Valyl-glycine (Val-Gly) is useful as a synthetic substrate of gamma-glutamyl-valyl-glycine (gamma-Glu-Val-Gly), which exhibits a strong taste of "kokumi." For efficient enzymatic synthesis of Val-Gly from valine methylester and glycine using (L)-amino acid esterase (LAE), we screened microorganisms producing LAE with synthetic activity toward Val-Gly. Among 17 isolates showing LAE activity, Elizabethkingia sp. TT1, which was identified by 16S rDNA sequence analysis, showed the highest synthetic activity toward Val-Gly. LAE from Elizabethkingia sp. TT1 (TT1LAE) was purified approximately 1300 times, resulting in a yield of 2.8% and specific activity of 118.8 mu mol/min/mg protein. SDS-PAGE analysis revealed a subunit molecular mass of 78 kDa. The molecular mass of the native enzyme determined by gel filtration was 103 kDa. The purified enzyme showed maximum activity at pH 9.0 and at a temperature of 25 A degrees C, and it was stable over the pH range of 5.0-8.5 and 25 A degrees C-40 A degrees C. No metal ions that were tested had a significant effect on enzyme activity, but the enzyme was slightly inhibited by EDTA.