Construction Of A Novel Genetically Engineered Strain Of E.coli With Extremely High Gamma-Glutamyltranspeptidase Activity And Use Of It For Efficient Production Of Theanine

Theanine(γ-glutamylethylamide)is the main unique amino acid component in green tea.It is a high economic value amino acid which has special flavour and multiple physiological beneficial effects.The demand for theanine continues to increase for the extensive application of it in the food and pharmaceutical industries.Gamma-glutamyltanspeptidase(GGT,EC 2.3.2.2)is considered to be one of the most efficient enzyme for industrial production of theanine,which can transfer the y-glutamyl of many y-glutamyl compounds to ethylamine.In this study,to obtain large amount bioactive GGT and decrease the cost of enzymatic synthesis of theanine,we co-expressed E.coli K-12 GGT and E.coli chaperone set in E.coli BL21(DE3)cells.A novel genetically engineered strain of E.coli(BL21/C4)with extremely high Gamma-Glutamyltranspeptidase activity was constructed.After coexpression,the activity of GGT in this recombinant E.coli cells reached 15500U/L which was approximately 380 fold higher than in the wild-type cells.Besides,to find a new breach of improving the reaction conversion rate,we firstly show that product theanine degraded by GGT while systhesis by GGT.Successfully we prohibited the degradation of product theanine by high molar ratio of ethylamine to theanine(20 to 1).Finally,0.127 M theanine was synthesised by whole cells of co-expressed BL21/C4 in the presence of glutamine(0.17 M)through stabilizing the ratio of ethylamine to theanine above 15 to 1 in the reaction.The conversion rate was up to 75.0%.