SU1392902A1 - Method of extracting and purifying bacterial endonuclease from cultural serratia marcescens - Google Patents

Abstract

The invention relates to the field of enzyme production, in particular extracellular bacterial endonuclease. The aim of the invention is to reduce the duration of the process, increase the yield and purity of the target product. The endonuclease was isolated directly from the culture fluid in a single step using ligand exchange chromatography on negligent sorbents containing iminodiacetic acid groups charged with Cu ions. Sorption of the endonuclease is carried out on a column or in bulk. After washing the chelating sorbent with the initial buffer of 0.1 M Na-acetate with pH 6.5-6.6, containing 0.3-0.5 M NaCl, the Endonuclease is desorbed with a yield of 65-95% with the same buffer, with pH 4.7-5.0. 1 tab. § (L

Description

113

The invention relates to the field of the production of enzymes, in particular extracellular bacterial endonuclease. Endonuclease is used in molecular biology, bioorganic quinine, as well as in veterinary medicine.

The aim of the invention is to reduce the duration of the process, increase the yield and purity of the target product. The endonuclease is isolated by the cultured VKPMV-2379 in a single step, with donar langandoben chromatography on chelate sorbents containing groups of iminodiacetic acid (IDA) charged with C ions. The preparation of such sorbents is described in the work, General formula

HE

I /

P-0-CHi-CH-CH -N

CHjCOO

Si

e

CHjCOO P - organic carrier

20

25

OR I

P, -0-Si- (CH4), - O-CH4-CH-CH,

./

CH, CO (G 30

about/:.

where P is an inorganic carrier.

The ligand exchange chromatography method is based on the ability of proteins to form complexes with transition metal ions. Some functional groups of amino acid residues of the protein surface, mainly imidazole and thiol, are involved in the complexation process. The stationary phase is a chelate sorbent charged with transition metal ions that interact with the separated compounds by forming coordination bonds. Unlike other types of chromatography, in this process the sorption of proteins may occur in the presence of high concentrations of salts, non-ionic detergents and some organic solvents, which allows the enzyme to be isolated directly from the culture fluid.

It turned out that when the RP 6.5-6.6 from the culture fluid

five

0

five

0

five

0

five

0

five

predominantly an nucleotide, and other proteins remain in solution. Therefore, with the extraction of INN enzyme, the pH of the culture liquid was adjusted to 6.5-6.6.

Sorption of the endonuclease on the chelate sorbent is carried out on a column with a relative protein: sorbent "6 units. cm at a speed of 15-20 ml / h or in volume with a protein: sorbent ratio of 10-12 units. After washing the chelate sorbent with the initial buffer of 0.1 M Na-acetate with a pH of 6.5-6.6, containing 0.3-0.5 M NaCl, the dendonuclease is desorbed with a yield of 65-95Z with the same buffer, but with pH A, 7-5.0. The presence of 0.3-0.5 M NaCl in the buffers used suppresses ion-exchange interactions. Using higher concentrations of NaCl is impractical.

Example 1. SSL IDC-agarose (29 1 mol / ml), charged with Cn ions, washed with 0.1 M Na-acetate buffer pH 6.5, containing 0.5 M NaCl, 30 ml of culture liquid was added ( 7.5 units A dd / ml and 7400 units act / ml) and pH 6.5 and transferred at room temperature on a rocking chair for 0.5 hours. Transfer to a column and wash at a rate of 17 ml / h 0, 1 M Na-acetate buffer pH 6.5, containing 0.5 M NaCl, until absorbed at 280 nm. The endonuclease is desorbed with the same buffer prn pH 5.0. The yield on activity 95Z, the degree of purification 50 times, the specific activity of 182000 units. act./ml protein.

Example 2. On a column containing 1 ml of chelating sorbent IDK-toyoperla HW-55 (36 µmol / ml), balanced with 0.1 M Na-acetate buffer pH 6.6 with 0.3 M NaCl at a rate of 15 ml / hr 10 ml of a cool liquid (5, Aiao units / ml and 5600 units of aCt) with a pH of 6.6 are washed with 0.1 M Na-acetate buffer pH 6.6, containing 0.3 M NaCl until no. Absorption prn 280 nm. Desorb annduclease with the same buffer at pH 4.7. The yield of activity is 80%, the specific activity of 200,000 units, act. / Mg of protein.

Example 3. Analogously to example 2, but as a chelate sorbent IDK-billow (19 mol / ml) is used. The activity yield is 63Z, the specific activity is 100,000 ate. act./ml protein.

Example 4. AtranorHMHO Example 2, but the sorption of the enzyme was carried out at pH 6.0, and desorption at pH 5.5. The yield of activity is 30%, the specific activity is 150000 units. act./mg protein.

Example 5. Analogously to example 2, but the sorption of the enzyme is carried out at pH 8.0 in 0.005 M Tris-HC buffer containing 0.5 M NaCl, and desorption at pH 4.0, O, I M Na-acetate buffer containing 0 , 5 M NaCl. Activity yield 28%, specific activity 60000 units. Aktoml protein.

Thus, carrying out the process of sorption of nuclease on the chelation sorbent in volume allows a significant reduction in the enzyme isolation time to 6-7 hours and with a yield of 65-95Z a preparation containing at least nuclease is obtained (determined by scanning gels after electrophoresis).

Claims (1)

Formula invention The method of isolation and purification of bacterial endonucleases from the culture fluid of the Serratia marcescene strain VKPM B-2379 using chromatography methods, characterized in that, in order to prolong the process, increase the yield and purity of the target product, the isolation and purification is carried out directly from the culture fluid ligand exchange chromatography on chelate sorbents containing iminodiacetic groups charged with Cu ions, while sorption is carried out at pH 6.5 - 6.6, and desorption at pH A, 7 - 5.0 sodium acetate, contains aschim 0.3 - 0.5 M sodium chloride. Comparative table Offer