-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 subunit 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 Co 2+ , Mn 2+ , Ni 2+ at 0.1 and 1.0 mmol . L -1 and by Cu 2+ , Cd 2+ and Zn 2+ at 0.1 mmol . L -1 but it was strongly inhibited by 1.0 mmol . L -1 phenylmethylsulphonyl fluoride, Hg 2+ , Cu 2+ , Cd 2+ and Zn 2+ 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, ArgPro-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.
Lactobacillus curvatus / X-prolyl-dipeptidyl aminopeptidase / purificationRésumé -Purification et caractérisation d'une X-prolyl-dipeptidyl aminopeptidase isolée à partir de Lactobacillus curvatus DPC2024. Une X-prolyl-dipeptidyl aminopeptidase (PepX) a été purifiée environ 176 fois à partir d'un extrait intracellulaire de Lactobacillus curvatus DPC2024. L'enzyme native est apparue être de la forme dimère avec des sous-unités de masse moléculairẽ 97,4 kDa déterminée par électrophorèse SDS-PAGE. L'activité optimale de l'enzyme purifiée, déterminée sur le fragment Ala-Pro-p-nitroanilide (pNA), a été obtenue à pH 7,5 et à 45 °C. L'enzyme Lait 80 (2000) 385-396 385