Purification and characterization of an aminopeptidase from Lactobacillus curvatus DPC2024

Abstract

Abstract An aminopeptidase was purified to homogeneity from a cell-free extract of Lactobacillus curvatus DPC2024, a component of the non-starter lactic acid bacterial flora of Cheddar cheese. Optimal aminopeptidase activity on leucyl- p -nitroanilide (Leu–pNA) was observed at pH 7.0 and 40°C. Molecular masses of ∼64 and ∼32 kDa were estimated for the enzyme by gel permeation chromatography and sodium dodecyl sulphate polyacrylamide gel electrophoresis, respectively, indicating that the native enzyme (64 kDa) exists as a dimer. The aminopeptidase was slightly activated by Ba 2+ , Ca 2+ and dithiothreitol but strongly inhibited by Hg 2+ , Ni 2+ , Cu 2+ , Cd 2+ , Zn 2+ , thiol-blocking agents (p-hydroxymercuribenzoate, p-chloromercuribenzoate and iodoacetic acid) at 1 m m and by EDTA, o-phenanthroline and phenylmethylsulphonyl fluoride at 10 m m . The enzyme was most active on Leu–pNA and a number of dipeptides but showed little or no activity against tri- and oligopeptides. The N-terminal amino acid sequence was determined for 20 residues and showed significant homology to a leucyl-aminopeptidase from Lb. delbrueckii subsp. lactis DSM7290 and prolinases from Lb. helveticus CNRZ32 and Lb. rhamnosus 1/6. Regarding its molecular mass, metal dependency and specificity, the enzyme was different, in most respects, from the general aminopeptidases (PepN and PepC) and glutamyl aminopeptidase (PepA) of lactic acid bacteria.