Enzymatic synthesis of theanine with Escherichia coli γ-glutamyltranspeptidase from a series of γ-glutamyl anilide substrate analogues

• Published in: Biotechnology and bioprocess engineering Vol. 18; no. 2; pp. 358 - 364
• Main Authors: Zhang, Hong-juan, Zhang, Wei-guo, Wang, Zhi-yuan, Zhan, Yue-ping, Xu, Li-sheng, Liu, Jun-zhong, Liu, Qian, Jiao, Qing-cai
• Format: Journal Article
• Language: English
• Published: Heidelberg Springer-Verlag 01.04.2013; The Korean Society for Biotechnology and Bioengineering; 한국생물공학회
• Subjects: acylation; anilides; Biotechnology; Chemistry; Chemistry and Materials Science; Escherichia coli; Industrial and Production Engineering; Research Paper; substrate specificity; temperature; theanine; 생물공학; γ-glutamyltranspeptidase; kinetic studies; γ-glutamyl anilide analogues; theanine
• ISSN: 1226-8372; 1976-3816
• DOI: 10.1007/s12257-012-0644-7

In order to investigate the catalytic mechanism of Escherichia coli γ-glutamyltranspeptidase, ten para- and meta-substituted γ-glutamyl anilides were chemically prepared and employed as substrates to synthesize L-theanine to assay the activity of γ-glutamyltranspeptidase. The reaction was optimized for γ-glutamyl-p-nitroanilide. Key factors such as substrate specificity, pH, temperature, and the substrate mole ratio were all investigated. Kinetic studies of the acyl transfer reaction were described and the Hammett plot was constructed. This study indicated that the ratelimiting acylation reaction of γ-glutamyltranspeptidase can apparently be accelerated by either the electron-withdrawing or electron-donating substituents of γ-glutamyl anilides. The reaction could be catalyzed by the general acid and carboxy of Asp-433 or phenolic hydroxyl Tyr-444 may be the acid by autodock simulation for all prepared γ-glutamyl anilides.