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.
Highlights
L-Theanine (γ-glutamylethylamide) is an amino acid derivative approved by the Food and Drug Administration, USA, as a food additive to stimulate the Umami taste
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
E. coli-W525D was selected as a best strain for immobilization study for L-theanine production
Summary
L-Theanine (γ-glutamylethylamide) is an amino acid derivative approved by the Food and Drug Administration, USA, as a food additive to stimulate the Umami taste. L-Theanine is naturally present in specific organisms such as Camellia sinensis (green tea) and the mushroom, Xerocomus badius [5] It can be obtained by chemical synthesis or extraction from green tea, but both procedures include time-consuming, cost-ineffective, and complicated operational processes [6]. GGT is a ubiquitous enzyme that catalyzes the hydrolysis of the γ-glutamyl linkages of γ-glutamyl compounds and the transfer of their γ-glutamyl moieties to acceptor substrates [9]. It exists in a wide range of organisms from mammals to bacteria [10]. The activity of GGTs produced by such cultured bacteria can be exploited to produce L-theanine when provided with L-glutamine and ethylamine as substrates
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