, the target product is isolated from the resulting biomass by known methods.

The Bav BII restriction enzyme, the isulisomer Asul, recognizes and hydrolyzes a region of the nucleotide sequence of DNA 5 ... G GNCC ... 3. The restriction enzyme Bav BII splits the DNA of phage I into 74 sections, the DNA of adenovirus Ad2 164, the DNA of monkey SV40 virus 11, the DNA of phage0X174 through 2 and the plasmid pBR322 through 15.

The search for the producer strain was carried out using a rapid method for determining the site-specific enedonuclease activity in toluene lysates of microbial cells, followed by electrophoretic separation of the infectious gel of the phage I DNA digestion products. A total of 18 strains of Bacillus alvei were examined.

The B.alvei BKM B-674 strain was obtained from the All-Union Collection of Microorganisms of the Academy of Sciences of the USSR, where it is stored under the number VKM B-674 NCI B8199 NRRLB-384

Characteristics of the strain

Morphological features. Rod-shaped cells, straight or nearly straight, 0.5-0.8 x 2-5 microns in size, motile (the sign varies). Arranged singly and in pairs, form spores. Gram-positive (non-permanent symptom, varies Gr - Gr).

Cult of rado-biochemical properties

On sloped agar - a weak, gentle coating. The colonies on the MPA are very small, shiny, convex, with even edges.

On mot-peptone broth and Hottinger broth - a noticeable uniform turbidity, greyish tender film.

Attitude to oxygen - aerobic.

Catalase - forms.

Utilization of carbohydrates - on glucose, lactose, sucrose form an acid; on arabinose, xylose and mannitol do not form acid. Starch - hydrolyzed.

Litmus milk - coagulation, peptonization.

Indole and dioxyacetone - form.

Casein is cleaved. 0 Tyrosine - do not split,

Phenylalanine - do not deaminate.

Optimum growing and storage conditions.

The B.alvei BKM B-674 culture grows well on liquid and solid nutrient media — Hottinger broth, LB medium, and s-septone broth.

The maximum and minimum growth temperatures of the culture are, respectively, 35-45 ° C and 15-20 ° C, and the optimum temperature is 37 ° C. The growth optimum is pH 7.0-7-2. At pH 10 culture does not grow.

The highest yield of biomass containing restrictase Bav BII is obtained in case 5 when the producer strain is grown at 37 ° C under aerobic conditions in a fermenter on Hottinger's nutrient medium, which contains in 1 liter: 250 ml of Hottinger's basic protein; 10rNaCI; 1 rK2HPO / i; 0 Н20 up to 1 l. Growing is stopped after 3-4 at the end of the logarithmic growth phase, when growth reaches 3-1.5. Biomass yield 6.5-8.5 g / l. On the medium, fish extract (based on kilka hydrolyzate) grows slowly, and the accumulation of restriction enzyme activity is 3-4 times lower than when cultivated on LB or Hottinger media.

Fermentation of the culture is carried out after 0 inoculum is added to the fermenter in a ratio of 1:10 to the volume of the medium.

Working cultures are subcultured on m soy peptone broth or Hottinger broth once every 7–10 days.

5 Cultures were stored in semi-liquid and septonic agar under liquid paraffin in lyophilized form in ampoules.

The culture is non-pathogenic for mice.

Isolation of Bav BII restriction enzyme was carried out by fractionation of cell-free extracts with isopropanol and chromatography on an ion exchanger and affinity sorbent.

The incubation mixture for determining the activity of restrictase Bav BII, volume 5 30-50 μl, contains: 0.01 M Tris-HCl buffer, pH 7.5, 0.01 M MgCl2, 0.001 M dithiothrethol (or 0.007 M 2-mercaptoethanol ), 100 μg / ml bovine serum albumin, 1 μg of phage A DNA and 0.1-1.0 μl of the enzyme. Incubation is carried out at 37 ° C in

for 60 minutes Phage-D digestion products are analyzed by electrophoresis in a 0.8-1% agarose gel, followed by staining of the gel in ethidium bromide. Per unit of Bav BII restriction enzyme activity is taken the amount of enzyme (in µl), which in 1 hour of incubation completely hydrolyzes 1 µg of phage DNA under optimal reaction conditions.

Example 1. Culture B.alvei BKMB-674 pre-adapted on the main nutrient medium containing in 1 l: 250 ml of Hottinger formula; 10 g NaCl; 1 g

K2HP04, up to 1 L of water.

The inoculum for the fermenter is prepared by replanting the adapted culture in 1 liter of fresh medium at a rate of 1:10 and propagated under aeration conditions.

The culture was grown after the inoculum was added to the fermenter in a ratio of 1:10 to volume at 37 ° C under aerobic conditions (1 liter of air per liter of nutrient medium), pH of the medium was 7.0. During cultivation, the pH of the culture liquid is maintained in the pH range 7.2-7.5.

Growing is continued until the end of the logarithmic growth phase is reached.

The yield of raw biomass is 6.5 to 8.5 g / l of culture fluid.

The content of restrictase Bav BII - 4 x 104 u / g biomass.

The allocation of restrictase Bav Bil

10 g of Bacillus alvei BKM B-674 biomass is suspended in a buffer solution and sonicated at + 2-4 ° C. The remnants of the cell membranes and intact cells are removed by ultracentrifugation, and the cell-free extract

fractionated with isopropacol. The restriction enzyme-enriched material is chromatographed in a potassium phosphate buffer in a KS gradient on a DEAE-cellulose DE-52 column, and then on a blue separose CL-6B column.

Get 12 x 104 units of Bavls restrictase activity in 10 ml of buffer with 50% glycerol. The yield of the purified enzyme preparation is 12 x 103 units of activity per 1 g of wet weight of biomass. The drug Bav BI is stable when stored for 8–10 months at a temperature of –20 ° C.

The enzyme preparation does not contain impurities of phosphatases, nonspecific endonucleases and exonucleases and is suitable for DNA structural studies and genetic engineering.

EXAMPLE 2 The cultivation of the Bacillus alvei strain BKM B-674 is carried out as described in Example 1, except that as a nutrient medium: use LB medium, which contains in 1 liter: 10 g of tryptone; 5 g yeast extract; 5 g of NaCi; up to 1 liter of water.

Biomass yield 6-7 g / l of culture fluid.

The content of resgriktazy Bav BII 3 x 104 u / g microbial mass.

After purification, as described above, the yield of restriction enzyme Bav BII is 8 x 10 units of activity / g wet weight of biomass,

Thus, according to the proposed method, a new restrictase is obtained.

Claims (1)

Formula inventive The bacterial strain Bacillus alvei BKM B- 674 - producing restrictase Bav BII.