Purification and characterization of an X-prolyldipeptidyl aminopeptidase from Lactobacillus curvatus DPC2024

An X-prolyl-dipeptidyl aminopeptidase (PepX) was purified ∼176-fold from the cell-free extract of Lactobacillus curvatus DPC2024. The native enzyme appeared to be a dimer with a sub-unit molecular mass of ∼97.4 kDa as determined by sodium dodecyl sulphate gel electrophoresis. Optimal activity of the purified enzyme on Ala-Pro-p-nitroanilide (pNA) was at pH 7.5 and 45°C. The enzyme retained more than 60% of its activity after pre-incubation for 30 min at 45°C but the activity decreased sharply following pre-incubation at temperatures above 45°C. The enzyme was activated by Co²⁺, Mn²⁺, Ni²⁺ at 0.1 and 1.0 mmol·L^-1 and by Cu²⁺, Cd²⁺ and Zn²⁺ at 0.1 mmol·L^-1 but it was strongly inhibited by 1.0 mmol·L^-1 phenylmethylsulphonyl fluoride, Hg²⁺, Cu²⁺, Cd²⁺ and Zn²⁺ and partially by ethylenediaminetetraacetic acid, o-phenanthroline and p-chloromercuribenzoate. The enzyme hydrolysed Ala-Pro-pNA, Arg-Pro-pNA, Gly-Pro-Arg, Val-Pro-Leu at a faster rate and slowly hydrolysed Ala-Ala-pNA but it was not active on Ala-Ala-Ala, Ala-Leu-Ala, Pro-Pro-Pro, Arg-Pro-Pro, dipeptides and N-terminally blocked p-nitroanilide derivatives and other peptides. The sequence of the first 20 amino acid residues was determined and showed 40% homology to X-prolyl-dipeptidyl aminopeptidases from Lactobacillus helveticus CNRZ 32, Lactobacillus helveticus 53/7, Lactococcus lactis ssp. cremoris P8-2-47 and Lactococcus lactis ssp. lactis NCDO 763 and 35% homology to a PepX from Lactobacillus delbrueckii ssp. lactis DSM 7290.