SU1678832A1 - Strain of bacteria lactobacillus plantarum - a producer of restrictase lpli - Google Patents

Abstract

The invention relates to biotechnology and can be used in molecular genetic studies, the microbiological industry, in the practice of research and development. The aim of the invention is to identify a strain of Lactobacillus plantarum with a higher yield of enzyme. The strain producing Lactobacillus plantarum BKM B-578 is grown under anaerobic conditions on a nutrient medium containing sources of carbon, nitrogen and mineral salts, the target product is isolated from the resulting biomass by known methods. The biomass of the strain Lactobacillus plantarum 578 contains a new Lp0l restriction endonuclease that recognizes and cleaves the nucleotide DNA sequence 5 A C G A T in quantities sufficient to produce a restriction enzyme on a preparative scale. The content of restrictase in biomass is 6-7x10 units / g of raw biomass. For the LpBI isosomer, restriction enzyme Clal, the yield of the purified enzyme is about 430 units / g of raw biomass. The yield of the purified enzyme Lp6 I is 1.6-1.7 x105 units / g of raw biomass, which is about 400 times higher than the known figures for restriction enzyme Clal (430 units) of the following

Description

The invention relates to biotechnology and can be used in molecular genetic studies, the microbiological industry, in the practice of research and development.

The purpose of the invention is the detection of a strain of Lactobacillus plantarum, a producer of a restriction enzyme, having a higher yield of the enzyme.

The producing strain Lactobacillus plantarum BKM B-578 is grown on a nutrient medium containing sources of carbon, nitrogen and mineral salts under anaerobic conditions, and the target product is isolated from the resulting biomass by known methods.

LpPl restriction enzyme, Clal restriction endonucleate isoschisomer recognizes a site of ATC G A T and cleaves the DNA of phage A in 15 sites, adenovirus 2 in two, pBR322 in one and does not hydrolyze the DNA of SV40 and 0X174.

LpC I restriction enzyme is a valuable tool for molecular genetic research. The enzyme has one cleavage site on the plasmid pBR322 in the gene responsible for tetracycline resistance. This circumstance

about vj

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with y

allows the pBR322 vector to be used to clone any DNA enzyme with 5 -pCG protruding ends. Such DNA fragments are formed by the restriction enzyme LpEl, and Tar, Hpa II, Asu I, HgiD I, ScIN I, Nar, Asu, and others.

The search for a producer strain was carried out using the developed express method for determining the activity of restriction enzymes in toluene extracts of bacterial cells, followed by electrophoresis of DIC cleavage products on an agarose gel. The bacterial strain Lactobacillus piamarum 578 was obtained from the All-Union Collection of Microorganisms of the Academy of Sciences of the USSR, where it is stored under the no. B of CM KM B-578 CCM 1845, 1904 ATCC 8014 NCIB 8030, 6376 MSTS 6376. Characteristics of the strain. Morphological properties. The sticks often form chains, immobile, gram-positive.

Cultivation and biochemical properties. On beveled agar, growth along the stroke is gentle. Colonies on agar are small, shiny, smooth. On m with-peptone broth and Hottinger broth - sediment, clear broth.

Attitude to oxygen - anaerobic. Utilization of carbohydrates - on glucose, lactose, maltose, mannitol, sucrose forms acid.

Indole and hydrogen sulfide - does not form. Gelatin injection - no dilution, sensitivity to antibiotics. The culture is sensitive to many antibiotics: penicillin, streptomycin, kanamycin, tetracycline, chloramphenicol, erythromycin, oleandomycin, neomycin, carbenicillin, lincomycin, gentamicin, methicycline, acycline, methacylin, methacylin, methyclycine, acyclin, tetracycline, lincomycin, gentamicin, erythromycin, oleandomycin, neomycin, carbenicillin, lincomycin, gentamicin, erythromycin, oleandomycin, neomycin, carbenicillin, lincomycin, gentamicin, erythromycin, oleandomycin; resistant to polymyxin. Optimum growing and storage conditions. Culture grows well on BCH, Hottinger broth, LB medium and medium No. 7, without aeration, at 30-37 ° C.

The highest yield of biomass containing LpBl restriction enzyme is obtained when grown on a nutrient medium, which contains a 1 l, g: yeast extract 5; peptone 10; glucose 5; NazHP04 12 H20 2.2; NaC 3; MgSO 0.5; 0.25.

A significant cultivating factor is anaerobic conditions. Under aerobic conditions, the enzyme yield is reduced. Isolation of the restriction enzyme is carried out by fractionation and column chromatography by known methods.

Example 1, the Culture of LactobaciHus piantarum strain 578 pre-adapted on the main nutrient medium, cpj-

holding in 1 l i. yeast extract 5; peptone 10; glucose 5; N32HP04 12H20 2.2; NaCl h; MgS04 0.5; CaCl2 0.25; at 37 ° C overnight. The inoculum for fermentation is prepared by replanting an adapted culture in 1 liter of fresh medium at a rate of 1:10, and propagated in the absence of aeration. The culture is fermented after the inoculum is added to the fermentor in a ratio of 1:10 to medium volume at 37 ° C no aeration; pH of 7.2 + 0.2. During fermentation, the pH of the culture liquid is maintained within the range of 7.4-7.6. Growing is continued until the end of the lag phase.

The biomass yield is 7 g / l of culture liquid. The content of restrictase Lpl I 6 x 105 units / r biomass.

Determination of enzyme activity is carried out using a rapid method in toluene lysates of bacterial cells, followed by electrophoresis of the products of DNA cleavage on an agarose gel.

Selection restrictase Lpfl. 3 g of Lactobacillus piantarum 578 biomass is suspended in a buffer solution and the cells are sonicated at 2-4 ° C. After removal of cell wall residues and undisturbed cells, the enzyme is fractionated with 1% polyethylenimine and then polyethylene glycol / dextran is fractionated in a two-stage system. The restriction-enriched extract is dialyzed and chromatographed in potassium phosphate buffer on a DEAE-cellulose column (DE-52, Whatman), then on a blue sepharose column.

Obtain 1.7 x 105 units. enzyme per g of biomass in 27.5 ml of glycerol buffer. The drug restrictase Lpl I stably retains activity for 9–12 months. The enzyme is suitable for analyzing DNA structure and molecular cloning,

Example 2. The cultivation of the strain Lactobacillus piantarum 578 is carried out analogously to example 1, except that the medium used is 1 medium: Hottinger broth 250 ml; glucose 20 g. Biomass yield 8 g / l of culture fluid, restriction enzyme content in biomass 7 x 10 units / r of raw biomass.

After purification, the yield of LpCl restrictase was 1.6 x 105 units / r of biomass.

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

The biomass of the strain Lactobacillus piantarum 578 contains the new restriction enzyme LpCl, which recognizes and cleaves nucleo5 16788326

the DNA DNA sequence is And G is 1.6-1.7 x 105 units of activity per 1

AT, in quantities sufficient to obtain raw biomass, which is about 400 times

neither the restriction enzyme in preparative scale exceeds the values indicated for limestone, the content of restrictase in the biomassenic restriction enzyme Clal (430 units).

Claims (1)

6-7 x 10 units / g raw biomass. For isoshi-5 formula zomera LpEl - restriction enzymes Clal, the exit of the strain-Strain bacteria Lactobaclllus enzyme enzyme per 1 g of biomass near plantarum VKM B-578 - producing restrictor430 units. The output of the purified restrictase Lptltazy LpBl.