SU691461A1 - Method of preparing ribulosodiphosphatecarboxylase-oxygenase - Google Patents

Description

3, Preparation of protein extract from isolated chloroplasts after their destruction.

4, Purification of the enzyme extract by fractionation with ammonium sulfate, dialysis and two-fold chromatography on Sephadex G-.200.

The disadvantage of this method is the low yield of the target product. The process of separating ribulozodiphosphatecarboxylase requires a large amount of leaf biomass, many of them, is difficult to reproduce the technical stage of chloroplast extraction. As a result, the enzyme has only a carboxylase function and a low specific activity.

The goal of the method is to develop a method for the simultaneous production of an enzyme with 2 catalytic functions — ribulozodiphosphate carboxylase and ribulozodiphosphate oxygenase — in one tehnolotic process with an increased activity of the enzyme and a high-energy prepared product from a cheap and available raw material with a high-tech raw material and a ready-made raw material from a cheap and available raw material with a high-tech raw material and a ready-to-use ready-made product from a cheap and available raw material with an increased activity of the enzyme and an increase in the use of the finished product from a cheap and available raw material with an increased activity of the enzyme and an increase in the use of the finished product from a cheap and available raw material with an increased activity of the enzyme and an increase in the use of the finished product from a cheap and available raw material with an increased activity of the enzyme, Said tsy s is achieved due to the fact Thu to give the title product isrolzuyut th in achestve volosyets Juncaceae feedstock comprising ribu lozodifosfatkarbs ssilazu-oksigenajy and proposed ksgmbinatssho minutes stud purification rnbulozodifosfatkdrbokSylazy-Oxygen .azy in conjunction with the following conditions and carrying out purification steps.

1. Homogenization of raw materials is carried out in a buffer with a pH of 8-8.2,

2 Purification of ribulozodiphosphatecarboxylae-oxygenase is carried out in the following steps:

a) gel-filtration of the extract from .via through Sephadex Q-50;

b) by treating an extremist with streptomycin sulfate; c) fractionation of the extract with ammonium sulfate; d) desalting on Sephadex; d) gel filtration; Sephadex;

According to the invention, a simplified process for the production of ributa 1-diphosphate carboxylase-oxygase (EC 4.1.1.39) from autotrophic organisms with a high yield of the target product with two catalytic functions from the hairline (Lomxus articulum) of the titanic EevmusCpsoith rostctNNjs janus, the report, the statement, enclosed by the statement, enclosed by with a pH of 8.0-8.2 and the subsequent isolation of the enzyme by successive stages: a) gel-filtering extract on Sephadex .Q 50; b) treatment with streptomycin sulfate; c) fractionation

ammonium sulfate; d) desalting on Sephadex G-50; e) gel filtration on Sephadex G-200,

Preferably, the process is carried out as follows: All enzyme purification operations are carried out in the cold. The leaves of the willower grass are homogenized in a buffer mixture following the composition: Tris-HSE 0.05 M, pH 8.0-8.2;

NaC 0.2 M; EDTA 0.005 M; MgCEj, 0.01M; dithiothreitol 0.01 M; the homogenate is centrifuged at 23000 g for 30-40 minutes. Further, in order to free the protein from low-yields. A market of impurities is gel-filtered on the obtained extract; through a Sephadex Q-50 column, equilibrated with the same buffer.

Nuclein removal target: Lp | syrup, processing protein fraction from c; prepared. The 10% nBM solution of the streptodavia splendid, which adds RI to the HJI9 concentration concentration, 1,% .. r-dad removes centrifuge (5 wye; 11 (eF1 and cDjugal fluid noiweprajptj. Kyyyyyon). aad "1JIJA ;, Fraktsyyu, obtained in .x, the center of fug1 p; ukj, ... about 1a pk. ra lied

In Minimum. koljeshtde buffer following sprint srstazh ;: tris-nse o, 025 m,

pH 8.0-8.2; WaCe 0.1 M; EDTA

0.0: 05 M; Mg; ce.jj. o, ag m; di; ty; otreitol o, 01 m. PactTSCip is cleared on

Sephadekaim column; . , balanced by the same.: the purification stage of the rrrvrdd / by chromatography of the exhausted protein friction on Sephadex, 0-200, equalized by that network by the buffer system in which both are carried out; ; Pry The fractions containing the enzyme are pooled. Homogenous preparation of enzyme is obtained, which can be stored.

in frozen or lyophilized sprays,

The hairs of rush in the greenhouse soil or in aquatic culture on a mixture of Pr Nišnikova,

A portion of the leaves (120 g) of 3-week-old seedlings of the rhizome mica were homogenized in a multiple volume of Tris-HC-buffer (pH 8.1) of low ionic strength (0.05 M) containing

0.2 M sodium chloride, 0.005 M

EDTA, 0.01 M magnesium chloride, 0.01 M dithiothreitol. . .

The resulting dark green mass is filtered through a sheet, then centrifuged for 40 minutes at 23000 ° -. For a more complete separation of soluble proteins, the precipitate is washed twice with stock buffer. After the first washing of the precipitate, an additional 5-6% protein is extracted. after the second 1-2% protein from the source,. The supernatant liquid (leaf extract), containing soluble leaf proteins, is further purified from low molecular weight non-proteinaceous substances by filtration through a column (100 x 4.0 cm) with Sepha dex G-50, balanced with buffer in which the wire t the destruction of the leaves. Filtration of the homogenate through Sephadex Q-50 distinguishes two components. All soluble proteins are eluted in the composition of the first component. To remove the nucleic acids, the protein fraction obtained after g filtration on Sephadex G-5C is gradually added 10% (freshly prepared) streptomycin sulfate to a final pe to 1% final precipitator concentration. Streptomycin sulphate is prepared in a buffer that is used to obtain the extra extract of the leaves of the hedgehog. After 30 minutes, the precipitate was separated by centrifugation for 20 minutes at 16,000 CJ-. . The precipitate is discarded and the precipitating liquid is used to fractionate the proteins with ammonium sulfate. Recrystallization and powdered ammonium sulfate are added to the protein solution to 36% saturation (the amount of ammonium sulfate is calculated by the nomogram), incubated for 60 minutes and centrifuged for 20 minutes at 160009-. Further, a protein fraction in the range of 36-44% saturation is obtained in the same way, and after the eifugation center for. removing ammonium sulfate from the enzyme preparation, the precipitate of proteins is dissolved in a minimum volume (2 ml) of 0.025 M Tris-nse-buffer (pH 8.0) containing 0.1 M chloride, sodium, 0.005 EDTA, 0.01 M magnesium chloride, 0.01 dithiothreitol, and filtered through a column (25 X 1.2 cm) with Sephadex G-50, equilibrated with the same buffer, at a rate of 0.1 ml / min. Protein fractions are collected. . Desalted on a column with Sephadex Q-50 protein solution in an amount of 3 ml (200 mg of protein) is layered on a column (100 x 2.2 cm), with Sephadex G-200, equilibrated with the same buffer, which was desalted. Elution of proteins from the column was carried out at a rate of 0 ml / min. Fractions of 2 ml are collected using an automatic collector of fractions. Fractions 35-44 with enzymatic activity are combined. The enzyme is stored at 0 ° C, -1G in the frozen state at -20 ° C or freeze-dried. Tab. Figure 1 illustrates the described example of enzyme release. 2 shows the characteristics of enzyme preparations during storage. . Freshly obtained ribulozodiphosphatecarboxylase in 0.025 M Tris-LSS buffer (pH 8.0) containing 0.1 M sodium chloride, 0.005 MEDTA, 0.01 M magnesium chloride, and 0.01 dithiothreitol was stable for weeks at 0 ° C and during 5-6 months. Lyophilized ribulozodydifosfatkarbok-. xylase can be stored at -20 ° C for 6 months, Ribulozodiphosphatoxygenase in 0.025 M Tris-HCt-buffer (pH 8.0) containing 1 Q, l M sodium chloride, 0.005 M EDTA, 0.01 M magnesium chloride and 0 , 01 M dithiothreitol, when stored for half a year at -20 ° C, loses its initial activity. However, the loading of the enzyme in the presence of 50 mM magnesium chloride and 0.1 M dithiotreitol during 5 min restores the initial activity. The composition of the enzyme mixture to determine the activity of ribulozodiphosphatecarboxylase (µmol / ml): magnesium chloride 10.0; dithiotreitol 10.0; KANS Oj 50.0 in 0.05 M Tris-HC buffer (pH 8.1); ribulose-1,5-diphosphate 0.5; ribulozodiphosphatecarboxylase not more than 0.05-0.1 mg of protein. The reaction mixture also serves as a control, but without the ryso ozo-1,5-diphosphate. The composition of the enzyme smsy To otipedeparation of the activity of ribu Brain phosphate oxygenase on the floor hormone LP-7 (µmol / ml): magnesium chloride dithiothreitol 0.4; ribulose-1,5-diphosphate 1,2; ribulose diphosphate oxygenase 0.71 mg; Tris-HSE buffer (pH 9.3-200.0). One unit of ribulozodiphosphate carboxylase carboxylates 1 μmol of ribulozodiphosphate d6 2 μmol of phosphoglynseric acid at 30 ° С in 1 min. One unit of ribulozodiphosphate oxygenase oxygenates 1 μmol of ribulozodiphosphate to 1 μmol of phosphoglycolate and 1 μmol of phosphoglycerate at 1 min. The homogeneity of the isolated ribulzodiphosphate carboxylase-oxygenase was proved by the following methods: 1) sedimentation equilibrium on a spinco analytical centrifuge, model E; in a small layer using shadow optics; 2) by electrophoresis in a 7.5% polyacrylamide gel; 3) using electron microscopy of a negatively colored drug.

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