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Method for synthesis of glutathione in vitro

A technology of glutathione and in vitro synthesis, applied in fermentation and other directions, can solve the problems of insufficient stability of acetyl phosphate, unfavorable industrial amplification, mass transfer obstacles, etc., and achieve the effects of eliminating mass transfer obstacles, reducing costs, and improving conversion rate.

Active Publication Date: 2014-02-05
BEIJING UNIV OF CHEM TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The disadvantage is that the acetyl phosphate used in the acetyl phosphate / acetate kinase ATP regeneration system is not stable enough and the price is expensive, which is not conducive to industrial scale-up
[0013] In summary, the problems faced by microbial enzymatic production of glutathione mainly include mass transfer obstacles caused by cell walls or biofilms and continuous and effective ATP energy supply system

Method used

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  • Method for synthesis of glutathione in vitro
  • Method for synthesis of glutathione in vitro
  • Method for synthesis of glutathione in vitro

Examples

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preparation example Construction

[0025] The preparation methods of the three enzymes adopt techniques well known to those skilled in the art; for example, Escherichia coli or Saccharomyces cerevisiae with high glutathione synthesis ability are used as host bacteria to construct genetically engineered recombinant bacteria to express and prepare glutathione. Three enzymes for peptide synthesis reactions. In the embodiment of the present invention, the gshA, gshB and ppk genes encoding γ-glutamylcysteine ​​synthetase, glutathione synthetase and polyphosphate kinase were respectively cloned using E.coli BL21 (D3) as the starting bacterium , and after homologous expression in E.coli BL21(D3) through the pET-28a vector, the crude enzyme solution was extracted by sonication. The amount of the three enzymes added in the synthesis reaction was calibrated by Coomassie brilliant blue protein detection method.

[0026] Second, the detection method of glutathione concentration:

[0027] The content of intracellular glut...

Embodiment 1

[0031] Prepare 5mM reaction substrate solution, including: 0.1M Tris-Cl, 5mM cysteine, 7.5mM glycine, 7.5mM glutamic acid, 10mM ADPNa 2 , 100mM (NaPO 3 )n, 20mM MgCl 2 ;Add 92.6mg / L of γ-glutamylcysteine ​​synthetase and 94.8mg / L of glutathione synthase, and 93.2mg / L of polyphosphokinase, react at 37°C for 3h, 5h, 7h Take 0.5mL samples and analyze the content of glutathione with the alloxan method at 9 hours, the results are shown in figure 1 .

Embodiment 2

[0033] The rest of the conditions for synthesizing glutathione are the same as in Example 1; only the amount of polyphosphokinase added is 186.4 mg / L. See the test results figure 1 .

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Abstract

The invention relates to a method for synthesis of glutathione in vitro, comprising the step of: generating ATP (adenosine triphosphate) from ADP (adenosine diphosphate) and polyphosphoric acid compound under the catalysis of polyphosphate kinase; under the catalysis of gamma-glutamylcysteine synthetase and glutathione synthetase, synthesizing glutathione in vitro by glutamic acid, cysteine and glycine with the energy provided by the ATP. Compared with the prior art, the method of the invention can bring enzymes into complete contact with a substrate so as to improve the mass transfer efficiency and the utilization rate of enzymes. And the reaction system of the method is simple and strong in controllability. In addition, the method can solve the problem of great investment of ATP during production and reduce the production cost.

Description

technical field [0001] The invention relates to a method for synthesizing glutathione in vitro, in particular to a method for enzymatically synthesizing glutathione. Background technique [0002] Glutathione (GSH), as a biologically active tripeptide compound, has a variety of important physiological functions, and is crucial for maintaining a suitable redox environment in organisms. At present, the industrial application value of glutathione lies in its application in the food field as a bioactive additive, and in the field of medicine and health care with its anti-oxidation, anti-radiation, anti-aging and other functions. There is a growing trend of commercial demand for it at home and abroad. [0003] At present, the production methods of glutathione mainly include extraction method, chemical synthesis method, fermentation method and enzymatic method. The preparation of glutathione by the extraction method is backward and the yield is low; the chemical synthesis method h...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P21/02
Inventor 谭天伟李璐陈阳
Owner BEIJING UNIV OF CHEM TECH
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