PARTIAL-PURIFICATION AND CHARACTERIZATION OF AN AMINOPEPTIDASE FROM LACTOBACILLUS-HELVETICUS CNRZ-32

An intracellular aminopeptidase (α-aminoacylpeptide hydrolase, E.C. 3. 4.11) which catalyzed hydrolysis of L-lysine-p-nitroanilide was partially purified from the cell-free extract of Lactobacillus helveticus CNRZ 32. The enzyme was purified about 55-fold by streptomycin sulfate precipitation, followed by several Chromatographic procedures. The partially purified enzyme had a molecular weight of 97,000 which was estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Optima for activity by the enzyme were pH 6.5 and 45°C. During incubation at 45°C for 15 min, the enzyme was activated by Co2+, Ca2+, Mg2+, and was inhibited by Cu2+, Mn2+, Zn2+, ethylene diaminetetraacetic acid (EDTA), 1, 10-phenanthroline and HgCl2. Aminopeptidase activity was strongly inhibited by p-chloromercuribenzoate, N-ethylmaleimide, iodoacetic acid and iodoacetamide but was not affected by dithiothreitol, diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride. Enzyme activity inhibted by 1.0 mM EDTA was partially restored by addition of Co2+, Ca2+, Mg2+, Mn2+ and Zn2+. Sodium chloride at concentrations above 50 mM (about 0.3%, w/v) and pH 5.0 markedly inhibited enzyme activity. The enzyme had broad substrate specificity and catalyzed release of unsubstituted, N-terminal amino acids from dipeptides and p-nitroanilide derivatives of amino acids, but was not active on peptides having proline as the N-terminal or C-terminal amino acid. The enzyme also lacked endopeptidase, carboxypeptidase and proteolytic activity toward casein. © 1990, Gustav Fischer Verlag, Stuttgart · New York. All rights reserved.