Partial purification and characterization of an X-prolyl dipeptidyl aminopeptidase from Lactobacillus sanfranciscensis CB1

An X-prolyl dipeptidyl aminopeptidase (PepX) from Lactobacillus sanfranciscensis CB1, a key sourdough lactic acid bacterium, was partially purified by five chromatographic steps. As estimated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration, the enzyme appeared to be a 49.2-56 kDa monomer. Optimal activity occurred at pH 6.0 and 30 °C, with Km of 0.6 mM and Vmax of 99.6 nmol mg−1 min−1. The D value, calculated at 45 °C, was ∼5.46 s. The enzyme hydrolyzed (almost exclusively) substrates with a X-Pro N-terminal sequence. It did not possess prolidase, aminopeptidase or endopeptidase activities. No hydrolysis of the Pro-rich 33-mer epitope (a potent inducer of gut-derived human T-cell lines in celiac patients) was found when it was treated with PepX alone. When the general aminopeptidase type N was combined with PepX, the hydrolysis of 33-mer peptide (0.2 mM) was complete after 24 h of incubation at 30 °C. Leucine and glutamine residues were liberated from the Pro-rich 33-mer peptide by aminopeptidase type N, thus favouring the subsequent PepX activity. PepX was inactivated by p-chloromercuribenzoate, 3,4-dichloroisocoumarin and phenanthroline which exerted a competitive mode of inhibition. Among divalent cations, only Zn2+, Hg2+ and Mn2+ markedly decreased the enzyme activity. Quadratic response surface methodology was used to study the individual and interactive effects of temperature, pH and NaCl on the PepX activity. The enzyme maintained considerable activity under the environmental conditions which characterize the sourdough fermentation.