1. Enzymatic synthesis of γ-glutamylmethylamide from glutamic acid γ-methyl ester and methylamine catalyzed by Escherichia coli having γ-glutamyltranspeptidase activity
Lisheng Xu, Guizhen Gao, Cao Wengen, Jigui Xu, Liang Zhao, Hongwei Shi, Xingtao Zhang Appl Biochem Biotechnol. 2014 Jun;173(4):851-6. doi: 10.1007/s12010-014-0877-3. Epub 2014 Apr 15.
A new method for the synthesis of γ-glutamylmethylamide is presented. Glutamic acid γ-methyl ester was used as substrate for γ-glutamylmethylamide synthesis catalyzed by Escherichia coli with γ-glutamyltranspeptidase activity. Reaction conditions were optimized by using 300 mM glutamic acid γ-methyl ester and 3,000 mM methylamine at pH 10 and 40 °C. Bioconversion rate of γ-glutamylmethylamide reached 87 % after 10 h. γ-Glutamyltranspeptidase was reversibly inhibited only when glutamic acid γ-methyl ester was above 300 mM.
2. Enzymatic synthesis of theanine from glutamic acid γ-methyl ester and ethylamine by immobilized Escherichia coli cells with γ-glutamyltranspeptidase activity
Fei Zhang, Qing-Zhong Zheng, Qing-Cai Jiao, Jun-Zhong Liu, Gen-Hai Zhao Amino Acids. 2010 Nov;39(5):1177-82. doi: 10.1007/s00726-010-0553-z. Epub 2010 Mar 19.
Theanine (γ-glutamylethylamide) is the main amino acid component in green tea. The demand for theanine in the food and pharmaceutical industries continues to increase because of its special flavour and multiple physiological effects. In this research, an improved method for enzymatic theanine synthesis is reported. An economical substrate, glutamic acid γ-methyl ester, was used in the synthesis catalyzed by immobilized Escherichia coli cells with γ-glutamyltranspeptidase (GGT) activity. The results show that GGT activity with glutamic acid γ-methyl ester as substrate was about 1.2-folds higher than that with glutamine as substrate. Reaction conditions were optimized by using 300 mmol/l glutamic acid γ-methyl ester, 3,000 mmol/l ethylamine, and 0.1 g/ml of immobilized GGT cells at pH 10 and 50°C. Under these conditions, the immobilized cells were continuously used ten times, yielding an average glutamic acid γ-methyl ester to theanine conversion rate of 69.3%. Bead activity did not change significantly the first six times they were used, and the average conversion rate during the first six instances was 87.2%. The immobilized cells exhibited favourable operational stability.
3. Synthesis of theanine from glutamic acid gamma-methyl ester and ethylamine catalyzed by Escherichia coli having gamma-glutamyltranspeptidase activity
Fei Zhang, Qing-Zhong Zheng, Qing-Cai Jiao, Jun-Zhong Liu, Gen-Hai Zhao Biotechnol Lett. 2010 Aug;32(8):1147-50. doi: 10.1007/s10529-010-0273-1. Epub 2010 Apr 11.
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 45 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.
