Purification and characterization of an aminopeptidase from Lactococcus lactis subsp. cremoris AM2
1989
Neviani, E. | Boquien, C.Y. | Monnet, V. | Thanh, L.P. | Gripon, J.C.
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 (Km) 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.
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