Endopeptidases of Lactobacillus helveticus CNRZ32: Identification and characterization.

摘要

Lactobacillus helveticus CNRZ32 when used as a starter adjunct has been shown to accelerate cheese flavor development, enhance flavor intensity, and reduce bitterness. Bitterness in cheese is believed to result from the accumulation of low molecular weight hydrophobic peptides, such as α _s1-casein (CN) (f1–9) and β-CN(f193–209). It is believed that CNRZ32 endopeptidase(s) are required for this strain to efficiently hydrolyze bitter peptides, thereby reducing bitterness. This dissertation reports the identification and characterization of two genetically similar endopeptidases (PepO and PepO2) in Lb. helveticus CNRZ32.; CNRZ32 PepO and PepO2 were identified by screening a genomic library with the synthetic substrates, N-benzoyl-Phe-Val-Arg-ρ-nitroanilide (NA), N-benzoyl-Pro-Phe-Arg-ρNA, and N-benzoyl-Val-Gly-Arg-ρNA, and a bitter peptide β-CN(f193–209) based substrate, N-acetyl-β-CN(f203–209)-ρNA, respectively. Nucleotide sequence analysis revealed a PepO open reading frame of 1941 by encoding a putative 71.2 kDa peptide and a PepO2 open reading frame of 1947 by encoding a putative 71.4 kDa peptide. Both endopeptidases contain a zinc-dependent protease motif, His-Glu-Xxx-Xxx-His, and hence were classified as metalloproteases. Protein homology searches revealed that PepO and PepO2 have 40% and 41% identity to Lactococcus lactis PepO, respectively, and PepO and PepO2 share 56% identity and 70% similarity.; The CNRZ32 PepO2 was found to have a substrate specificity distinct from previously reported endopeptidases from lactic acid bacteria. All of the bonds hydrolyzed in α_s1-CN(f1–9) and β-CN(f193–209) by PepO2 were either Pro-Val or Pro-Ile, indicating that PepO2 is a post-proline endopeptidase. The hydrolysis of peptide bonds involving Pro is likely to be particularly important in the hydrolysis of CN-derived peptides as Pro constitutes 16.7% of β-CN and 8.5% of α_s1-CN. Additionally, CN-derived bitter peptides have been observed to contain relatively large amounts of Pro. These results suggest that PepO2 has a central role in CNRZ32's demonstrated ability to reduce bitterness in cheese when used as a starter adjunct.; Six promoters from Lb. helveticus CNRZ32 were examined for their level of expression of the reporter β-glucuronidases in Lb. helveticus, Lb. casei and Lc. lactis. Significant differences were observed between the promoters examined. By selecting from these six promoters, the level of expression of a desired enzyme can be modulated.