Characterization of alkaline Bacillus amyloliquefaciens γ-glutamyltranspeptidase expressed in Bacillus subtilis and its application in enzymatic synthesis of L‑Theanine

L-Theanine (L-Th) is a non-protein amino acid in the tea leaves, has particular health benefits and is widely used as a dietary supplement, pharmaceutical, nutritional and cosmetic ingredient, which can be synthesized by γ-glutamyltranspeptidase (GGT, EC 2.3.2.2) with L-glutamine (L-Gln) and ethylamine as substrate. In this study, the gene of GGT from Bacillus amyloliquefaciens SK11.001 controlled by P43 promoter was cloned into Bacillus subtilis WB800 incubation to obtain a highly active alkaline GGT. Further research on targeting mutations and promoter screening can be conducted to construct more favorable L-Th producing engineered bacteria. The properties of GGT were determined and the results showed that the activity of the enzyme was optimal at pH 10.5 and the optimal temperature was 35 °C. GGT was kept below 40 °C for 1 h, with more than 90% of initial activity, and remained more than 80% of initial activity after 12 h at pH 4.0–11.5. The optimal reaction system was incubated in 50 mM carbonate buffer (pH 10.5) with 200 mM L-Gln, 1600 mM ethylamine hydrochloride and 2 U/mL GGT for 5 h at 35 °C, 200 rpm in a shaker. The conversion rate of L-Th was 95%, which was higher than in B. amyloliquefaciens BH072 (40%).