Purification and characterization of an aminopeptidase from Lactobacillus sake

An aminopeptidase was purified from the cell-free extract of Lactobacillus sate IATA115 by ammonium sulfate fractionation and several chromatographic procedures including hydrophobic interaction, gel filtration, and anion exchange chromatography. The purified enzyme was a 35-86 kDa monomer. Activity was optimal at 37 degrees C and pH 7.5, and the K-m values estimated for Leu- and Met-AMC (7-amido-4-methylcoumarin) were 0.091 and 0.174 mM, respectively. The aminopeptidase exhibited maximal activity against Leu- and Ala-AMC, while Lys- and Arg-AMC were not hydrolyzed. Among peptides, highest activity was observed against Ala-Ala, Ala-Leu, and Leu-Ala, while dipeptides containing basic amino acids at the N terminus were not hydrolyzed. Serin and aspartic proteinase inhibitors had no effect on the activity. However, the enzyme was inhibited by puromycin, amastatin, bestatin, arphamenine B, and sulfhydryl group reagents but activated by reducing reagents. The presence of Hg2+, Cu2+, Cd2+, and Ni2+ as well as high concentrations of chelator agents caused inhibition, while other divalent cations such as Ca2+, Sn2+, Mg2+, Ba2+, and Mn2+ stimulated the activity.