Secretion of Bacillus amyloliquefaciens γ-Glutamyltranspeptidase from Bacillus subtilis and Its Application in Enzymatic Synthesis of l-Theanine

In this study, the gene of γ-glutamyltranspeptidase (GGT) from Bacillus amyloliquefaciens (BaGGT) controlled by the P_lac promoter was cloned into Bacillus subtilis to construct two recombinant vectors with either one or two signal peptides to drive extracellular secretion. After optimization, 90 ± 0.2 mg/L BaGGT was obtained when the inducing conditions were 24 h and 80 μM (IPTG). The properties of BaGGT were measured, showing that the optimal reaction conditions were 40 °C and pH 9.0 with 55.0 ± 0.5 U/mg enzymatic activity. K_m and V_max were 0.214 mM and 88.13 μmol/min/mg. BaGGT could be stored for 72 h with 90% of the initial activity at 40 °C and retained more than 50% of the initial activity after being maintained at different pH values for 24 h. Finally, enzymatic synthesis of l-theanine was performed with the optimal conditions: 20 mM l-Gln, 100 mM ethylamine HCl, 0.5 U/mL BaGGT, incubated at 40 °C for 6 h, 200 rpm.