An extracellular γ-glutamyl transpeptidase (GGT) produced from Bacillus altitudinis IHB B1644 was purified to homogeneity employing ion-exchange chromatography. GGT comprised two subunits of 40 and 22 kDa determined by SDS-PAGE. The maximum enzyme activity was optimal at pH 9 and 37 °C. The purified enzyme was stable from pH 5-10 and <50 °C. Steady-state kinetic studies revealed a Km value of 0.538 mM against γ-GpNA. For substrate specificity, GGT showed highest affinity for l-methionine. The inhibitors' effect demonstrated that serine or threonine and tryptophan residues are essential for enzyme activity. l-Theanine production was optimized by employing a one-variable-at-a-time approach with 60-65% conversion rate. The final reaction consisted of 20 mM l-glutamine, 200 mM ethylamine hydrochloride, and 10 U mL-1 enzyme concentration at 37 °C in Tris-Cl (50 mM, pH 9) for 5 h. l-Theanine was purified using a Dowex 50W X 8 hydrogen form resin and confirmed by HPLC and 1H NMR spectroscopies.
Keywords: Bacillus altitudinis IHB B1644; L-theanine; characterization; purification; γ-glutamyl transpeptidase.