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Chemical enzymatic process of synthesizing glutathion

A glutathione, chemical enzymatic technology, applied in the direction of transferase, fermentation, etc., can solve the problems of side reactions, low substrate concentration, affecting enzyme utilization, etc. The effect of efficiency

Inactive Publication Date: 2006-05-17
NANJING UNIV OF TECH
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  • Abstract
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Problems solved by technology

[0007] But the substrate that this method adopts is the precursor of L-cysteinyl L-glycine or group protection and L-glutamic acid or L-glutamine, and the water solubility of these substrates is not high, causes substrate concentration If it is too low, it will affect the utilization of enzymes. The enzymes used are also crude enzymes. During the enzyme conversion reaction process, some impurities in the crude enzymes will affect the reaction and cause side reactions, so that the purity of the final product is not high. Use crude enzymes Enzyme reaction, its conversion efficiency is not high, will cause the waste of reaction substrate, and some impurities will affect the separation of enzyme reaction products, thereby affecting the yield; Further, impurities are added in the reaction process, which limits the popularization and use of this method

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Examples

Experimental program
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Effect test

Embodiment 1

[0030] Take 20g of sucrose, 30g of corn steep liquor, 10g of peptone, K 2 HPO 4 15g, MgSO 4 0.5g, pH 7.5, add tap water to prepare 1000mL liquid culture medium, prepare 3.5L according to this formula, put it into a 5L fermenter and sterilize it by high-pressure steam at 0.1MPa for 25 minutes for later use. At the same time, divide 50mL of medium into 500mL, add 8 layers of gauze, wrap it in kraft paper, and sterilize it together with the fermenter for later use.

[0031]After taking out the bacterial species preserved in the refrigerator—Bacillus subtillis NX-2 (preservation number is CGMCCNo.0833), it was connected to fresh slant medium (slant medium (g / L): peptone 10, beef extract 3 , NaCl5, agar 20), activated culture for 24 hours, access to seed medium (seed medium (g / L): glucose 20, corn steep liquor 10, peptone 5, K 2 HPO 4 2, MgSO4 0.25) at 32.5°C, 220rpm shaking culture for 20 hours. At the end of the cultivation, connect it to a fermenter, add Sixin organic si...

Embodiment 2

[0040] The preparation and purification methods of γ-glutamyl transpeptidase and the preparation method of the substrate S-benzyl-cysteinylglycine dipeptide are the same as in Example 1.

[0041] Dissolve 0.134g of S-benzyl-cysteinylglycine dipeptide in a 50mL Erlenmeyer flask, add 0.09g of L-glutamine, 10mL of deionized water, adjust the pH to 11.0 with 5mol / L NaOH, and add 1.0U of γ-glutamyl transpeptidase, after mixing evenly, stir and react for 10 hours, and the reaction temperature is 39°C. After the reaction, the reaction solution was micro-filtered to remove the enzyme, and the silica gel thin-layer preparative chromatography, the mobile phase was n-butanol-glacial acetic acid-water (4:1:1, v / v / v), to obtain S-benzyl-glucose Satithione 0.095g, the yield is 48%, and the purity is 95%. Prepare S-benzyl-glutathione in the same way for later use.

[0042] Put 2.0g of S-benzyl-glutathione in the newly prepared Na / liquid ammonia solution, shake slightly until the solution n...

Embodiment 3

[0044] The preparation and purification methods of γ-glutamyl transpeptidase and the preparation method of the substrate S-benzyl-cysteinylglycine dipeptide are the same as in Example 1.

[0045] Dissolve 0.2g of S-benzyl-cysteinylglycine dipeptide in a 50mL Erlenmeyer flask, 0.08g of L-glutamine, 10mL of deionized water, adjust the pH to 10.5 with 5mol / L NaOH, and add 1.0U of γ-glutamyl transpeptidase, after mixing evenly, stir and react for 10 hours, and the reaction temperature is 38°C. After the reaction, the reaction solution was micro-filtered to remove the enzyme, and the silica gel thin-layer preparative chromatography, the mobile phase was n-butanol-glacial acetic acid-water (4:1:1, v / v / v), to obtain S-benzyl-glucose Satithione 0.16g, the yield is 55%, and the purity is 96%. Prepare S-benzyl-glutathione in the same way for later use.

[0046] Put 2.5g of S-benzyl-glutathione in the newly prepared Na / liquid ammonia solution, shake slightly until the solution no longe...

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Abstract

The present invention discloses chemical enzymatic process of synthesizing glutathion. The chemical enzymatic process includes the following steps: A. synthesizing substrate chemically; B. preparing gamma-glutamyl transpeptidase; and C. adding gamma-glutamyl transpeptidase into the substrate solution to produce transpeptidase reaction preparing glutathion. The said substrate is S-benzyl-cysteinyl glycine and L-glutamine. The process is simple, low in cost and high in glutathion converting rate, and may be used in industrial production.

Description

technical field [0001] The invention relates to a method for synthesizing glutathione, in particular to a method for synthesizing glutathione by chemical enzymatic method using gamma-glutamyl transpeptidase. Background technique [0002] Reduced glutathione is an active tripeptide with important physiological functions. It is formed by condensation of glutamic acid, cysteine ​​and glycine through peptide bonds. It has a wide range of uses in the fields of medicine, food and health products. For example, in medicine, it can be used to detoxify and protect the liver, treat cataract and other diseases, and in food, it can strengthen nutrition, enhance the flavor of food and prevent browning of food. [0003] At present, the production methods of glutathione mainly include extraction method, chemical synthesis method, fermentation method and enzymatic method. Extraction method prepares glutathione, its raw materials are limited, growth route is long and the product purity is no...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/02C12N9/10
Inventor 范伟平欧阳平凯荀志金徐虹
Owner NANJING UNIV OF TECH
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