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, beta-CN f193-209. Aminopeptidase and endopeptidase genes of Lb. helveticus WSU19 were cloned in Escherichia coli DH5 alpha. The beta-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 beta-CN f193-209. Complete degradation of beta-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 beta-CN f193-209. PepE cleaved beta-CN f193-209 only when combined with PepN or PepO-like endopeptidases. Aminopeptidase and post-proline endopeptidase activities contributed to the initial degradation of beta-CN f193-209. (c) 2007 Elsevier Ltd. All rights reserved.