Purification and Characterization of an Aminopeptidase from Lactococcus lactis subsp. cremoris Wg2

Abstract

An aminopeptidase was purified to homogeneity from a crude cell extract of Lactococcus lactis subsp. cremoris Wg2 by a procedure that included diethyl-aminoethane-Sephacel chromatography, phenyl-Sepharose chromatography, gel filtration, and high-performance liquid chromatography over an anion-exchange column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme showed a single protein band with a molecular weight of 95,000. The aminopeptidase was capable of degrading several peptides by hydrolysis of the N-terminal amino acid. The peptidase had no endopeptidase or carboxypeptidase activity. The aminopeptidase activity was optimal at pH 7 and 40�C. The enzyme was completely inactivated by the p -chloromecuribenzoate mersalyl, chelating agents, and the divalent cations Cu 2+ and Cd 2+ . The activity that was lost by treatment with the sulfhydryl-blocking reagents was restored with dithiothreitol or β-mercapto-ethanol, while Zn 2+ or Co 2+ restored the activity of the 1,10-phenantroline-treated enzyme. Kinetic studies indicated that the enzyme has a relatively low affinity for lysyl- p -nitroanilide ( K m , 0.55 mM) but that it can hydrolyze this substrate at a high rate ( V max , 30 μmol/min per mg of protein).