SU1044631A1 - Method for preparing (desaminating) enzymes of l-glutamate: nad p+-oxy-doreductase - Google Patents

Abstract

METHOD FOR OBTAINING ENZYMES 1 .i.1.3, 11GLUTALT: NAO (P) +. AND 1.1. OXIDOR1DUCKTASE. 1.1, 1-GLUTAMAT; MAO P -OXIDOREDUCTASE (DEAMINATING) by extracting unicellular green algae with phosphate buffer followed by fractionation of the extract, so that, in order to accelerate the process and increase the yield of each enzyme, manganese chloride is added to the extract to a final concentration of 0.025-0.030 M, manganese phosphate precipitate is sorbing 1..1.3 1-glutamate: NAD (P) -oxidoreductase is separated from the liquid phase containing 1L.1. t-glutamate: NAD. acid reductase, centrifugation and 1. it.1.3, L-glutamate: NAD (P) -oxidoreductase elute with a precipitate of 0.1 - O, 2M phosphate buffer. O 4 O) SB

Description

The invention relates to physico-chemical biology and biotechnology, in particular, to methods for producing and purifying enzymes.

A known method of producing iso: enzymes. By affinity chromatography LI. The above method is rather laborious and time consuming.

The closest to the proposed technical essence and reachable (the effect is the method of separation of the enzymes 1.A.1.3,1-glutamate: NAD (P) -oxidation. Reductase and 1.4.1.4, L-glutamate: NAD P-oxyoxidoreductase on unicellular green algae, including the release of an extract from algal C23 cells,

The separation of glutamate dehydrogenase by a known method involves preparing a column with DEAE-cellulose, which consists in treating an ion exchanger with 0.5 M HC1, and then with 0.5 M NaOH, followed by washing on a Buchner funnel with a large volume of distilled water to neutrality; filling the column with charged 1DAAE cellulose and balancing it with a starting buffer; pre-purification of cell-free extract from interfering ion-exchange chromatography of chlorophyll-lipoprotein complexes, which is carried out by precipitation of proteins with ammonium sulphate from 35 to 5% of saturation with subsequent desalting of the protein preparation on a column with Sephadex C-25, applying the protein preparation containing the section enzymes, on a finished column with DEAE-cellulose; washing the column with starting buffer in order to remove the non-DEAE-cellulose protein; stepwise elution with seven buffer solutions of the increasing ionic resin, since NAD (P) -glutamate dehydrogenase (1.4. CZ, L-glutamate: NAD (P) oxidoreductase) is washed off from the column with 0.08-0.1 M phosphate buffer, and NAD Rglutamate dehydrogenase-azene-azane. 1.4; 1 GluTaMaT: NAD P-o-sidoreductase) -0.2 M NaCl in 0.1 M phosphate buffer.

The whole procedure takes 58-60 hours. The data on the separation of glutamate dehydrogenases from Chlorella pyrenoidosa 82 T using 10 ion-exchange chromatography are presented in Table. one.

Table

Cell-free iBKCTpaKT

Salting out with ammonium sulphate, fraction 35-65 of saturation

30.4 0.0

36.7

202

0,212

1,100

0.02.1

0.028

0,257

1.25 50

12.6 0.0

0.413

14.3 24

0.0 5,500

26

56

0.0 to the non-covalent method is that the separation of NAD (p) tlutamate dehydrogenase and NAD P-glutamate dehydrogenase is long, time-consuming, requires a compulsory pre-treatment and gives a relatively low yield of enzymes. In addition, DEAE-cellulose is a scarce imported material. The purpose of the invention is to accelerate the method and increase the yield of enzymes. The goal is achieved by the fact that according to the method of obtaining enzymes 1 .4.1.3.1 - glutamate: MAO (P) oxidoreductase and 1.4.1.4.1-glutamate: NAD P oxidoreductase (deaminating) by extracting unicellular green algae with phosphate buffer. with the subsequent fractionation of the extract, manganese chloride is added to the extract to a final concentration of 0.025-0.030 M, a precipitate of manganese phosphate sorbing 1 .V. 1.3, L glutamate: MAO (P) -oxidoreductase, separated from the liquid phase containing 1.4.1.4, L-glutamate: NAO P / oxidoreductase, centrifugation and 1 .. L-glutamate: NAD (P) oxidoreductase to elute from the residue O, 1-0.2 M phosphate buffer. I PRI me R. ml of extracts cooled to 5-7 ° С in 0.05 M phosphate buffer (pH 7.4) from unicellular

Cell-free

3790 496 extract

0.0 779 0.0 2290

170 0.0 0.47 1010

948 0.13

0.25

1,100

0.34

1.4 82

3.6 95

0.0 31. .4 of the green alga Ankistrodesmus braunii (in the case of another alga, the temperature of the extract can be increased to) containing NAD (P) glutamate dehydrogenase and NAD P-glutamate dehydrogenase, MnCl 2 solution is added dropwise with vigorous stirring (at a rate of 2.5 ml of 1 M MnS 12 solution - on 100 ml of extract). Under these conditions, a manganese phosphate precipitate is formed and formed during 20 minutes, on which NAD (P)) glutamate dehydrogenase is adsorbed. The precipitate formed is separated from the supernatant containing NAD P-. Glutamate dehydrogenase, by centrifuging at 8000 g for 30 min and to remove excess proteins captured by the precipitate, is washed with 100 ml of 0.01 m phosphate buffer pH 7.4. Elute NAD (P) -glutamate dehydrogenase from the precipitate is carried out with O, G phosphate buffer pH 7, 4 in two 100 ml portions. The combined eluate contains almost the entire NAD (P) -glutamate dehydrogenase, and more than 80 NAD P-glutamate dehydrogenase is stored in the supernatant. Su the procedure takes 2.5 hours. The results of the separation of glutamate. hydrogenases by the proposed method are presented in table 2. T a b l and c a 2 i

Thus, the proposed methods (according to a known method, the yield after the separation of glutamate dehydrogenase separation 56 and for NAD (P) does not require glutamate dehydrogenase and NAD P-glut for the preliminary purification of enzymes, i.e., dehydrogenase, respectively, but not

allows to divide them already at the stages of the proposed method 90 and 82);

di cellulose extract; Significantly reduced labor intensity

it reduces the time required and reduces the cost of the entire separation process (from 58 to 2.5 hours, i.e. separation by eliminating more than 20 times); In addition, the enhanced ion-exchange chromatographic yield of the separable enzyme.

Claims (1)

METHOD FOR PRODUCING ENZYMES 1.4.1.3, ^ GLUTAMATE: NAD (P) +. OXIDOR | DUCTASES AND 1.4.1.4, · L-GLUTAMATE; NAD P ⁴ -oxidoreductases (deaminating) by extraction of unicellular green algae with phosphate buffer, followed by fractionation of the extract, with the exception that in order to accelerate the process and increase the yield of each enzyme, manganese chloride is added to the extract to a final concentration of 0,025-0,030 M phosphate precipitate of manganese, the sorbent 1.4.1.3,1glutamat: YA0 (P) ⁺ -oksidoreduktazu is separated from the liquid phase, _in soder- zhaschey 1.4.1.4 ', L-glutamate: NAD P * Oak S sidoreductase, centrifugation and 1.4.1.3, L-glutamate: NAD (P) ⁺ oxidoreductase elute with sediment 0.1 - 0.2 M phosphate buffer. > ι to