Glutamic acid gamma-methyl ester (GAME) was used as substrate for theanine synthesis catalyzed by Escherichia coli cells possessing gamma-glutamyltranspeptidase activity. The yield was about 1.2-fold higher than with glutamine as substrate. The reaction was optimal at pH 10 and 45A degrees C, and the optimal substrate ratio of GAME to ethylamine was 1:10 (mol/mol). With GAME at 100 mmol, 95 mmol theanine was obtained after 8 h.