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

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 DH5α. 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 Pro206-Ile207 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.