γ-Glutamyltranspeptidase from Bacillus amyloliquefaciens: transpeptidation activity enhancement and L-theanine production

L-theanine, a unique amino acid in green tea with health benefits, can be enzymatically synthesized by gamma-glutamyltranspeptidase (gamma-GT; EC 2.3.2.2). Here, a salt-tolerant gamma-glutamyltranspeptidase from a marine bacterium Bacillus amyloliquefaciens was expressed in Escherichia. coli BL21 (DE3) and was shown to be optimally active at 55 degrees C, pH 8.5 and alkali stable. A mutant, with higher transpeptidation activity, was obtained following two rounds of directed evolution using error-prone PCR and site-saturation mutagenesis. The mutation increased the ratio of transpeptidation to hydrolysis from 1.6 to 35.6. Additionally, Kinetic analysis exhibited 17.5% decrease of K-m, 13.0-fold increase of K-cat, and 16.3-fold increase of K-cat/K-m in mutant V319A/S437 G versus the wild-type. The 3-D modelling analysis revealed a tighter binding pocket in mutant V319A/S437 G. The frequency of hydrogen bond between donor substrate and two residues in the catalytic pocket (Gly437 and Thr375) was enhanced, which stabilized the ligand binding and thus improved the catalytic efficiency. The optimal conditions for the biocatalytic synthesis were determined as pH 10.0, 20 mu g mL(-1) BaGT, 200 mM L-glutamine, 2 M ethylamine, and a reaction time of 5 h. The V319A/S437 G mutant was shown to increase the percentage yield of L-theanine from 58% to 83%. These results indicate the great potential of V319A/S437 Gin L-theanine production after further study.