Efficient Production of L-Theanine Using Immobilized Recombinant <i>Escherichia coli</i>Cells Expressing a Modified γ-Glutamyltranspeptidase Gene from <i>Pseudomonas nitroreducens</i>

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.

Heterogeneous Expression and Purification of the Wheat VRN1 K-Box Domain Suggest the Formation of a Tetramer of the VRN1 Protein

In cereal species such as wheat (Ttiticum aestivum) and barley (Hordeum vulgare), many studies have indicated that VERNALIZATION 1 (VRN1) functions as a flowering promoter, which activates florigen gene expression. The wheat florigen gene Wheat FLOWERING LOCUS T (WFT, which is identical to VRN3) is an integrator of the vernalization, photoperiod and autonomous pathways in wheat flowering, and the WFT expression is correlated with the VRN1 expression. VRN1 encodes an APETALA1/FRUITFULL-like MADS-box transcription factor which expression is induced by vernalization, leading to flowering thorough up-regulation of WFT. In Arabidopsis, it has been reported that protein-protein interactions are keys for MADS-box protein function and MADS-box transcription factors must dimerize to bind to the target gene. In this study, by using gel permeation chromatography (GPC) with purified VRN1 protein, we indicated the possibility that VRN1 protein exists as tetramer-like as flowering homeotic MADS-box proteins in Arabidopsis.