Method for preparing glutathione through enzyme method

A technology for glutathione and enzymatic preparation, applied in fermentation and other fields, can solve the problems of unsolved enzyme activity stability, affecting production speed, and unable to solve mutual inhibition and other problems

Active Publication Date: 2014-07-16
ANHUI GSH BIO TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But this method has the following problems, which directly limits the industrial application of this technology:
[0007] 1) The two enzymes GSH I and GSH II are immobilized together, which cannot solve the mutual inhibition between the two-step reactions, which affects the production speed;
[0008] 2) Immobilized enzyme technology does not solve the stability problem of enzyme activity, which increases the cost of enzyme production; and
[0009] 3) The biggest problem of enzymatic synthesis is the source of ATP. Due to the high price of ATP, the cost of enzymatic synthesis of GSH is much higher than that of traditional fermentation methods

Method used

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  • Method for preparing glutathione through enzyme method
  • Method for preparing glutathione through enzyme method
  • Method for preparing glutathione through enzyme method

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Experimental program
Comparison scheme
Effect test

Embodiment 1G

[0108] The preparation of embodiment 1GSH I enzyme and GSH II enzyme

[0109] The GSH I enzyme and GSH II enzyme in the method of the present invention can be obtained commercially, or are artificially modified enzymes having the same catalytic function.

[0110] The preparation process of GSH I enzyme and GSH II enzyme is as follows:

[0111] According to the gsh I and gsh II gene sequences (GenBank: X03954.1 and X01666), two pairs of amplification primers were designed and synthesized by Zhongmei Taihe Biotechnology Co., Ltd. The primer sequences are as follows:

[0112] gshI sense primer: 5'-C CCATGG TCCCGGACGTATCACAGGCGCTG-3';

[0113] gshI antisense primer: 5'-C GGATCC TCAGGCGTGTTTTTCCAGCCACAC-3';

[0114] gsh II sense primer: 5'-C CCATGG TCAAGCTCGGCATCGTGATGG-3';

[0115] gsh II antisense primer: 5'-C GGATCC TTACTGCTGCTGTAAACGTGC-3'.

[0116] The DNA of Escherichia coli (Escherichia coli) K12 strain (purchased from Tiangen Biochemical Technology Co., Ltd.) wa...

Embodiment 2

[0121] The determination of embodiment 2 reaction time

[0122] see figure 1 , prepare the process flow sheet of GSH according to the present invention and carry out following reaction:

[0123] (1) Generate γ-glutamylcysteine ​​(γ-GluCys) in reaction tank A:

[0124] In reaction tank A, the reaction system 1 of 100L sterile water is the solution that contains substrate 600g glutamic acid and 400g cysteine, and 600g Tris, 1100g potassium chloride, 870g sodium chloride and 800g magnesium chloride, in reaction Add 5 g of GSH I enzyme to system 1, add potassium hydroxide to adjust the pH value to 8.5, and add ATP to start the reaction. During the reaction, the pH was controlled to be constant at 8.5, and the temperature was 40°C.

[0125] Use high-performance liquid chromatography (HPLC) to detect the production of γ-glutamylcysteine ​​at 0, 1, 2, 3, 4, and 5 hours of the reaction, see image 3 , Figure 4 and Figure 5 . image 3 It is the HPLC collection of illustrative ...

Embodiment 3

[0133] The preparation of embodiment 3 glutathione

[0134] see figure 1 , prepare glutathione according to the following steps according to the process flow chart of the present invention for preparing GSH:

[0135] (1) Generate γ-glutamylcysteine ​​(γ-GluCys) in reaction tank A:

[0136] In reaction tank A, the reaction system 1 of 100L sterile water is the solution that contains substrate 600g glutamic acid and 400g cysteine, and 600g Tris, 1100g potassium chloride, 870g sodium chloride and 800g magnesium chloride, in reaction Add 5 g of GSH I enzyme to system 1, add potassium hydroxide to adjust the pH value to 8.5, and add ATP to start the reaction. During the reaction, the pH was controlled to be constant at 8.5, and the temperature was 40°C. After 3 hours, the amount of ATP used was about 1200g.

[0137] High-performance liquid chromatography (HPLC) detects that the production of γ-glutamylcysteine ​​is about 6.6g / L, and more than 90% of ATP is converted into ADP (A...

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Abstract

The present invention provides a method for preparing glutathione (GSH) through an enzyme method. According to the method, two steps of reactions for GSH synthesis are respectively performed in different reaction tanks, and enzymes used in each reaction are separated after each reaction is performed, such that enzyme activities of GSH I and GSH II are utilized to the maximal degree, and mutual inhibition between the two enzymatic reactions are reduced, wherein the two steps of the reactions comprise a gamma-glutamylcysteine generation reaction ??and a GSH generation reaction, and the enzymes used in each reaction comprise gamma-glutamyl cysteine synthetase (GSH-I) and glutathione synthetase (GSH-II). With the method, recycling of GSH I and GSH II is achieved, an ATP regeneration system required by the GSH preparation reaction is adopted, production cost for GSH preparation is reduced, GSH yield is improved, large-scale continuous production is achieved, and economic benefits are significant.

Description

technical field [0001] The invention relates to a method for preparing glutathione, in particular to a method for enzymatically preparing glutathione. Background technique [0002] Glutathione is widely found 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 formed by glutamic acid (Glu), cysteine ​​(Cys) and glycine (Gly) through peptide bonds, and there is a special γ-peptide bond in the molecule, that is, γ-COOH of glutamic acid and cysteine The peptide bond formed by the condensation of α-NH2 is different from the ordinary peptide bond in the protein molecule. GSH is a white...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P21/02
Inventor 刘珊珊秦永发
Owner ANHUI GSH BIO TECH CO LTD
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