Enzymic method for simultaneously preparing glutathione and adenylate

A technology of glutathione and adenosine, which is applied in the direction of peptides and fermentation, can solve the problems of adenosine triphosphate consumption, high cost, and increased difficulty in purification, and achieve the effects of saving reaction time, easy separation, and fast reaction rate

Active Publication Date: 2016-05-25
BEIJING TIANKAI YIDA BIOLOGICAL SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] The biggest problem of enzymatic synthesis of GSH is the large consumption of adenosine triphosphate (ATP). At least 3-5kg of ATP is needed to produce 1kg of GSH, which is too expensive
However, the use of yeast will introduce impurities such as pigments into the reaction system, which will increase the difficulty of further purification. The use of enzymes to regenerate ATP is a research direction in recent years.

Method used

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  • Enzymic method for simultaneously preparing glutathione and adenylate
  • Enzymic method for simultaneously preparing glutathione and adenylate
  • Enzymic method for simultaneously preparing glutathione and adenylate

Examples

Experimental program
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Embodiment 1

[0051] The preparation of embodiment 1GshF enzyme

[0052] The GshF enzyme in the method of the present invention can be obtained commercially, or it can be artificially modified to have the same catalytic function.

[0053] The preparation process of GshF enzyme is as follows:

[0054] According to the gshF gene sequence (GenBank: NC_008532), a pair of amplification primers were designed and synthesized by Zhongmei Taihe Biotechnology Co., Ltd. The primer sequences are as follows:

[0055] GshF sense primer: 5'-CCATATGACATTAAACCAACTTCTTCAAAAACTG-3'; and

[0056] GshF antisense primer: 5'-CGAATTCTTAAGTTTGACCAGCCACTATTTC-3';

[0057] Extract the DNA of Streptococcus thermophilus (CGMCC1.6472) strain (CGMCC1.6472), use it as a template, amplify the gshF gene fragment by PCR, and connect it to the pET22b vector (purchased from Invitrogen Company) respectively, after the sequence is correct, They were transferred into E.coliBL21 (DE3) strain (purchased from Tiangen Biochemical ...

Embodiment 2

[0064] The preparation of embodiment 2Adk enzyme

[0065] The Adk enzyme in the method of the present invention can be obtained commercially, or it can be artificially modified to have the same catalytic function.

[0066] The preparation process of Adk enzyme is as follows:

[0067] According to the adk gene sequence (GenBank: NC_000913), a pair of amplification primers were designed and synthesized by Zhongmei Taihe Biotechnology Co., Ltd. The primer sequences are as follows:

[0068] ADK sense primer: 5'-CCATATGCGTATCATTCTGCTTGGCGCTCCGG-3'; and

[0069] ADK antisense primer: 5'-CGGATCCTTAGCCGAGGATTTTTTTCCAGATC-3';

[0070] Extract the DNA of Escherichia coli (Escherichiacoli) K12 strain (purchased from Tiangen Biochemical Technology Co., Ltd.), and use it as a template to amplify the adk gene fragment by PCR, and connect it to the pET22b vector (purchased from Invitrogen Company). The sequence is sequenced After being correct, transfer to E.coliBL21 (DE3) strain (purchas...

Embodiment 3

[0077] Embodiment 3 uses free enzyme to prepare GSH and AMP

[0078] image 3 Process flow diagram for the preparation of GSH using free enzymes for the process of the present invention. see image 3 , prepare GSH according to the process flow chart of the present invention and use free enzyme to prepare GSH and AMP according to the following steps:

[0079] (1) Generate GSH and AMP in the reaction tank:

[0080] In the reaction tank, the reaction system of 100L sterile water contains substrates 2.8kg glutamic acid, 1.8kg cysteine ​​and 2.0kg glycine, and 5.1kgATP, 1.2kg disodium hydrogen phosphate, 0.7kg potassium chloride, The solution of 0.6kg sodium chloride, 0.1kg ammonium chloride and 1.0kg magnesium chloride hexahydrate should be uniformly stirred during preparation to prevent precipitation. Adjust the pH value to about 7.0, add 0.01kg GshF enzyme and 0.001kg Adk enzyme to the reaction system to start the reaction. During the reaction, the pH value was controlled t...

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Abstract

The invention discloses an enzymic method for simultaneously preparing glutathione and adenylate. The enzymic method comprises the following steps: (1) generating glutathione and adenylate from GshF enzyme and Adk enzyme in a reaction tank; (2) separating the immobilized GshF enzyme and Adk enzyme in the reaction tank, or separating the free GshF enzyme and Adk enzyme by virtue of filtering equipment; and (3) separating products GSH and AMP. By virtue of the preparation method, the reaction condition in production of the GSH is optimized, the generation concentration of GSH reaches 30g/L-50g/L, and the utilization rate of amino acid is relatively high; meanwhile, by carrying out partial regeneration on ATP consumed in reaction by virtue of the Adk enzyme, the consumption of the ATP is reduced; and furthermore, the GSH and the AMP can be simultaneously prepared by virtue of the preparation method.

Description

technical field [0001] The invention relates to a preparation method of glutathione, in particular to a method for simultaneously preparing glutathione and adenylic acid by enzymatic method. 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 water, low-concentration ethanol...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/02C12P19/32
CPCC07K5/0215C12P19/32
Inventor 刘珊珊于铁妹黄庆军秦永发
Owner BEIJING TIANKAI YIDA BIOLOGICAL SCI & TECH
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