Purification and Characterization of a Tripeptidase from Lactobacillus sake.

A tripeptidase was purified to homogeneity from the cell extract of Lactobacillus sake by ammonium sulfate precipitation, hydrophobic interaction chromatography, gel filtration chromatography, and two steps of anion exchange chromatography. After SDS-PAGE a single band of protein was detected of approximately 55 kDa. A similar molecular mass was estimated by gel filtration. The tripeptidase activity was optimal at pH 7.0 and at 40 degrees C. The enzyme was strongly inhibited by metal chelators, reducing agents, and bestatin while thiol group reagents, serine proteinase inhibitors, and aspartic proteinase inhibitors had no effect on the activity. The enzyme was activated by Mn(2+) and almost totally inhibited by Zn(2+) and to a lesser extent by Sn(2+). The enzyme only exhibited activity against tripeptides, and those hydrolyzed at higher rates were Ala-Ala-Ala, Ser-Ser-Ser, and Leu-Gly-Gly.