TITLE:
Efficient Production of L-Theanine Using Immobilized Recombinant Escherichia coli Cells Expressing a Modified γ-Glutamyltranspeptidase Gene from Pseudomonas nitroreducens
AUTHORS:
Putthapong Phumsombat, Chiharu Sano, Hiroki Ikezoe, Junji Hayashi, Takafumi Itoh, Takao Hibi, Mamoru Wakayama
KEYWORDS:
L-Theanine, γ-Glutamyltranspeptidase, Pseudomonas nitroreducens, Escherichia coli, Immobilization
JOURNAL NAME:
Advances in Biological Chemistry,
Vol.10 No.6,
December
8,
2020
ABSTRACT: 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.