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Method for preparing glutathione using hydantoinase

A glutathione and hydantoinase technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problems of easy hydrolysis, autorotation peptide, low yield of GSH, high production cost, etc., to achieve Avoid the use of energy ATP, avoid protection and deprotection steps, ensure site-specific effects

Inactive Publication Date: 2011-08-31
NANJING TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The extraction method is a classic method for the production of GSH. It mainly uses GSH-rich animal and plant tissues and yeast as raw materials, and then separates and refines them by adding appropriate solvents or combining amylase and protease treatment. However, this method has a high yield of GSH. Low cost, high cost, serious organic solvent pollution, low purity and many other problems, so it is rarely used
Fermentation production is to increase the yield of the product by selecting (constructing) microorganisms with strong GSH synthesis ability and high intracellular product content, screening and optimizing the culture formula, establishing and optimizing the fermentation control strategy, improving and improving the downstream process technology, etc. and quality, the main problems of the fermentation method are low conversion efficiency, long fermentation cycle, too low GSH content requires complex separation process, large equipment investment, high production cost, and relatively low product purity and yield, which limits the production of this product. further extension of the law
The chemical synthesis method usually needs to protect the α-amino group and α-carboxyl group of L-glutamic acid step by step, and only the γ-carboxyl group is freed for peptide grafting reaction. After the peptide grafting is completed, the α- The protecting group of the amino group and α-carboxyl group is removed, so that in the repeated protection and deprotection process, it is easy to cause peptide bond breakage, racemization, etc., resulting in a decrease in yield and optical purity
There are two kinds of enzymes that can be used to synthesize GSH: GSH synthetase (GSH I and GSH II) and γ-glutamyl transpeptidase (GGT), but the former needs to consume energy ATP while performing catalytic conversion to synthesize GSH , it is necessary to build an ATP regeneration system, and although the latter does not need to consume ATP, it is also prone to various side reactions such as hydrolysis and autorotation

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  • Method for preparing glutathione using hydantoinase
  • Method for preparing glutathione using hydantoinase
  • Method for preparing glutathione using hydantoinase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Synthesis of 5-carboxyethylhydantoin.

[0037] L-glutamic acid (0.1mol) and potassium cyanate (0.12mol) were dissolved in 50mL of water, with 0.5mol·L -1 Potassium hydroxide solution was used to adjust the pH to 9.0, and the reaction was stirred at 75°C for 2 hours to generate the intermediate product N-carbamoyl L-glutamic acid. Add 0.5mol·L -1 Adjust the pH of the solution to 1-2 with hydrochloric acid, raise the temperature to 100°C and heat to reflux for 0.5 h, cool, filter and dry to obtain a white solid, recrystallize with water, and obtain 13.7 g of white crystal 5-carboxyethylhydantoin, with a yield of 79.5%. m.p.179~181℃; 1 H NMR (DMSO-d 6 , 500MHz) δ: 12.14(s, 1H, COOH), 10.59(s, 1H, NH), 7.91(s, 1H, NH), 4.03~4.00(m, 1H, CH), 2.33~2.30(m, 2H , CH 2 ), 1.95~1.68 (m, 2H, CH 2 ); HRMS(ESI) calcd for C 6 h 8 N 2 NaO 4 [M+Na] + 195.0376, found 195.0374.

Embodiment 2

[0038] Example 2: Synthesis of 5-carboxyethylhydantoin.

[0039] Sodium monohydrogen L-glutamate (0.1mol) and sodium cyanate (0.14mol) were dissolved in 50mL of water, with 0.1mol·L -1 Sodium hydroxide solution was used to adjust the pH to 8.5, and the reaction was stirred at 80°C for 2 hours to generate the intermediate product N-carbamoyl-L-glutamic acid. Add concentrated hydrochloric acid to adjust the pH of the solution to 1-2, raise the temperature to 100°C and heat to reflux for 1.5 hours, cool, filter and dry to obtain a white solid, recrystallize with water to obtain 14.2 g of white crystal 5-carboxyethylhydantoin, the yield is 82.7 % (m.p.179-181°C).

Embodiment 3

[0040] Example 3: Synthesis of N-hydantoyl-S-benzyl-L-cysteine.

[0041] Add 200 mL of DMF to 5-carboxyethylhydantoin (0.1 mol) obtained in Example 1, stir and dissolve at room temperature, add DCC (0.1 mol) and HOBt (0.1 mol), react for 2 hours, filter to remove DCU (dicyclohexyl urea), Take the filtrate. Add S-benzyl-L-cysteine ​​(0.13 mol) and react overnight. Pour into water, add ethyl acetate to extract three times, the extract was dried and then concentrated to obtain a white solid, which was recrystallized from methanol to obtain 33.4 g of white crystal N-hydantoylpropionyl-S-benzyl-L-cysteine , yield 91.4%, m.p.81~83℃; 1 H NMR (DMSO-d 6 , 500MHz) δ: 12.80(s, 1H, COOH), 10.62(s, 1H, NH), 8.25(d, J=8.0Hz, 1H, NH), 7.89(s, 1H, NH), 7.32~7.24( m, 5H, PhH), 4.45~4.41(m, 1H, CH), 4.05~4.03(m, 1H, CH), 3.75(s, 2H, CH 2 ), 2.81~2.63 (m, 2H, CH 2 ), 2.31~2.17 (m, 2H, CH 2 ), 1.96~1.70 (m, 2H, CH 2 ); HRMS(ESI) calcd for C 16 h 19 N 3 NaO 5 S[M+Na] + 388.0938, found...

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Abstract

The invention discloses a method for preparing glutathione using a hydantoinase. The method comprises the following steps: adopting glutamic acid and cyanate as raw materials for preparing 5-carboxyethyl hydantoin; further adopting a peptide synthesis reagent for sequentially inoculating S-R group-L-cysteine and aminoacetic acid for preparing N-hydantoin propionyl-S-R group-L-cysteinyl-aminoacetic acid; then using wet bacteria containing L-hydantoinase and L-carbamoylase as a catalyst, and carrying out catalytic hydrolysis on the N-hydantoin propionyl-S-R group-L-cysteinyl-aminoacetic acid for preparing S-R group-glutathione; and finally removing S-R group for preparing the glutathione. A hydantoin ring is adopted in the method for co-protecting alpha-amino and alpha-carboxyl in glutamic acid, and only gamma-carboxyl is exposed, thereby ensuring the specificity of the position of the peptide synthesis reaction. The method can avoid the repeated protection and the de-protection steps in a plurality of chemical synthesis reactions, the operation is simple, and the side reactions are reduced.

Description

technical field [0001] The invention relates to a method for preparing glutathione by hydantoin enzymatic method. Background technique [0002] Glutathione, namely γ-L-glutamyl-L-cysteinyl-glycine (γ-L-glutanmy-L-cysteinyl-glycine, glutathione), is composed of L-glutamic acid, L-cysteine It is a biologically active tripeptide compound with γ-glutamyl and sulfhydryl groups formed by the condensation of amino acid and glycine through peptide bonds. As early as 1888, French scientist de Rey-Pailhade first discovered glutathione in the ethanol extract of baker's yeast and named it "Philothion". In 1921, British biochemist Hopkins isolated glutathione from yeast extract and officially named it "Glutathione". Thereafter, the chemical molecular structure of glutathione was finally determined after chemical analysis, acid-base titration, degradation and synthesis studies (Wieland T.Chemistry and Properties of Glutathiones Colowick S, Schwarz DR, Lazarow A, et al. al. Glutathione....

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P21/02C12R1/07
Inventor 曹飞周佳栋王月霞张芙蓉黄荣醒韦萍
Owner NANJING TECH UNIV