Method for preparing glutathione with enzymatic method

A technology for glutathione and enzymatic preparation, which is applied in the preparation methods of peptides, chemical instruments and methods, peptides, etc., to achieve the effects of simple operation, low price and low cost

Active Publication Date: 2019-01-04
ANHUI GSH BIO TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method still needs to add a certain amount of ATP to carry out the enzymatic reaction

Method used

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  • Method for preparing glutathione with enzymatic method
  • Method for preparing glutathione with enzymatic method
  • Method for preparing glutathione with enzymatic method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] The preparation of embodiment 1 crude enzyme

[0053] The GshF enzyme, ATP regenerating enzyme and AK enzyme in the method of the present invention can be obtained commercially, or are artificially modified enzymes having the same catalytic function.

[0054] The enzyme preparation process is as follows:

[0055] Primers were designed according to the gene sequences of GshF enzyme, PPK enzyme, ADK enzyme, PAP enzyme and AK enzyme, and the gene fragments were respectively amplified by PCR and connected to the pET22b vector (commercially available). Transformed into E.coliBL21(DE3) strain (commercially available).

[0056] Insert the transformed E.coli BL21(DE3) monoclonal into LB medium, culture to the logarithmic phase, add 1mM isopropyl-β-D-thiogalactopyranoside (IPTG) for induction, induce The cells were collected after 5 hours, and high-expression strains were screened by sodium dodecylsulfonate-polyacrylamide gel electrophoresis (SDS-PAGE).

[0057] Insert the sc...

Embodiment 2

[0061] Embodiment 2 uses free enzyme to prepare GSH

[0062] figure 2 Process flow diagram for the preparation of GSH using free enzymes for the process of the present invention. Such as figure 2 Shown:, prepare GSH according to the following steps using free enzyme according to the process flow diagram of the present invention's preparation GSH:

[0063] (1) Generate GSH in the reaction tank:

[0064] In the reaction tank, the 100L reaction system contains substrates 2.5kg glutamic acid, 2.5kg cysteine, 1.5kg glycine, 0.3kg adenosine, 2.5kg sodium hexametaphosphate, 0.2kg ammonium sulfate, 0.3kg Sodium chloride, 0.5kg magnesium chloride hexahydrate, 0.1kg manganese chloride monohydrate and 0.5kg disodium hydrogen phosphate solution should be uniformly stirred to prevent precipitation during preparation. Adjust the pH value to 7.0, add GshF enzyme 800U / L, PPK enzyme 500U / L, ADK enzyme 500U / L, PAP enzyme 200U / L and AK enzyme 600U / L to the reaction system to start the reac...

Embodiment 3

[0076] Embodiment 3 uses free enzyme to prepare GSH

[0077] Such as figure 2 As shown, the process flow diagram for preparing GSH according to the present invention uses free enzyme to prepare GSH according to the following steps:

[0078] (1) Generate GSH in the reaction tank:

[0079] In the reaction tank, the 100L reaction system contains substrates 2.0kg glutamic acid, 2.0kg cysteine ​​hydrochloride, 2.0kg glycine, 0.2kg adenosine, 1.5kg sodium hexametaphosphate, 0.2kg chloride The solution of ammonium, 0.2kg potassium chloride, 0.8kg magnesium chloride hexahydrate and 0.3kg Tris should be uniformly stirred to prevent precipitation during preparation. Adjust the pH value to 7.4, add GshF enzyme 1000U / L, PPK enzyme 300U / L, ADK enzyme 300U / L and AK enzyme 500U / L to the reaction system to start the reaction, and the added enzymes are all crude enzyme solutions. During the reaction, the pH value was controlled to be 7.4, and the temperature was 40°C.

[0080] After react...

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Abstract

The invention discloses a method for preparing glutathione with an enzymatic method. The method includes the following steps that (1), the glutathione is generated in a reactor through GshF enzymes, ATP regenerated enzymes and AK enzymes; (2), the fixed GshF enzymes, ATP regenerated enzymes and AK enzymes are separated in the reactor, or unbonded GshF enzymes are separated through filtering equipment; (3), filtrate obtained in the step (2) is separated, coarse GSH and a small amount of ATP, ADP and AMP are obtained; (4), the GSH obtained in the step (3) is concentrated, crystallized and dried,and finished GSH is prepared. The method includes the following steps that adenosine is used for replacing the ATP or the AMP, and a large amount of cost is reduced for industrial production; reaction conditions of GSH production are optimized, and the reaction rate is high; a stable enzyme recovery system is established, and energy saving and environmental protection are achieved; byproducts canbe directly used for a circulating reaction, or collected in a centralized mode to be used for producing the ATP, and the operation is simple.

Description

technical field [0001] The invention relates to a method for preparing glutathione, in particular to a method for preparing glutathione by an enzymatic method, specifically an enzymatic method for preparing glutathione by using adenosine instead of ATP. 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 as a biologically active additive and antioxidant in the fields of food and cosmetics. [0003] GSH is a tripeptide formed by condensation of glutamic acid (Glu), cysteine ​​(Cys) and glycine (Gly) through peptide bonds. The relative molecular weight is 307.32, the isoelectric point is 5.93, and it is a wh...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K5/037C12P21/02C07K1/34C07K1/18C07K1/30
CPCC12P21/02C07K5/0215A61K38/06C07K1/306
Inventor 刘珊珊刘辉周稳文秦永发
Owner ANHUI GSH BIO TECH CO LTD
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