PURIFICATION AND CHARACTERIZATION OF AN AMINOPEPTIDASE FROM LACTOBACILLUS-SAKE

An aminopeptidase was purified from the cell-free extract of Lactobaci llus sate IATA115 by ammonium sulfate fractionation and several chroma tographic procedures including hydrophobic interaction, gel filtration , and anion exchange chromatography. The purified enzyme was a 35-86 k Da 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) we re 0.091 and 0.174 mM, respectively. The aminopeptidase exhibited maxi mal 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 ac ids at the N terminus were not hydrolyzed. Serin and aspartic proteina se inhibitors had no effect on the activity. However, the enzyme was i nhibited by puromycin, amastatin, bestatin, arphamenine B, and sulfhyd ryl 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.