Purification and characterization of a dipeptidase from Lactobacillus curvatus DPC2024

A dipeptidase was purified to homogeneity from a cell-free extract of Lactobacillus curvatus DPC2024 by chromatography on diethylaminoethyl-sephacel, phenyl sepharose, chelating sepharose fast flow and MonoQ. The purified dipeptidase was a monomer of ∼52 kDa as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis and gel filtration chromatography. The enzyme was optimally active at pH 8 and 50°C and retained ∼10% of its maximum activity after pre-heating for 10 min at 70°C. The enzyme was a metallopeptidase, strongly inhibited by 0.1 mM ethylenediaminetetraacetic acid and o-phenanthroline and reactivated by a number of divalent metal ions. The enzyme was also inhibited by p-chloromercuribenzoate and β-mercaptoethanol. The enzyme was a strict dipeptidase, capable of hydrolysing a range of dipeptides but not tri-, tetra- or pentapeptides, p-nitroanilide derivatives of amino acids nor N- and C-terminal-blocked dipeptides. The N-terminal amino acid sequence of the first 20 residues showed significant homology with dipeptidases from Lactobacillus delbrueckii subsp. lactis DSM 7290 and Lactococcus lactis subsp. cremoris MG1363.