Purification and partial characterization of a tripeptidase from Pediococcus pentosaceus K9.2

Abstract

A tripeptidase was purified from the cytoplasm of Pediococcus pentosaceus K9.2 by anion-exchange chromatography, gel filtration chromatography, and high-performance liquid chromatography. The molecular mass of the enzyme was estimated by gel filtration at 100,000 Da. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified peptidase showed one protein band of 45,000 Da. Optimal enzyme activity was obtained at pH 7.0 and at 50°C. The peptidase hydrolyzed all tripeptides tested. Cleavage was not observed with dipeptides, oligopeptides, or amino acid-p- nitroanilide derivatives. Strong inhibition of activity was caused by EDTA, 1,10-phenanthroline, dithiothreitol, and β-mercaptoethanol, whereas phenylmethylsulfonyl fluoride and sulfur-reactive reagents had no effect on peptidase activity. Mg2+, Mn2+, and Ca2+ stimulated the hydrolyzing activity of the enzyme. The 20 N-terminal amino acids of the tripeptidase from P. pentosaceus had 84% identity with those from the corresponding N- terminal region of the tripeptidase from Lactococcus lactis subsp. cremoris Wg2.