Characterization of a d-Stereoselective Aminopeptidase (DamA) Exhibiting Aminolytic Activity and Halophilicity from Aspergillus oryzae

β-Aminopeptidases exhibit both hydrolytic and aminolytic (peptide bond formation) activities and have only been reported in bacteria. We identified a gene encoding the β-aminopeptidase homolog from a genome database of the filamentous fungus Aspergillus oryzae . The gene was overexpressed in A. oryzae , and the resulting recombinant enzyme was purified. Apart from bacterial homologs [β-Ala-para-nitroanilide ( p NA)], the enzyme preferred d -Leu- p NA and d -Phe- p NA as substrates. Therefore, we designated this gene as d - stereoselective aminopeptidase A ( damA ). The purified recombinant DamA was estimated to be a hexamer and was composed of two subunits with molecular masses of 29.5 and 11.5 kDa, respectively. Optimal hydrolytic activity of DamA toward d -Leu- p NA was observed at 50 °C and pH 8.0. The enzyme was stable up to 60 °C and from pH 4.0–11.0. DamA also exhibited aminolytic activity, producing d -Leu- d -Leu-NH 2 from d -Leu-NH 2 as a substrate. In the presence of 3.0 M NaCl, the amount of p NA liberated from d -Leu- p NA by DamA was 3.1-fold higher than that in the absence of NaCl. Thus, DamA is a halophilic enzyme. The enzyme was utilized to synthesize several hetero-dipeptides containing a d -amino acid at the N-terminus as well as physiologically active peptides.