PepN-like aminopeptidase from Lactobacillus curvatus DPC2024: Purification and characterization

Abstract

An enzyme with a PepN-like aminopeptidase activity was purified 127-fold with 4.3 % recovery from a cell-free extract of Lactobacillus curvatus DPC2024, a component of the non-starter lactic acid bacterial flora of Cheddar cheese. The purified enzyme exhibited maximum activity on leucyl-p-nitroanilide (Leu-pNA) at pH 7.0 and 40 °C. The enzyme had a molecular mass of ∼98 kDa as estimated by gel permeation chromatography, and showed one band corresponding to a molecular mass of ∼95 kDa on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), indicating that the native enzyme existed as a monomer. The aminopeptidase appeared to be a metalloenzyme since it was strongly inhibited by ethylenediaminetetraacetic acid (EDTA) and o-phenanthroline at 0.1 mmol·L-1 concentration and was partially reactivated by Zn2+ and Co2+. The enzyme was also partially inhibited by p-chloromercuribenzoate and iodoacetic acid, suggesting the involvement of sulphydryl groups in the reaction mechanism. The enzyme had a broad substrate specificity, hydrolysing a number of p-nitroanilide derivatives of amino acids and peptides and di-, tri-, tetra-and pentapeptides. The N-terminal amino acid sequence of the first 20 amino acid residues was determined (AELMRFYQSFQPEHYQVFLD), and showed 40-55 % homology with Zn2+-dependent aminopeptidases from Streptococcus thermophilus NCDO53, Lactobacillus delbrueckii subsp. lactis DSM7290, Lactococcus lactis subsp. lactis MG1363 and Lactococcus lactis subsp. cremoris Wg2. © Inra/Elsevier, Paris.