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

Abstract

-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

Characterization of protease activities in a crude extract of germinated cacao

“…The serine peptidase blockers, AEBSF and pefabloc, at higher concentrations, inhibited 52 and 56% of enzymatic activity, respectively, suggesting that serine residues may participate in enzyme catalysis, as occurs with other microbial Xaa-ProDAPs (Magboul & McSweeney, 2000). However, another serine peptidase inhibitor, PMSF (at 0.1 mM) reduced Xaa-Pro-DAP2 activity by only 24%; leupeptin, which also inhibits cysteine peptidases, had the same effect on enzymatic activity.…”

“…Likewise, we assessed the Ala-pNA substrate, finding a lower degree of hydrolysis as compared with Leu-pNA and Lys-pNA at 10 mM. The non-specific Type N APE identified in a large number of lactic bacteria demonstrated Lys-pNA, Leu-pNA (Gómez De La Cruz, 1996) and Ala-pNA (Magboul & McSweeney, 2000) activities. In fermented cacao, the aminopeptidase activity is considered as the second most important enzyme after aspartyl peptidase (AP) (Hansen et al, 1998).…”

Lactobacilli possess inhibitory activity against dipeptidyl peptidase-4 (DPP-4)

“…PepX is a proline-specific peptidase with enzymatic activity almost identical to that of DPP-4, i.e., removal of N-terminal dipeptide residues from peptides containing a proline in the penultimate position (Meyer-Barton et al 1993). The PepX gene or PepX activity have been reported on a few occasions in lactic acid bacteria, including L. acidophilus (Bockelmann and Fobker 1991), L. casei (Habibi-Najafi and Lee 1994), L. curvatus DPC2024 (Magboul and McSweeney 2000), L. sanfranciscensis, L. lactis, L. delbrueckii, L. helveticus, L. rhamnosus and Streptococcus thermophilus (Savijoki et al 2006). The existence of an enzyme such as PepX provides a possible explanation as to why DPP-4 inhibitory activity can be found in bacteria, in that it may be produced to regulate enzymatic activity or bacterial metabolism, or to help the bacterium compete with other bacterial species.…”

Lactic Acid Bacteria as a Potential Source of Enzymes for Use in Vinification

“…The activities of greatest interest are those conferred by a single enzyme, ideally one with an extracellular localization. Such (294) Tripeptides (168) N-terminal pentapeptide of bradykinin (294,295) CBZ-amide-p-NA (294) Tetrapeptides (168) X-Pro-p-NA derivatives (171) Pentapeptides (168) X-Pro-X tripeptides (171) AMC derivatives (78,242) WC Casein-FITC (78,242) Cheddar cheese slurry peptides (197) ArAm derivatives (213,270) Milk protein (221) AMC derivatives (242,295) Casein ( Cheddar cheese slurry peptides (197) ArAm derivatives (270) Milk protein (8,58,64,221) Caprine and ovine curdled milk substrates (93) Caprine and ovine curdled milk protein (93) Caprine and ovine curdled milk peptides (93) Lactobacillus pentosus AMC derivatives (295) Dipeptides (294) Gluten (107) Tripeptides (2) Pro-AMC (295) ABZ-peptide-p-NA (294) N-terminal pentapeptide of bradykinin (294,295) BZ-peptide-p-NA (294) Peptide-p-NA derivatives (123) CD Casein (70,107) p-NA derivatives …”