Improving pH stability and catalytic activity of γ-glutamyltranspeptidase by site-directed mutation

γ-Glutamyltranspeptidase (GGT) is a key enzyme in glutathione metabolism that can synthesize a series of γ-glutamyl compounds. However, a variety of environmental conditions (especially pH>9.0) can restrain GGT and hence the application of GGT in biocatalysis. The 3D structure of γ-glutayltranspeptidase from B. subtilis NX-2 (wt_GGT) was predicted by a homologous model in this study. In order to improve hydrophobic interaction between two subunits of wt_GGT and enhance GGT pH tolerance, residues Asp46, Thr201, Tyr280, Gln322, Glu502 and His543 were chosen as the candidates, which were substituted with valine by site-directed mutagenesis after analyzing the spatial position, conservation and properties of those residues on the surface of wt_GGT. Six mutations were successfully expressed in E. coli and the catalytic properties and stabilities of the obtained mutations were investigated. The results show that substitution of Asp46, Tyr280, Glu502 or His543 with Val result in a slight decrease of K_m compared with that of wt_GGT. The k_cat of mutation Y280 is significantly increased. Moreover, the pH stability of mutations D46V, Y280V and E502V are all improved and Y280V is the best. The 3D models of D46V, E502V and Y280V show that V-280 could form a stronger hydrophobic interaction with four hydrophobic residues (Y-283, W-277, I-501 and I-497) within the scope of 10 Å, which may be the main reason for the better tolerance of Y280V under higher pH values.