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 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 microkat mg(-1), and 0.16 mM and 0.31 microkat 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.
Basten
Daniëlle E J W
DEJW
Section Molecular Genetics of Industrial Micro-organisms, Wageningen University, Dreijenlaan 2, 6703 HA, Wageningen, The Netherlands1.
Visser
Jaap
J
Section Molecular Genetics of Industrial Micro-organisms, Wageningen University, Dreijenlaan 2, 6703 HA, Wageningen, The Netherlands1.
Schaap
Peter J
PJ
Section Molecular Genetics of Industrial Micro-organisms, Wageningen University, Dreijenlaan 2, 6703 HA, Wageningen, The Netherlands1.
D000626:Aminopeptidases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001234:Aspergillus niger / Q000201:enzymology*; D003001:Cloning, Molecular; D005800:Genes, Fungal; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
D000626:Aminopeptidases; C056860:lysyl aminopeptidase