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. 1989 Sep;55(9):2308–2314. doi: 10.1128/aem.55.9.2308-2314.1989

Purification and Characterization of an Aminopeptidase from Lactococcus lactis subsp. cremoris AM2

E Neviani 1,†, C Y Boquien 1, V Monnet 1,*, L Phan Thanh 1, J-C Gripon 1
PMCID: PMC203073  PMID: 16348010

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°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 (Vmax) were, respectively, 4.5 mM and 3,600 pkat/mg for the substrate l-histidyl-β-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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Selected References

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