title: 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 degrees C. The enzyme was completely inactivated by the p-chloromecuribenzoate mersalyl, chelating agents, and the divalent cations Cu and Cd. The activity that was lost by treatment with the sulfhydryl-blocking reagents was restored with dithiothreitol or beta-mercapto-ethanol, while Zn or Co 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 mumol/min per mg of protein).
authors.affiliation: Department of Microbiology, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands.
authors.forename: P S
authors.initials: PS
authors.lastname: Tan
authors.forename: W N
authors.initials: WN
authors.lastname: Konings