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Lysine aminopeptidase of Aspergillus niger

Section Molecular Genetics of Industrial Micro-organisms, Wageningen University, Dreijenlaan 2, 6703 HA, Wageningen, The Netherlands¹

Author for correspondence: Peter Schaap. Tel: +31 317 485142. Fax: +31 317 484011. e-mail: peter.schaap{at}algemeen.mgim.wag-ur.nl

Conserved regions within the M1 family of metallo-aminopeptidases have been used to clone a zinc aminopeptidase from the industrially used fungus Aspergillus 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 similarity, respectively), two members of the M1 family of metallo-aminopeptidases. The encoding gene was successfully overexpressed in A. niger and the overexpressed product was purified and characterized. Aminopeptidase A was found to be active towards a number of amino acid p-nitroanilide (pNA) substrates, 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 µkat mg^-1, and 0·16 mM and 0·31 µkat 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 inhibited by the metal chelators EGTA, EDTA and 1,10-phenanthrolin. Bestatin was also able to inhibit the activity.

Keywords: metallopeptidase

Abbreviations: pNA, p-nitroanilide

The EMBL accession number for the sequence reported in this paper is AJ292570.

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