GGT Enzymatic Synthesis Of ?-glutamylcysteine

Gamma-Glutamine(y-l-glutamyl-l-cysteine,GGC)has a gamma-glutamine acyl and a live wave of sulfhydryl,which is condensed from L-glutamic acid and L-cysteine by peptide bond.GGC is an intermediate for the synthesis of glutathione,which is generally used as a substrate in vivo for the synthesis of glutathione by glutathione synthase(GSH ?).Studies have shown that direct supplementation of glutathione or cysteine prodrugs was not effective on increasing intracellular GSH concentrations.GGC can be directly absorbed by many cells and can effectively increase intracellular GSH concentration.Therefore,GGC has important application value in the fields of food,health care,cosmetics and medical treatment,and it has practical significance to study the industrialized production of GGC.Gamma-glutamine transferase(?-glutamyltransferase,GGT)is the key enzyme in the glutamine cycle in the organism,which can transfer gamma-glutamine from donor to receptor and form a new compound containing gamma-glutamine,which has been widely used in the production of glutamine-acyl products.For the excellent catalytic properties of GGT,in this paper,the GGT from Escherichia coli K-12 was used as a catalyst to synthesize Bis-?-glutamylcysteine using L-glutamine(L-Gln)and cysteine(Cys-Cys)as substrates,and get the final product GGC after dithiothreitol(DTT)reduction.The main content are as follows:(1)Finding the GGT gene sequence of Escherichia coli K-12 in NCBI,and designing primers to construct expression vectors.After SDS-PAGE electrophoresis analysis,the molecular weight of the recombinant GGT was about 59 kDa,the molecular weight of large subunit was about 39 kDa,and the molecular weight of the small subunit was about 20 kDa.(2)Analyzing the basic properties of recombinant GGT,the optimum temperature was 40 ?,the optimum pH was around 10.In the process of synthesis of the GGC,the affinity parameter of GGT to glutamine was Km=4.005mmol/L,Vmax=0.067 mmol/(min-L).(3)Preliminary study on the process of GGT enzymatic synthesis of GGC indicated that the maximal production of GGC would be 6.13g/L and the conversion rate of L-glutamine would be 24.5%,when the initial reaction conditions of 0.1 mol/L of L-glutamine,0.2 mol/L of cystine and 0.024 U/mL of GGT,and the reaction is carried out at pH 10 and 40? for 12 hours.Increasing the donor concentration can increase the yield of the product,but it will reduce the conversion rate of the donor.Increasing the enzyme concentration in the reaction system will only increase the rate of product synthesis and shorten the fermentation time,but there is no significant effect on the conversion rate of the product.It is helpful to restrain autotranspeptidation and hydrolysis by batch supplement Gln,which can improve the conversion rate of the product.(4)Identifying the product,the fermentation product was analyzed as 67%by HPLC after preliminary separation and purification,and it was determined by LC-MS.