PURIFICATION AND CHARACTERIZATION OF AMINOPEPTIDASE-A FROM LACTOCOCCUS-LACTIS SUBSP LACTIS NCDO-712

An aminopeptidase A (EC 3.4.11.7) was purified from Lactococcus lactis subsp. lactis NCDO 712. Of the 18 aminoacyl-alanine dipeptides tested, the enzyme hydrolysed Asp-Ala, Glu-Ala and Ser-Ala. It was also active against tripeptide substrates but did not hydrolyse Ala-Asp or Ala-Glu. The kinetics of dipeptide hydrolysis were allosteric with positive cooperativity of substrate binding. The Hill constants were 1.52 for Asp-Ala, 1.51 for Glu-Ala and 1.61 for Ser-Ala. The M(r) was found to be 245000 by gel-filtration chromatography. A single band corresponding to an M(r) of 41000 was detected by SDS-PAGE of the purified enzyme which indicated that the enzyme is hexameric. Activity against glutamate p-nitroanilide was optimum at 65-degrees-C and pH 8. Activity was inhibited by 1 mM-EDTA, implying that the enzyme is metal-containing. Cu2+, Mn2+ and Zn2+ (all at 1 mM) inhibited activity and Co2+ was stimulatory.