Title: Efficient synthesis of L-theanine in recombinant Escherichia coli containing γ-Glutamyltranspeptidase gene from Pseudomonas nitroreducens (PnGGT)

Biography:

PHUMSOMBAT Putthapong is currently Ph.D. student at Ritsumeikan University, supported by Japan under JICA innovative Asia scholarship. He received both Bachelor and Master degree from Kasetsart University, Thailand before continue his research at enzyme technology laboratory, under the guidance of Prof. MAMORU Wakayama. He is working on project of improving enzyme activity of recombinant GGT. His field of interest is enzymology, biotechnology, molecular genetics, protein technology and lactic acid bacteria.

Abstract

L-Theanine (γ-glutamylethylamide) is a non-protein amino acid present in tea. It contributes to the umami taste and unique flavor to green tea infusion. It was reported to have many beneficial physiological effect, especially anti-stress and improvement of concentration for learning ability. It can be obtained by chemical synthesis or extraction from green tea, but both processes involve time consuming, cost-ineffective and complicated operational process. Hence, biotechnological production of L-theanine become interesting by using microbial enzyme such as glutamyltranspeptidase (GGT). GGT (EC 2.3.2.2), a heterodimeric enzyme found in various sources from bacteria to mammals catalyzes the transfer of the γ-glutamyl moiety of γ-glutamyl compound to γ-glutamyl acceptors such as amino acids and peptides. Bacterial GGTs have been reported in Escherichia coli (EcGGT), Bacillus licheniformis (BlGGT), and Pseudomonas nitroreducens (PnGGT). Our previous study on site directed mutagenesis of PnGGT suggested that Trp525 was a key amino acid residue in determining the preference of acceptor substrate in the reaction. Moreover, E. coli producing W525D mutant of PnGGT (E. coli-W525D mutant) showed low hydrolysis activity with high transfer activity, causing the increase of L-theanine production from ethylamine and L-glutamine. Improving the efficiency of L-theanine production using E. coli -W525D mutant was also achieved by whole cell calcium-alginate immobilization with determination of optimal pH, temperature, and molar substrate ratio. Under the suitable condition, pH 11, 40ºC, and substrate molar ratio of 1:10, L-glutamine: ethylamine, E. coli W525D mutant exhibited the highest theanine production compared to P. nitroreducens, E. coli-wild type, and other recombinant mutant strains.