Purification and Characterisation of an Aminopeptidase A from Cytoplasm of Lactococcus lactis subsp cremoris AM2

An aminopeptidase A has been purified 387-fold with a yield of 23.6% from cytoplasm of Lactococcus lactis subsp, cremoris AM2. The native enzyme had a molecular mass of 240000 Da and was composed of six equal subunits of molecular mass 39800 Da. Enzyme activity was most effectively inhibited by chelating agents, by dithiothreitol or by captopril (all at 1 mM), while bestatin, amastatin, phenylmethylsulphonylfluoride (PMSF), iodoacetamide and N-ethylmaleimide had little or no effect at the same concentration. Of the divalent cations tested with the enzyme, Ba2+ and Mg2+ had little effect, whereas Zn2+ and Co2+ were stimulatory while Ca2+, Cu2+ and, to a lesser extent, Mn2+ were inhibitory. The enzyme was assayed on a wide variety of dipeptides and hydrolysed only those with an aspartyl, glutamyl or seryl residue at the N-terminal position. These residues were also released from longer peptides by the action of this enzyme, although no hydrolysis was observed with peptides containing asparagine, glutamine, succinate, glutarate or pyroglutamate residues at the N-terminal position. Kinetic studies revealed that the affinity of the enzyme for substrate increases with increasing length of peptide, and with increased hydrophobicity of the penultimate residue from the N-terminus. Seryl dipeptides were found to have lower affinity. for the enzyme than either glutamyl or aspartyl dipeptides. The enzyme was reasonably heat stable, with no loss of activity being recorded after incubation at 50 degrees C for 120 min.