Characterization of a novel bifunctional glutathione synthetase derived from Enterococcus italicus

Glutathione is a tripeptide containing a -amide bond and a sulfhydryl group, which is composed of glutamic acid, cysteine, and glycine.It possesses various important biological functions such as antioxidation.Its biosynthesis initially occurs through the action of glutamate cysteine ligase, which catalyzes the linkage of glutamic acid and cysteine to form -glutamylcysteine, and then glutathione synthetase catalyzes the addition of glycine to -glutamylcysteine to form -glutamylcysteine glycine.In this study, a strain with high glutathione production was isolated and identified as Enterococcus italicus through 16S rRNA gene sequencing.The 1600 bp bifunctional glutathione synthetase encoding gene gshF was obtained by PCR and heterogeneously expressed in Escherichia coli BL21(DE3) using a pET-22b(+) vector.After purification by Ni column, the molecular weight of bifunctional glutathione synthetase GshF was determined to be 60 kDa.Enzymatic analysis showed that the optimum reaction temperature of GshF was 37 C and the optimum pH was 8.0.Moreover, 30 mmol/l Mg 2+ significantly enhanced enzyme activity.These results provided a basis for understanding the mechanism of high glutathione production in E. italicus and offered a new enzyme source for glutathione biosynthesis.