Method for purifying double-function glutathione synthetase

A technology of glutathione and purification method, applied in biochemical equipment and methods, enzymes, ligases, etc., can solve the problems of high cost, long time, low enzyme recovery rate, etc. Easy-to-operate effects

Active Publication Date: 2016-01-27
ANHUI GSH BIO TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In order to obtain highly purified enzymes, methods such as ion exchange chromatography and gel chromatography are generally required, but such methods are time-consuming, costly and have low enzyme recovery rates, and are not suitable for large-scale industrial production of GshF

Method used

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  • Method for purifying double-function glutathione synthetase
  • Method for purifying double-function glutathione synthetase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] (1) Take the thallus containing GshF enzyme and dissolve it in water, the thalline concentration is about 100g / L, and homogeneously crush under high pressure.

[0030] (2) Use hydrochloric acid to adjust the pH value of the crushed enzyme solution to 5.0, place it at 25°C for 30 minutes, then use sodium hydroxide to adjust the pH value of the enzyme solution to 7.0, and centrifuge to remove the precipitate.

[0031] (3) Add 0.05M calcium chloride to the enzyme solution, place it at 4-10°C for 30 minutes, and centrifuge to remove precipitated impurities;

[0032] (4) Finally, adjust the pH to 6.0, then add 0.02M copper chloride, 0.01M manganese chloride and 0.02M zinc sulfate, place at 4-10°C for 30 minutes, and centrifuge to collect the precipitate, which is GshF enzyme.

[0033] Dissolve a small amount of purified solid GshF enzyme and prepare a solution of 1 mg / ml. Using the known method for measuring GshF enzyme activity recorded in the prior art, it is detected that...

Embodiment 2

[0035] (1) Take the thallus containing GshF enzyme and dissolve it in water, the thalline concentration is about 100g / L, and homogeneously crush under high pressure.

[0036] (2) Use hydrochloric acid to adjust the pH value of the crushed enzyme solution to 3.0, place it at 4°C for 30 minutes, then use sodium hydroxide to adjust the pH value of the enzyme solution to 7.0, and centrifuge to remove the precipitate.

[0037] (3) Add 0.001M calcium chloride and 0.001M ferrous chloride to the enzyme solution, place it at 4°C for 30 minutes, and centrifuge to remove precipitated impurities;

[0038] (4) Finally, adjust the pH to 4.0, then add 0.005M copper chloride, 0.005M manganese chloride and 0.4M zinc sulfate, leave it at 4°C for 30 minutes, and centrifuge to collect the precipitate, which is GshF enzyme.

[0039] Dissolve a small amount of solid GshF enzyme and prepare a solution of 1 mg / ml. Using the known method for measuring GshF enzyme activity recorded in the prior art, it...

Embodiment 3

[0041] (1) Take the thallus containing GshF enzyme and dissolve it in water, the thalline concentration is about 100g / L, and homogeneously crush under high pressure.

[0042] (2) Use ammonia water to adjust the pH value of the crushed enzyme solution to 9.0, place it at 40°C for 10 minutes, then use phosphoric acid to adjust the pH value of the enzyme solution to 5.0, and centrifuge to remove the precipitate.

[0043] (3) Add 0.5M magnesium chloride to the enzyme solution, place it at 40°C for 10 minutes, and centrifuge to remove precipitated impurities;

[0044] (4) Finally, adjust the pH to 9.0, then add 0.4M copper chloride and 0.005M manganese chloride, place at 40°C for 10 minutes, and centrifuge to collect the precipitate, which is GshF enzyme.

[0045] Dissolve a small amount of solid GshF enzyme and prepare a solution of 1 mg / ml. Using the known method for measuring GshF enzyme activity recorded in the prior art, it is detected that the GshF enzyme activity is about 50...

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Abstract

The invention discloses a method for purifying double-function glutathione synthetase. The method includes the steps of firstly, preparing crude extract containing the double-function glutathione synthetase; secondly, adding divalent metal ions with concentration of 0.001-0.5M into the crude extract to remove sediment so as to obtain supernate; adjusting the pH of the supernate to 4-9, adding one or more of divalent copper ions (Cu<2+>), divalent manganese ions (Mn<2+>) or divalent zinc ions (Zn<2+>) with concentration of 0.005-0.4M, and collecting sediment, namely the double-function glutathione synthetase. The method for purifying the double-function glutathione synthetase has the advantages that the method is simple, easy to operate, short in time consumption and low in cost; the double-function glutathione synthetase (GshF) produced by the method is insoluble in water and reusable, and the method is suitable for large-scale industrial production of GshF.

Description

technical field [0001] The invention relates to a purification method for bifunctional glutathione synthetase, in particular to a purification method for large-scale purification of bifunctional glutathione synthetase. Background technique [0002] Glutathione is widely present in animals, plants and microorganisms, and is one of the most important non-protein sulfhydryl compounds in organisms. It has reduced glutathione (GSH) and oxidized glutathione (GSSG), which are abundant in GSH exists and plays a major role, which is widely used in the treatment of liver diseases, tumors, oxygen poisoning, aging and endocrine diseases, and is used in the food field as a biologically active additive and antioxidant. [0003] GSH is a tripeptide formed by condensation of glutamic acid (Glu), cysteine ​​(Cys) and glycine (Gly) through peptide bonds. The relative molecular mass is 307.32, the isoelectric point is 5.93, and it is a white crystal at room temperature, easily soluble in wate...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/00
CPCC12N9/93
Inventor 于铁妹刘珊珊秦永发
Owner ANHUI GSH BIO TECH CO LTD
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