Enzymatic Production Of Glutathione, Theanine Through The Transpeptidation Reaction Of γ-glutamyltranspeptidase From Escherichia Coli K-12

Glutathione is a necessary material which 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 in 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 research 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.Theanine,which was first separated from green tea in 1950,is not only the main free amino acid component of tea,but also the major umami component of tea.It possesses high physiological and medical benefits.The preparation of theanine is a research hot point.It is very difficult and more expensive to extract a high purity theanine directly from tea leaves.The technics of chemosynthesis theanine is complicated and the yield of theanine by tissue culture is low.So,the biosynthesis of theanine is getting more and more attention.γ-glutamyltranspeptidase(GGT,EC 2.3.2.2)catalyzes not only the hydrolysis of theγ-glutamyl linkages ofγ-glutamyl compounds,but also the transfer of theirγ-glutamyl moieties to other amino acids or peptides.In this paper,we report an effective enzymatic method for synthesizing theanine and glutathione involving Escherichia coliγ-GGT.The E.coliγ-GGT encoding fragment was correctly inserted into the prokaryotic expression vector pET28a(+),pET32a(+)and pGEX-4T-1,while with lactose as inducer,E.coli BL21(DE3)harboring pGEX-4T- 1/γ-ggt can express the best solubility and specific activity ofγ-glutamyl transpeptidase protein.Over 80～90 g/L theanine was successfully achieved and glutathione synthesis needs to further study. It has been reported that Hsp72 inhibits both the stress-induced activation of ASK1 and ASKl-involved apoptosis.To better understand the mechanism by which Hsp72 modulates stress-activated sig-naling,we construct the eukaryotic expression vector for human induced heat shock protein 70(HSPT0)gene and investigate the possible effects of Hsp72 on the protein or mRNA level of ASK1.The 90kDa Heat Shock Protein(HSPg0)is one of the most abundant cytosolic proteins in eukaryotes.It normally functions as a molecular chaperone to participate in folding of newly synthesized proteins,refolding of denatured proteins after stress and regulating of client proteins' stability and activity.Up to now,over 100 HSP90 client proteins have been found,which mainly include transcription factors,protein kinases,polymerases and so on.Transforming growth factor(TGF)-β-activated kinasel(TAK1),which was originally identified as a member of MAP kinase kinase kinase(MAPKKK)family mediating TGF-βsignaling,has been shown to be involved in the IL-1 -induced signal pathway.We have now demonstrated that HSP90 physically associates with TAK1, thereby affects the proteasome/ubiquitin and protein stability of this kinase induced by IL-1.Furthermore,we construct HSP90 and TAK1 truncation mutants,including HSP90(1-232aa),HSP90(233-732aa),HSP90(402-732aa),TAK1(1-299aa),and TAK1(301-579aa),and also identified that the N-terminal domain(amino acids 1-401)of HsPg0 is required for interaction of HSP90 and TAK1.