SU1449583A1 - Strain of bacillus subtilis bacteria as producer of restriction endonuclease bsu 151 - Google Patents

Abstract

This invention relates to the microbiological industry. The strain deposited in VKPM Institute of genetics and selection of industrial microorganisms under the number B-3667. The strain produces the restriction endonuclease Bsu 15 1, which is an isoschizomer of retictate C1a 1. Bsu 15 1 cleaves the 5-ATCGAT-3 nucleotide sequence of the double-stranded DNA. The strain biomass is grown by aeration in nutrient broth based on yilka hydrolyzate until it reaches an early stationary; growth phase. Cells are sonicated and restrictase isolated by chromatography on Biogel A g.O., 5 m, DE-52 cellulose, and F-cellulose P-11. The output of the enzyme 300000 .. units / 1 g of raw biomass. 1 tab. With S W SP 00: o

Description

{The invention relates to microbio and is a strain, which Topijift can be used to obtain; restriction endonuclease. (restriction, tazy) recognizing and cleaving the sequence of nucleotides of 5 -ATCCAT- 3 J & composition of double-stranded DNA, ° -

The purpose of the invention is to obtain a strain I producing a restriction enzyme of a given specificity, Q, in high yield, growing on normal nutrient media.

Itamm produces a restriction enzyme called .Bsu 15 1, possessing the ability of the J5 network to identify and cleave the sequence of nucleotides 5 of AGSSAT-3. Deep cultivation

spend at

30 ° С

and aeration

20

1-2 volumes in t-shnuty on nutrient medium containing, g / l of water; sprat hydrolyzate 40, NaCl 20, until the growth is slowed down, the restriction enzyme restriction enzyme 300,000 units per gram bis mass.25

| Used strain B, subtilis (15) BKI | M B-36.67 was obtained as a result of the search for strains - products of restriction enzymes,

1Stamma .. Bacillus subtilis (15) jQ BKI B-3667 has the following characteristics,

iMorphological features, In young culture (16-18 g), the cells are stick: 5idnye |, 0.7-0.8 μm in size in 1449583

7.4 After incubation, they form large, whitish colonies, convex in shape, mucous consistency. Prolonged incubation (2-4 days) leads to the disappearance of mucus, the colonies are large, flat, with little wrinkling. The medium is used to revitalize the culture.

Wednesday 2. Composition as in medium 1 only without agar. The medium is used for preparative cleavage of cells, At 30 ° C and aeration-2 volumes per minute, 200 rpm bashchalki. The turbidity of the culture is even.

The resulting restriction enzyme Bsu 15 1 is characterized by the following properties: it recognizes and specifically cleaves the nucleotide sequence in the DNA molecule 5 -ATCC, AT-3, the optimum pH value

6.5-7.5 ,. the optimum temperature is 30-37 seconds. Restriction activity does not vary in a wide range of NaCl concentrations up to 100 mM.

A comparison of the characteristics of the proposed method and the known one is presented in the table, in which the restriction enzymes obtained from Bacillus subtilis B-3667 and Bacillus species are shown.

ne;) janitor and 2-3, sometimes 3 microns, in length, are located singly, in pairs silt: 1 form chains. Moving, burning laterally. They form endospores of elliptical form, located mainly centrally, not 6uhee one per cell-sporangium. Distinct bloating of sporanga is not observable. Do not form okolosporol-tel, gram-positive,

Physiological and biochemical characteristics,, catalase positive. When glucose is mixed, they form acid, gas. Voges-Proskauer reactions are susceptible, Indole is not formed, nitrates are reduced to nitrites, Hydrolysis T starch and casein, and liquefied gelatin.

Cultural features. Grow well on common nutrient media ((|; PA, BCH), ptiiCTa is optimal at 37 ° C, but grows at 50 ° C,

Medium 1: agar-15 g / l, sprat hydrolyzate 40 g / l, NaCl 20 g / l, pH 7.2- .35

Cleaning steps

40

45

Output, u / g

50

Precipitated in strep-Chromatotomycin sulphate and a decanter on the following: sulfate-biogel; ammonium precipitation; A-0.5 mol.; Chromatocellulose; a decanter on orange-DE-52; sepharose; heparinsepharose P-11

12,000 .. 300,000 1. Getting restricts55

Example basins Bsu 151,

Strain B, subtilis (15) is maintained in test tubes on agar medium under liquid paraffin. For liveliness, the strain is subcultured on Petri dishes with agar medium containing, g / l: sprat hydrolyzate 40 ,. NaCl 20 (medium 1), agar 15, and incubated at 37 ° C during the day. Grown cells The resulting restriction enzyme Bsu 15 1 is characterized by the following properties: it recognizes and specifically cleaves the nucleotide sequence in the DNA molecule 5 -ATCC, AT-3, the optimum pH value

6.5-7.5 ,. the optimum temperature is 30-37 seconds. Restriction activity does not vary in a wide range of NaCl concentrations up to 100 mM.

A comparison of the characteristics of the proposed method and the known one is presented in the table, in which the restriction enzymes obtained from Bacillus subtilis B-3667 and Bacillus species are shown.

Cleaning steps

Output, u / g

Precipitated in strep-Chromatotomycin sulphate and a decanter on the following: sulfate-biogel ammonium precipitation - A-0.5 m, reduction, chromatocellulose decanter on orange-DE-52, sepharose, phosphor-phocellulose cell R-11 lulose heparinsepharose R-11

12000 .. 300000 1. Receiving restricts Example of basins Bsu 151,

Strain B, subtilis (15) is maintained in test tubes on agar medium under liquid paraffin. To recover, the strain is subcultured on Petri dishes with agar medium containing, g / l: sprat hydrolyzate 40 ,. NaCl 20 (medium 1), agar 15, and incubated at 37 ° C during the day. Grown cage 1A495834

ki are transferred to a liquid medium 1. Inoculum activity. Selected fractions to:; alGG :: GzGG combined with t and dialysis nonv night buffer. Gradient chromatography overnight. The resulting inoculum is introduced in the same way as before. Samples

possessing restriction activity, are combined and stored at -20 ° C in buffer B with 50% glycerol.

Received 10 ml of the drug Bsu 15 1 with an activity of more than 500 units / μl, i.e. 300,000 units per gram of weed biomass.

The restriction enzyme is stably stored in

, j -D1AIIJU-1 hypil ll lirUJtZ

to the fermenter and pulling are carried out under the same conditions with aeration of 2 volumes per minute in the nutrient medium 1 until reaching the early stationary growth phase. The culture fluid is oh- and ohlazhd and the cells are precipitated by centrifugation.

30 g of raw biomass of B. subtilis

(15) is suspended. In 80 ml of 50 mM tris-for a month.

al yKor -MSE- SS r p. and meter 2. determination of active elimination by centrifugation. Supernatant is applied on a column with Biogel A - 0.5 m, pre-equilibrated with buffer A (NaCl 0.8 M; Triton, p.X-100cO, 1%, 20 mM, pI 7.5, EDTA 0.5 mM, 2-mercaptoethanol 0.1 No. 1, Fractions are eluted with the same buffer and tested for the presence of restriction enzyme activity by the ability to specifically cleave ft ph DNA when incubated in buffer: Tris-HC1 20 mM, pH 7.5, MgCl 1t3 mm, NaCl 50 mM, 2-mercaptoethanol 10 mM at 3.7 s.

Restrictions of Bsu 15.1.

The enzyme is incubated for 60 min at 37 ° C in 40 μl of the mixture: DNA of phage A 1 μg, Tris-HC 0.1 M, pH 7.0; MaCl 0.05 Mj MgCl 0.01 M. Then electrophore is carried out on a 0.7% agarose gel. Per unit of activity, the amount of enzyme is taken for the complete specific cleavage of 1 µg of phage L DNA within 1 hour.

Thus, in accordance with the invention, the obtained and

 characterized by highly efficient

Fractions containing the restriction enzyme strain - producer of restrstase oLr ™ r «: p. ° rs: pT -gyr

EDTA 0.5 mM, 2-mercaptoethanol 0.1 mM). The precipitate was removed by centrifugation, and the supernatant was applied to a DE-52 cellulose column equilibrated with buffer B. Proteins eluted

35

--... h / yjcjj

coping 25 times the production of a known strain.

Claims (1)

Invention Formula 0-1 .M NaCl gradient, 10 ml fractions are analyzed for restriction enzymes. Bacillus subtilis strain VKPM B-3667 is a producer of Bsu 151 restriction endonuclease. P p. and mr. 2. definition of active Restrictions of Bsu 15.1. The enzyme is incubated for 60 min at 37 ° C in 40 μl of the mixture: DNA of phage A 1 μg, Tris-HC 0.1 M, pH 7.0; MaCl 0.05 Mj MgCl 0.01 M. Then electrophore is carried out on a 0.7% agarose gel. Per unit of activity, the amount of enzyme is taken for the complete specific cleavage of 1 µg of phage L DNA within 1 hour. Thus, in accordance with the invention, the obtained and characterized by highly efficient   -ggyr 35 --... h / yjcjj coping 25 times the production of a known strain. Invention Formula The bacterial strain Bacillus subtilis VKPM B-3667 - producing restriction endonuclease Bsu 151.