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Method for preparing glutathione by means of enzymic method

A technology for glutathione and enzymatic preparation, which is applied in fermentation and other directions, can solve the problems of increased difficulty in purification, and achieve the effects of saving reaction time, reducing production costs, and reducing ATP consumption

Active Publication Date: 2016-01-06
ANHUI GSH BIO TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

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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  • Method for preparing glutathione by means of enzymic method
  • Method for preparing glutathione by means of enzymic method
  • Method for preparing glutathione by means of enzymic method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] The preparation of embodiment 1GshF enzyme

[0061] 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.

[0062] The preparation process of GshF enzyme is as follows:

[0063] 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:

[0064] GshF sense primer: 5'-CCATATGACATTAAACCAACTTCTTCAAAAACTG-3'; and

[0065] GshF antisense primer: 5'-CGAATTCTTAAGTTTGACCAGCCACTATTTC-3';

[0066] 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

[0073] The preparation of embodiment 2Ppk, Adk and Pap enzyme

[0074] The Ppk, Adk and Pap enzymes in the method of the present invention can be obtained commercially, or are artificially modified enzymes having the same catalytic function.

[0075] The preparation process of Ppk, Adk and Pap enzyme is as follows:

[0076] According to the GshF gene sequence (GenBank: NC_008532), three pairs of amplification primers were designed and synthesized by Zhongmei Taihe Biotechnology Co., Ltd. The primer sequences are as follows:

[0077] ppk sense primer: 5'-CCATATGGGTCAGGAAAAGCTATACATCG-3';

[0078] ppk antisense primer: 5'-CGGATCCTTTATTCAGGTTGTTCGAGTGATT-3';

[0079] adk sense primer: 5'-CCATATGCGTATCATTCTGCTTGGCGCTCCGG-3';

[0080] ADK antisense primer: 5'-CGGATCCTTAGCCGAGGATTTTTTTCCAGATC-3';

[0081] PAP sense primer: 5'-GCCATGGATACAGAAACGATCGCCAGTGCAG-3'; and

[0082] pap antisense primer: 5'-CGGATCCTTAATCCGTGTCGCGATCCGCTT-3';

[0083] Extract the DNA of Escherichia coli...

Embodiment 3

[0090] Embodiment 3 uses free enzyme to prepare glutathione

[0091] 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 diagram of the present invention and use free enzyme to prepare glutathione according to the following steps:

[0092] (1) Generate glutathione GSH in the reaction tank:

[0093]In the reaction tank, the reaction system of 100L sterile water contains substrates 800g glutamic acid, 800g cysteine ​​and 600g glycine, as well as 2000g ATP, 600g disodium hydrogen phosphate, 1100g potassium chloride, 870g sodium chloride and 800g Magnesium chloride solution, potassium hydroxide is added to adjust the pH to about 7.0, and 100 g of GshF enzyme solution is added to the reaction system to start the reaction. During the reaction, the pH value is controlled to be 6.5-7.5, and the temperature is 35-38°C.

[0094] (2) Regenerate ATP in the reacti...

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Abstract

The invention discloses a method for preparing glutathione by means of an enzymic method. The method comprises the following steps that 1, Gsh F is utilized for generating glutathione in a reaction kettle; 2, regenerating enzyme is added into the reaction kettle, and ADP and / or AMP generated through a reaction is regenerated to be ATP; 3, the Gsh F and the regenerating enzyme which are fixed are separated in the reaction kettle or a filter device is used for separating dissociative Gsh F and the regenerating enzyme; 4, the product GSH and ATP, ADP and AMP in a reaction solution are separated. According to the preparation method, Gsh F enzyme is used for replacing Gsh I enzyme and Gsh II enzyme, a two-step reaction is changed into the one-step reaction, time is saved, and the feedback inhibition of the GSH for the two-step reaction is lowered; an ATP regenerating system for GSH production is established, the ATP consumption is lowered, and the cost is lowered; impurities such as pigment introduced by ATP regenerated by the yeast are avoided, and purification is easy; the enzyme recycling system is built, and large-scale production can be achieved.

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 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 aqueous solution, liquid ammonia and di...

Claims

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Application Information

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IPC IPC(8): C12P21/02
Inventor 刘珊珊秦永发
Owner ANHUI GSH BIO TECH CO LTD
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