SU1440919A1 - Strain of kurthia zopfii bacteria as producer of restrictive endonuclease - Google Patents

Description

4 4i

About WITH

WITH

This invention relates to microbiology, in particular to the preparation of restriction enzyme producers.

The purpose of the invention is to identify the strain-Maj simplification and cheapening of the process of obtaining its restrictase, which has the ability to recognize and cleave the GATC nucleotide sequence,

The invention provides for the use of the kepathogenic strain Kurthia Zopfii 9 of VKLM B-3668 - a producer of restriction enzymes, the cultivation is carried out in a nutrient medium LB under aeration conditions, the cells are destroyed by ultrasound, then the chromatographic purification of the enzyme is carried out.

The used strain of Kurthia zopfii 9 was isolated as a result of a search for strains producing restriction enzymes among microorganisms living in the Aral Sea, and is stored in the VKPM VNII of Genetics under registration number B-3668.

The strain has the following morphological, cultural and physiological properties,

In young cultures (18-24 h) - regular unbranched sticks with rounded ends, found in chains that are often parallel, the diameter of sticks is about 0.8 microns, the mouth often varies depending on the growth stage, but usually 2-8 microns. Older cultures (3-7 days) consist of cocci-shaped cells formed during the fragmentation of rods; the rods are driven by the peritrichous flagella. Endospores do not form, Gram-positive, non-acid-resistant chemo-organotrophs. Metabolism is respiratory, not fermentative. Catalase-positive oxidative. Do not restore nitrates. Do not form acid from carbohydrates or polyatomic alcohols, alkalinize the medium. - Strict aerobes. Temperature optimum 25-, limits. Good growth in the presence of 4-6% NaCl. The resulting restriction enzyme has the following properties: it recognizes and specifically cleaves the GATC nucleotide sequence; the optimum pH for restriction enzyme action is 7.5-9.0; optimal temperature for restriction enzyme activity; Mg ions are required; optimal concentration is 5-15 mM.

Example. Receiving restriction enzyme Kzo 9 1 from Kurthia zopfii 9. Cells. Kurthia zopfii 9, stored in 50% glycerol in L-broth (LB) composition, g: tryptone 10, yeast extract 3, NaCl 10, water to 1 l at 20 ° C, plated on a L-agar plate. After 2 days of incubation at 30 ° C, the cells are collected in a loop and added to 100 ml of LB and grown to a density of 1.5 at.

The culture is then introduced into 2 L of LB and extruded to a density of 2.5. After cooling, the cells are harvested by centrifugation. Biomass INPUT 2 g wet weight with i l medium.

0

five

thirty

35

40

45

50

55

4 g of cells are suspended in 20 ml of 0.02 M buffer, pH 7.5, containing 10 MyS-mercaptoethanol and 0.1% Triton X-100 and destroyed in an ultrasonic disintegrator. Cell debris was removed by centrifugation (18,000 rpm, 30 min at) and the supernatant was applied to 30 ml of Whatman phosphocellulose P-11 (equilibrated with buffer A) (0.02 M K-phosphate, pH 7.5, 0.001 M -mercaptoethanol, EDTA) 5 and then the column is washed with buffer A until the UV absorbing material disappears from the eluate. The adsorbed material was eluted; from a solution of 1 M NaCl —in buffer A. 60 ml of the resulting protein wash off the column was dialyzed overnight against 2 liters of buffer A and then applied to 10 ml of DEAE - cellulose (DE-52, VJhatman, England), equilibrated with buffer A, and eluted with 100 ml gradient of 0-1.0 M Nad in buffer A. The volume of each fraction is 5 ml. Aliquots from each fraction (1 μl) are incubated for 1 h with 1 μl of DNA. Phase L and analyzed in agarose gel. Phage (8-P) DNA cleavage fractions were pooled, dialyzed for 3 hours against 2 liters of buffer A and applied to 4. ml of P-1.1 phosphocellulose (Whatman, England) and 40 ml of 0-1 gradient were applied. , 0 in buffer A. The volume of each fraction is 2 ml. The fractions containing Kzo 9 1 are determined in the same way as in the chromatography on a daAE cellulose. Fractions 12–13, containing Kzo9 1, are combined and dialyzed against buffer A, containing: 0.1 M NaCl and 50% glycerol. The resulting enzyme preparation (I ml) has a specific activity of 0000ed / ml.

3 and D

Per unit of activity, take the minimum amount of enzyme needed to completely cleave 1 µg of phage L DNA for 1 hour at.

The enzyme preparation is stored at. From I g of biomass, 2500 units of Kzo 9 1 restrictase are obtained.

Determining the sequence of nucleotides recognized by the restriction enzyme Kzo 9 1.

The dp of nucleotide sequence recognition, recognized by the restriction enzyme Kio 9 1, is carried out by hydrolysis of the DNA of phage and restrictase kzo 9 I, Sau 3AI separately, as well as joint hydrolysis by these restrictases. The observed picture of the complete coincidence of the fragments obtained during the hydrolysis of DNA restrictases Kzo 9 1 and Sau 1 separately and together indicates that the restriction enzyme Kzo 9 1 is an isoshizomer of restrictase Sau 3AI, recognizes and splits the nucleotide sequence of GATC.

Restr1Ptazas from Kurthia- zopfii. 9 is named Kzo 9 according to the generally accepted nomenclature.

Claims (1)

Invention Formula The bacterial strain Kurthia zopfii 9 BKTIM B-3668 is a producer of the restriction endonuclease Kzo 9 1,