title: Purification and Characterization of an Aminopeptidase from Lactococcus lactis subsp. cremoris AM2.
abstract: An aminopeptidase was purified from cell extracts of Lactococcus lactis subsp. cremoris AM2 by ion-exchange chromatography. After electrophoresis of the purified enzyme in the presence or absence of sodium dodecyl sulfate, one protein band was detected. The enzyme was a 300-kilodalton hexamer composed of identical subunits not linked by disulfide bridges. Activity was optimal at 40 degrees C and pH 7 and was inhibited by classical thiol group inhibitors. The aminopeptidase hydrolyzed naphthylamide-substituted amino acids, as well as dipeptides and tripeptides. Longer protein chains such as the B chain of insulin were hydrolyzed, but at a much slower rate. The Michaelis constant (K(m)) and the maximal rate of hydrolysis (V(max)) were, respectively, 4.5 mM and 3,600 pkat/mg for the substrate l-histidyl-beta-naphthylamide. Amino acid analysis showed that the enzyme contained low levels of hydrophobic residues. The partial N-terminal sequence of the first 19 residues of the mature enzyme was determined. Polyclonal antibodies were obtained from the purified enzyme, and after immunoblotting, there was no cross-reaction between these antibodies and other proteins in the crude extract.
authors.affiliation: Station de Recherches Laitières, Institut National de la Recherche Agronomique, 78350 Jouy-en-Josas, Laboratoire de Génie des Procédés Biotechnologiques Agro-alimentaires, Institut National de la Recherche Agronomique, 78850 Thiverval-Grignon, and Station de Pathologie Porcine et d'Immunologie, Institut National de la Recherche Agronomique, Nouzilly, 37380 Monnaie, France.
authors.forename: E
authors.initials: E
authors.lastname: Neviani
authors.forename: C Y
authors.initials: CY
authors.lastname: Boquien
authors.forename: V
authors.initials: V
authors.lastname: Monnet
authors.forename: L P
authors.initials: LP
authors.lastname: Thanh
authors.forename: J C
authors.initials: JC
authors.lastname: Gripon