Cloning and characterization of debittering peptidases, PepE, PepO, PepO2, PepO3, and PepN, of Lactobacillus helveticus WSU19

Abstract

Peptidases of Lactobacillus helveticus WSU19 are important for debittering aged Cheddar–type cheese. Our objective was to determine specificities of aminopeptidase N (PepN) and endopeptidases E, O, O2, and O3 (PepE, PepO, PepO2, and PepO3) of Lb. helveticus WSU19 on the bitter peptide, β–CN f193–209. Aminopeptidase and endopeptidase genes of Lb. helveticus WSU19 were cloned in Escherichia coli DH5 α. The β–CN f193–209 peptide was digested by cell–free extracts from peptidase–positive clones under cheese ripening conditions. The degradation pattern was analyzed qualitatively using matrix–assisted laser desorption/ionization time–of–flight mass spectrometry. Proline residues precluded PepN activity on β–CN f193–209. Complete degradation of β–CN f193–209 by PepN required post–proline endopeptidases, particularly PepO and PepO3. PepO–like endopeptidase activities on Pro 206–Ile 207 prevented formation of bitter peptides from the C–terminus of β–CN f193–209. PepE cleaved β–CN f193–209 only when combined with PepN or PepO–like endopeptidases. Aminopeptidase and post–proline endopeptidase activities contributed to the initial degradation of β–CN f193–209.