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Method for synthesizing glutathione by enzymatic catalysis

A technology of glutathione and synthetase, which is applied in the direction of peptides and fermentation, can solve the problems of reduced fluidity and activity, and achieve the effects of reduced feeding amount, short reaction time and obvious effect

Active Publication Date: 2016-11-09
KAIPING GENUINE BIOCHEM PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Generally, the immobilized enzyme itself is still soluble in water, but the enzyme is combined with a water-insoluble macromolecule carrier or embedded in it by physical or chemical methods, so that the fluidity of the enzyme is reduced.
Although the current immobilized enzyme has increased stability, is easy to separate from the reaction system, and is convenient for transportation and storage, but its activity decreases

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Follow the steps below to synthesize glutathione:

[0035] 1. Mix the γ-glutamylcysteine ​​synthetase solution and the glutathione synthetase solution at a ratio of 1:1 to obtain the mixed solution A, and then mix the mixed solution A with the acetate kinase solution by enzyme Mix protein at a ratio of 1:1 to obtain mixture B;

[0036] 2. Add the immobilized carrier to the mixture B, stir and immobilize, the conditions are: temperature 5°C, time 3h, filter to obtain immobilized enzyme; the amount of immobilized carrier is: add 1g of immobilized carrier for every 40mg of protein; immobilized The carrier was prepared according to the following method: Weighed 1 part by weight of chitosan and added it to 150 parts by weight of 1 wt% acetic acid solution, stirred and dissolved, then added 10 parts by weight of silica gel particles with a particle size of 0.1-0.5 mm, stirred for 12 hours, and added 100 parts by weight after filtering. Parts by weight of 0.5mol / L sodium hydr...

Embodiment 2

[0040] Follow the steps below to synthesize glutathione:

[0041] 1. Mix the γ-glutamylcysteine ​​synthetase solution and the glutathione synthetase solution at a ratio of 1:1 to obtain the mixed solution A, and then mix the mixed solution A with the acetate kinase solution by enzyme Mix protein at a ratio of 1:1 to obtain mixture B;

[0042]2. Add the immobilized carrier to the mixture B, stir and immobilize, the conditions are: temperature 5°C, time 3h, filter to obtain immobilized enzyme; the amount of immobilized carrier is: add 1g of immobilized carrier for every 40mg of protein; immobilized The carrier was prepared according to the following method: Weighed 1 part by weight of chitosan and added it to 200 parts by weight of 0.5 wt% acetic acid solution, stirred and dissolved, then added 20 parts by weight of silica gel particles with a particle size of 0.1-0.5 mm, stirred for 8 hours, filtered and added 120 parts by weight of 0.5mol / L sodium hydroxide solution soaked fo...

Embodiment 3

[0046] Follow the steps below to synthesize glutathione:

[0047] 1. Mix the γ-glutamylcysteine ​​synthetase solution and the glutathione synthetase solution at a ratio of 1:1 to obtain the mixed solution A, and then mix the mixed solution A with the acetate kinase solution by enzyme Mix protein at a ratio of 1:1 to get mixture B;

[0048] 2. Add the immobilized carrier to the mixture B, stir and immobilize, the conditions are: temperature 5°C, time 3h, filter to obtain immobilized enzyme; the amount of immobilized carrier is: add 1g of immobilized carrier for every 40mg of protein; immobilized The carrier was prepared according to the following method: Weighed 1 part by weight of chitosan and added it to 100 parts by weight of 2wt% acetic acid solution, stirred and dissolved, then added 5 parts by weight of silica gel particles with a particle size of 0.1-0.5 mm, stirred for 15 hours, and added 80 Parts by weight of 0.8mol / L sodium hydroxide solution soaked for 1h, so that c...

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Abstract

The present invention discloses a method for synthesizing glutathione by enzymatic catalysis, comprising the following steps: S1, mixing Upsilon-glutamylcysteine synthetase liquid and glutathione synthetase liquid to obtain mixed liquid A, mixing the mixed liquid A with acetate kinase to obtain mixed liquid B; S2, adding an immobilized carrier in the mixed liquid B, stirring for immobilizing, and filtering to obtain an immobilized enzyme; S3, blending glutamic acid, L-cysteine, glycine, magnesium sulfate, ATP (adenosine triphosphate) and acetyl phosphate into a reaction liquid, adding the immobilized enzyme, and stirring for reacting; S4, after reacting, filtering the reaction liquid, and extracting and refining filtrate to obtain the glutathione. The method of the invention has high synthetic efficiency, the production content of the reaction liquid is higher than 10 g / L, and substrate conversion rate reaches higher than 90%; by introducing acetate kinase to construct an ATP regeneration and coupling system, ATP charge is greatly reduced and production cost is significantly reduced; the immobilized enzyme is reusable and highly stable.

Description

technical field [0001] The invention relates to a method for preparing glutathione, in particular to a method for enzymatically synthesizing glutathione. Background technique [0002] Glutathione (GSH) is a biologically active tripeptide compound containing γ-2 glutamyl and thiol, which is condensed from L-glutamic acid, L-cysteine ​​and glycine. It plays a direct or indirect role in important biological phenomena such as the synthesis of amino acids, the transport of amino acids, and the protection of cells. It is mainly distributed in animals, plants, and microbial cells. It is one of the most important non-protein sulfhydryl compounds in organisms. It is widely used in the treatment of liver diseases, tumors, oxygen poisoning, aging, and endocrine diseases, and has become an important medical regulator. One of the drugs for human immune function and auxiliary anti-cancer. The antioxidant properties of GSH make its application in the food industry attract people's attent...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/02
CPCC07K5/0215C12P21/02
Inventor 刘桂祯黄炯威周华润莫世艺劳伟明梁剑锋
Owner KAIPING GENUINE BIOCHEM PHARMA
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