Purification and partial characterization of a tripeptidase from Pediococcus pentosaceus K9.2.
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 degrees 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 beta-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.
Simitsopoulou
M
M
Faculty of Agriculture, Laboratory of Food Chemistry and Biochemistry, Aristotle University of Thessaloniki, Salonika, Greece.
Vafopoulou
A
A
Choli-Papadopoulou
T
T
Alichanidis
E
E
D000595:Amino Acid Sequence; D000626:Aminopeptidases; D002413:Cations, Divalent / Q000494:pharmacology; D002611:Cheese / Q000382:microbiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D013294:Lactococcus lactis / Q000201:enzymology / Q000235:genetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009842:Oligopeptides / Q000737:chemistry; D010376:Pediococcus / Q000201:enzymology* / Q000235:genetics; D010447:Peptide Hydrolases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013379:Substrate Specificity; D013696:Temperature
D002413:Cations, Divalent; D004791:Enzyme Inhibitors; D009842:Oligopeptides; D010447:Peptide Hydrolases; D000626:Aminopeptidases; C023191:PepT tripeptidase