Application of recombinant Bacillus subtilis γ-glutamyltranspeptidase to the production of L-theanine

L-Theanine, which has seen increasing use in the functional food industry, can be prepared via enzymatic synthesis using gamma-glutamyltranspeptidase (GGT; EC 2.3.2.2). In this study, the GGT from Bacillus subtilis 168 was cloned and expressed as a secreted protein using Escherichia coli BL21(DE3). The enzymatic properties of the GGT and the optimal conditions for the enzymatic synthesis of L-theanine were investigated in detail. The activity of the enzyme was optimal at pH 10; the optimal temperature was 50 degrees C. Desirable pH stability was observed between pH 5 and pH 12, and adequate thermostability was seen at 50 degrees C. In 5 h at 37 degrees C, the enzyme converted 200 mM L-glutamine and 2.2 M ethylamine to L-theanine with a final yield of 78%. Yields of L-theanine decreased to 58% when using 500 mM Gin and 45% when using 1 M Gln. The yield of L-theanine obtained at high substrate concentration provides the basis for the industrial-scale production of L-theanine. (C) 2014 Elsevier Ltd. All rights reserved.