An aminopeptidase with broad substrate specificity was purified to homogeneity (123.7-fold) with a yield of 3.43% from chicken (Callus gallus) intestine using a combination of chromatographic separation strategies. The enzyme was identified as alanyl aminopeptidase or aminopeptidase N (APN) by Peptide Mass Fingerprinting. The molecular weight of the enzyme was estimated to be similar to 180 kDa by SDS-PAGE and gel filtration chromatography. The enzyme was found to be a glycoprotein, having 40% sugar residue and a molecular mass of 108 kDa after deglycosylation. The enzymatic activity was optimal at 60 degrees C and pH 6.0. The enzyme preferentially hydrolyzed Leu-beta-NA (K-m = 0.1 mM) followed by Ala, Phe, Tyr and Gly at N-terminal. The enzyme activity was completely inhibited by 1,10 phenanthroline (1 mM) and bestatin (I mM) confirming it as a metalloprotease. Potential of this enzyme in combination with other endoproteases for the production of debittered protein hydrolysates has been discussed. (C) 2010 Elsevier Ltd. All rights reserved.