Purification and properties of an extracellular leucine aminopeptidase from the Bacillus sp. N2

Abstract

A halophilic bacterium was isolated from fermented anchovy sauce and identified as Bacillus species. An extracellular leucine aminopeptidase from Bacillus sp. N2 was purified to homogeneity using four successive purification steps. The enzyme has a native molecular mass of about 57 000 Da using FPLC gel filtration analysis and a molecular mass of 58 000 Da using SDS-polyacrylamide gel electrophoresis. This monomeric leucine aminopeptidase showed maximum enzyme activity at pH 9.5. The optimum temperature was 50 °C when L-Leu-p-nitroanilide was the substrate. The leucine aminopeptidase was inactivated by 1,10-phenanthroline, dithiothreitol and sodium dodecyl sulphate. Enzyme activity was increased by addition of Co2+. It is likely that Co2+ plays an important role in the catalysis or stability of the Bacillus sp. N2 leucine aminopeptidase. Sodium chloride (0-4.5 mol l-1) increased the hydrolytic activity towards L-Leu-p-nitroanilide. The N- terminal amino acid sequence was Glu-Arg-Glu-Leu-Pro-Phe-Lys-Ala-Lys-His- Ala-Tyr-Ser-Thr-Ile. The purified enzyme had a high specificity for L-Leu-p- nitroanilide.