Degradation of αs1-CN f1-23 by aminopeptidase N and endopeptidases E, O, O2, and O3 of Lactobacillus helveticus WSU19 under cheese ripening conditions

This study determined specificities of aminopeptidase N (PepN), endopeptidase E (PepE), endopeptidase O (PepO), endopeptidase O2 (PepO2), and endopeptidase O3 (PepO3), from Lactobacillus helveticus WSU19 on the αs1-CN f1-23 peptide, formed by residual chymosin during cheese ripening. Cell-free extracts (CFEs) were prepared from Escherichia coli DH5α derivatives expressing peptidase genes of Lb. helveticus WSU19. The αs1-CN f1-23 peptide was digested by CFEs under cheese ripening conditions. Degradation pattern was analyzed qualitatively using MALDI-TOF mass spectrometry. PepN exhibited activity on αs1-CN f1-23 only in the presence of an endopeptidase, particularly PepO-like endopeptidases. PepO, PepO2, and PepO3 cleaved αs1-CN f1-23 predominantly at Glu14–Val15, forming the bitter peptide αs1-CN f1-14. PepE cleaved αs1-CN f1-23 primarily at Lys3–His4, suggesting a role for PepE in degrading bitter peptides from the N-terminus of αs1-CN f1-23. Combinations of PepE/PepO and PepE/PepO2 were determined to have the potential to decrease the accumulation of αs1-CN f1-14.