Study On The Application Of γ-Glutamyltranspeptidase In The Synthesis Of A Precusor Of Glutathione

Glutathione is a necessary material wihich maintains the environment of the body. It is the coenzyme of some enzyme in the metabolism. Its reduced thiocytidine makes the body bring about immunity by participating the important oxidoreduction reaction of the body. Glutathione has wide use in the medicine and it has transparent effect especially in the treatment of toxic disease and liver disease. So it is thought highly of by many researchers in the medical field in the world. But the preparation of glutathione is a very difficult problem in the rearch and it is a hot spot up to now.From the process of the production of glutathione, there are mainly extraction, chemical synthesis , microorganism zymotechnics and Chemical-enzymatic synthesis. In 1989, Hidehiko Kumagai utilyzed v -glutamyltranspeptidase from Escherichia coli K-12 for enzymatic production of S-benzyl-glutathione. Glutathione could be obtained from S-benzyl-glutathione by the deprotection of benzyl. This method developed a new way to synthesize glutathione.In this paper, using γ-glutamyltranspeptidase from Bacillus subtilis NX-2 as catalyst, the transpeptide reaction to obtain S-benzyl-giutathione was carried out with L-glutamine as γ-glutamyl donor and S-benzyl-cysteinylglycine as the substrate. This method could avoid the complicated disposal steps of radical protection and deprotection in the chemical synthesis, simplify the procedur, improve the yield of the reaction.First, the technic conditions of the synthesis of S-benzyl-cysteinylglycine methyl ester was optimized . Using S-benzyl-cysteine as raw material , S-benzyl-cysteinylglycine methyl ester can be obtained by protection of amidogen, reacting with glycine methyl ester and deprotection of amidogen. The result showed that the transformation efficiency of the three reactions could reach 84. 2%, 88. 9% and 91%.Second, the author fermented Bacillus subtilis NX-2 from the laboratory to obtain γ -glutamyltranspeptidase. After purification, some of the property of the γ-glutamyltranspeptidase was studied, including effect of pH on the stability, effect of pH on the activity, thermal stability, effect of temperature on the activity and effect of metal ions on the activity. The result was: Y -glutamyltranspeptidase is stable at pH7.0~ 10.0, its optimum reaction pH is pH 8.0, it has good stability below 40℃, its optimum reaction temperature is 40℃, potassium ion, sodium ion, zinc ion, calcium ion, magnesium ion and copper ion don't inhibit the activity of Y -glutamyltranspeptidase, ferrous ion, iron ion and mercury ion inhibit the activity of Y -glutamyltranspeptidase obviously.Finally, the paper studied the enzymatic transpeptide synthesis technique of S-benzyl-glutathione. There is no same report in our nation. Under the optimum pH and optimum temperature of y -glutamyltranspeptidase, effect of mole ratio of reactant on the yield of the enzymatic reaction was studied. After seperation and preparation by TLC and HPLC, the structure of the S-benzyl-glutathione was identified by analysis of MS and NMR.The result of the paper confirmed the feasibility of this method. But the yield of S-benzyl-glutathione was very low due to the purity of Y -glutamyltranspeptidase and the specificity of this enzyme. It is necessary to utilize genetic engineering technique to reform the strain and study the purification technique of Y -glutamyltranspeptidase to improve the productivity and purity of the product.