Purification And Enzymatic Properties Of ?-glutamyl-transpeptidase From Toona Sinensis Leaves

Toona sinensis(TS),which originates in China,is a well-known deciduous tree belonging to the family of Meliaceae.Its shoot and young leaves have a unique and appealing aroma,and have been consumed as seasonal top vegetables for more than 2000 years.The unique aroma and special flavor are the most important quality characteristics of TS,however,the formation mechanism of aroma is not clear up to now,which is a limiting step to breed fine variety and develop keeping flavor technique.Volatile sufur compounds were found to play a key role on the emergence of TS unique aroma in our recent research.A flavor-related enzyme,?-glutamyl-transpeptidase(GGT),is an important member molecular in metabolic pathway.Through ammonium sulphate precipitation,dialysis and gel filtration chromatography,GGT with electrophoresis grade were obtained,its enzymatic properties were studied for the first time.The main results and conclusion are as follows:GGT were purified 40.67-fold with 13.40%recovery using ammonium sulphate precipitation,dialysis and gel filtration chromatography from lyophilized powder of TS.Gel filtration chromatography and SDS-PAGE revealed that GGT is a dimer protein with molecular weight of 90kDa,and the molecular size of two subunits is 26 kDa and 64 kDa,respectively.The apparent Km and Vmax of GGT were 3.623?mol/mL and 2.831?mol/mL·min of protein,respectively,and the optimum reaction temperature was 60?,while the optimum pH point was 7.0.The effect of metal ions on GGT catalysis activity showed that Cu2+ and Zn2+ had strong inhibition,Na+,Ca2+ and Mg2+ had only a slight inhibition,while Fe3+ had strong activation,K+ and Mn2+ had only a slight activation.LC-MS analysis indicated that the purified GGT catalyzed?-glutamyl-(cis-S-1-propenyl)-thioglycine,a proposed flavor precursor of TS,to release glutamic acid moiety.