Lysine aminopeptidase of Aspergillus niger

Conserved regions within the M1 family of metallo-aminopeptidases have been used to clone a zinc aminopeptidase from the industrially used fungus Aspe rgillus niger. The derived amino acid sequence of ApsA is highly similar to two yeast zinc aminopeptidases, LAPI and AAPI (53.3 and 50.9% overall simi larity, respectively), two members of the M1 family of metallo-aminopeptida ses. The encoding gene was successfully overexpressed in A. niger and the o verexpressed product was purified and characterized. Aminopeptidase A was f ound to be active towards a number of amino acid p-nitroanilide (pNA) subst rates, viz. K-pNA, R-pNA, L-pNA, M-pNA, A-pNA and F-pNA. The most preferred N-terminal amino acid is lysine and not leucine, arginine or alanine, the N-terminal amino acids preferred by the yeast homologues. The K-m and K-cat for K-pNA and L-pNA were 0.17 mM and 0.49 mu kat mg(-1), and 0.16 mM and 0 .31 mu kat mg(-1), respectively. The pH optimum of the enzyme is between 7. 5 and 8, whereas the enzyme is stable between pH 5 and 8. The enzyme is inh ibited by the metal chelators EGTA, EDTA and 1,10-phenanthrolin. Bestatin w as also able to inhibit the activity.