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 & gamma;-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 degrees C. GGT was kept below 40 degrees 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 degrees C, 200 rpm in a shaker. The conversion rate of L-Th was 95%, which was higher than in B. amyloliquefaciens BH072 (40%).

Automated summary

This study cloned the GGT gene from Bacillus amyloliquefaciens SK11.001 into Bacillus subtilis WB800 to obtain a highly active alkaline GGT. Further research can be done to construct more favorable L-Theanine producing engineered bacteria through targeted mutations and promoter screening. The optimal activity of GGT enzyme was observed at pH 10.5 and 35 degrees C. GGT remained stable with more than 90% initial activity below 40 degrees C for 1 hour and retained more than 80% initial activity after 12 hours at pH 4.0-11.5. The conversion rate of L-Theanine reached 95% in the optimal reaction system containing 50 mM carbonate buffer (pH 10.5), 200 mM L-Gln, 1600 mM ethylamine hydrochloride, and 2 U/mL GGT, incubated for 5 hours at 35 degrees C and 200 rpm in a shaker, higher than B. amyloliquefaciens BH072 (40%).