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 Plac 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. Km and Vmax 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.