SU1486516A1 - Strain of bacillus megaterium bacteria as producer of endonuclease of bme 12 restriction - Google Patents

Description

The invention relates to biotechnology. The purpose of the invention - to obtain | non-pathogenic strain, producing a restriction enzyme of a given specificity in high yield. Restrictase VIII 12 I is the isoschizomer of the rete * zyxtaz Za and 3те, Bte 12 I cleaves the sequence of 5 '-CATS-3 as part of double-stranded DNA. Biomass is grown with aeration in a nutrient medium containing sprat and yeast hydrolyzate extract, to a delayed growth phase. The cells are disrupted by ultrasound and are isolated by restriction chromatography on phosphocellulose and heparinsepharose. The output of the enzyme 2400 u / g biomass.

The invention relates to biotechnology, logic, and is a strain that can be used to obtain a restriction enzyme (restrictase) that recognizes and cleaves the nucleotide sequence 5 -CATS ~ 3 ', as part of a double-stranded DNA molecule.

The purpose of the invention is to obtain a non-pathogenic strain that produces a re- strictase of a given specificity with a high yield.

The Vaschiz strain over aseparate of VKPM B-3677 (12) was isolated as a result of a search in the study of soil samples. After the selection of the strain, clonal analysis is performed in order to select a highly productive and stable strain.

4 ^

00

ABOUT

sd

The strain has the following features.

Morphological signs. Large mobile rod-shaped bacilli of 0.8-1.5 * 2.0-7.0 microns in size. The cells are arranged singly, in pairs, form long chains. Peritrichous flagellates. Forms endospores. The location of the endospores is predominantly central, the shape of the spores is elliptical.

3

1 A 865 16

four

kaya. Visible bloating sporangia is not observed.

Gram-positive cells show gramvariability as they age.

Physiological and biochemical signs. Catalase-positive cells hydrolyze starch and casein, dilute gelatin. Acetylmethylcarbinol does not form. A wide range of organic substances is used as sources of carbon and nitrogen. Cells grow in the temperature range from 4 to 42 C. The optimal growth temperature is 37 ° C. The optimum pH of the medium is 7.2- '.

7.4. The culture is stored in a lyophilized form. Strain B. te ^ aSehhit 12 contains endonuclease VIII 12 I, isoshizomer 5ai and 3ΑΙ,

Cultural features. On solid nutrient medium, the shape of the colonies is round. The size of the colonies is 2-5 mm, the colony is opaque, does not fluoresce, does not emit pigment in the medium. The surface is smooth, the profile is convex, the edge of the colony is smooth. The structure is homogeneous, oily consistency. It grows well on ordinary nutrient media, for example, on a 3.5 ^ ^ spa or 2-4 ^ hydrolyzate sprat. In liquid nutrient media gives abundant growth with intensive aeration.

Storage of the strain is carried out under liquid paraffin or in glycerin at -70 ° C. Strain retains its properties for more than 4 years.

The invention is illustrated by the following examples.

Example 1. Cultivation of biomass and release of restrictase above 12 I For the cultivation of biomass use fish hydrolyzate and yeast extract of domestic production.

The seed material is prepared on the medium of the following composition, g / l: fish hydrolyzate 40, yeast extract 2. The medium is sterilized at 1.2 atm, 30 min. The inoculum is grown in one-liter flasks on a rocking chair at 200 rpm. Fermentation is carried out in a fermenter with a working volume of 4 liters. 400 ml of inoculum is introduced in the fermentation medium of the same composition as for the inoculum.

. Cultivation is carried out in a periodic manner under the following conditions: growing temperature 37 ° C, stirring speed 200 rpm, aeration 1 ° & min.min "* *. Growing wire 10

15

20

25

thirty

35

40

45

50

55

to achieve an optical density of D _{? 50} 4, 6 opt./ml (growth retardation phase). Cells are harvested by centrifugation and stored at -20 ° C. Biomass yield 4 g / l of culture fluid.

The biomass is suspended in 0.05 M Tris buffer and treated with an ultrasonic disintegrator. The obtained 'cell extract is subjected to gel filtration on A-05M biogel. The fractions containing the enzyme are combined and precipitated with ammonium sulfate, then centrifuged. The pellet is resuspended in 0.02 M potassium phosphate buffer pH '

7.5, containing 0.0005M EDTA and Ο, ΟΟΙΜ 2-mercaptoethanol (buffer A) and dialyzed against the same buffer for 2 hours. The removed enzyme solution is applied on a P-11 phosphocellulose column, equilibrated with buffer A. Fractions containing the enzyme is collected and further purified on a column with heparin-sepharose. The enzyme is eluted with a linear gradient from 0 to 1.M PaCl in buffer A. The fractions containing the enzyme are combined and dialyzed for 12 hours against 300 ml of buffer A containing 50 ^ glycerol.

Endonuclease activity is determined by cleaving the DNA of phage C 1 857. The enzyme is incubated for 1 hour at 37 ° C in 10 µl of the mixture: 01 µg of phage β DNA,

01 M Tris-NA! pH 8.0, 0.1 M DaC1,

0.01 M mdsB ^. This is followed by electrophoresis of the hydrolyzate in the gel of 0.7 ^ agarose. Per unit of activity, they take an amount of enzyme sufficient to completely break down 1 μg of phage DNA. The output of the enzyme 2400 u / g 'biomass.

Example 2. A nutrient medium is prepared of the following composition, g / l: yeast extract from "ϋχ £ οο" 5, tryptone from '' £ οο '' 10, IAS! 5. Cultivation is carried out as in. Example 1, in addition, the biomass is collected at an optical density of D ₃ ^ ₀ 6.0 opt./ml (stationary growth phase). The biomass yield of 5 g / l, the yield of the enzyme 1500 units / g of biomass.

Example 3. The cultivation is carried out as in example 1, except that the biomass is collected at an optical density of D $ 5 -2.5. The biomass yield of 3 g / l, the output of the enzyme 600 u / g biomass.

five

1486516

6

Example 4. The cultivation is carried out as in example 1, but before the optical density D _? 6.0. The biomass yield of 5 g / l, the output of the enzyme 2000 units / g of biomass.

Example 5. The cultivation of V. tezabehit carried out as in example 1, but maintain the temperature.

30 ° C. The biomass yield is 4 g / l, the enzyme yield is 300 units / g biomass.

Thus, in accordance with the present invention, a non-pathogenic effective restriction enzyme producing strain was obtained and characterized, providing enhanced production

enzyme under optimized conditions

five

yah cultivation.

Claims (1)

Claim The Bacterial Strain WasgNiv sheath ^ Segit VKPM B-3677 - the producer of the restriction endonuclease Vse 12 I.