Purification and Characterization of an Aminopeptidase from Lactococcus lactis subsp. cremoris AM2.
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.
Neviani
E
E
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.
Boquien
C Y
CY
Monnet
V
V
Thanh
L P
LP
Gripon
J C
JC