PepN-like aminopeptidase from Lactohacillus 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 oc.The enzyme had a molecular mass of -98 kDa as estimated by gel permeation chromatography, and showed one band corresponding to a molecu- lar 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 met- alloenzyme since it was strongly inhibited by ethylenediaminetetraacetic acid (EDTA) and o-phenan- throline at 0.1 mmol-Lr ' concentration and was partially reactivated by Zn2+ and C02+. The enzyme was also partially inhibited by p-chloromercuribenzoate and iodoacetic acid, suggesting the involve- ment of su1phydryl 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-tenninal amino acid sequence of the first 20 amino acid residues was determined (AELMRFYQSFQPEHYQVFLD), and showed 40-55 % homology with Zn2+-dependent aminopep- tidases from Streptococcus thermophilus NCDu53, Lactobacillus delbrueckii subsp. lactis DSM7290, Lactococcus lactis subsp. lactis MG 1363 and Lactococcus lactis subsp. cremoris W g2. © Inra/Elsevier, Paris.