Author(s)

Putthapong Phumsombat¹, Chiharu Sano¹, Hiroki Ikezoe¹, Junji Hayashi², Takafumi Itoh³, Takao Hibi³, Mamoru Wakayama^1*

¹Department of Biotechnology, College of Life Sciences, Ritsumeikan University, Shiga, Japan.
²Graduate School of Technology, Industrial and Social Sciences, Tokushima University, Tokushima, Japan.
³Department of Bioscience and Biotechnology, Fukui Prefectural University, Fukui, Japan.

L-Theanine (γ-glutamylethylamide) is a naturally occurring amino acid derivative known to have several beneficial physiological effects as a diet supplement, and to give an umami taste when used as a food additive. The compound is industrially produced by γ-glutamyltranspeptidase from Pseudomonas nitroreducens (PnGGT). Using recombinant PnGGT, we have shown previously that Trp385, Phe417, and Trp525 are key amino acid residues for recognition of acceptor substrates at the PnGGT active site. Here, we demonstrate that a recombinant W525D mutant of PnGGT produces L-theanine from ethylamine and L-glutamine more efficiently than wild-type PnGGT, attributable to an increased ratio of transfer activity to hydrolysis activity. An efficient production of L-theanine was achieved by immobilizing Escherichia coli cells expressing the W525D PnGGT mutant (E. coli-W525D) using 2% alginate as the supporting material. The highest L-theanine production using immobilized E. coli-W525D, representing a conversion rate of 90%, was achieved in optimal reaction conditions of pH 10, 40°C, and a substrate molar ratio of L-glutamine to ethylamine of 1:10. The immobilized E. coli-W525D retains 85% and 78% relative activity after storage for a month at 4°C and room temperature, respectively. Immobilized E. coli-W525D thus has strong potential for use in the future commercial production of L-theanine on a large scale.

L-Theanine, γ-Glutamyltranspeptidase, Pseudomonas nitroreducens, Escherichia coli, Immobilization

Phumsombat, P. , Sano, C. , Ikezoe, H. , Hayashi, J. , Itoh, T. , Hibi, T. and Wakayama, M. (2020) Efficient Production of L-Theanine Using Immobilized Recombinant Escherichia coli Cells Expressing a Modified γ-Glutamyltranspeptidase Gene from Pseudomonas nitroreducens. Advances in Biological Chemistry, 10, 157-171. doi: 10.4236/abc.2020.106012.