Identification of Endopeptidase Genes from the Genomic Sequence of Lactobacillus helveticus CNRZ32 and the Role of These Genes in Hydrolysis of Model Bitter Peptides

Abstract

Genes encoding three putative endopeptidases were identified from a draft-quality genome sequence of Lactobacillus helveticus CNRZ32 and designated pepO3, pepF, and pepE2. The ability of cell extracts from Escherichia coli DH5␣ derivatives expressing CNRZ32 endopeptidases PepE, PepE2, PepF, PepO, PepO2, and PepO3 to hydrolyze the model bitter peptides, ␤-casein (␤-CN) (f193-209) and ␣ S1 -casein (␣ S1 -CN) (f1-9), under cheese-ripening conditions (pH 5.1, 4% NaCl, and 10°C) was examined. CNRZ32 PepO3 was determined to be a functional paralog of PepO2 and hydrolyzed both peptides, while PepE and PepF had unique specificities towards ␣ S1 -CN (f1-9) and ␤-CN (f193-209), respectively. CNRZ32 PepE2 and PepO did not hydrolyze either peptide under these conditions. To demonstrate the utility of these peptidases in cheese, PepE, PepO2, and PepO3 were expressed in Lactococcus lactis, a common cheese starter, using a high-copy vector pTRKH2 and under the control of the pepO3 promoter. Cell extracts of L. lactis derivatives expressing these peptidases were used to hydrolyze ␤-CN (f193-209) and ␣ S1 -CN (f1-9) under cheese-ripening conditions in single-peptide reactions, in a defined peptide mix, and in Cheddar cheese serum. Peptides ␣ S1 -CN (f1-9), ␣ S1 -CN (f1-13), and ␣ S1 -CN (f1-16) were identified from Cheddar cheese serum and included in the defined peptide mix. Our results demonstrate that in all systems examined, PepO2 and PepO3 had the highest activity with ␤-CN (f193-209) and ␣ S1 -CN (f1-9). Cheese-derived peptides were observed to affect the activity of some of the enzymes examined, underscoring the importance of incorporating such peptides in model systems. These data indicate that L. helveticus CNRZ32 endopeptidases PepO2 and PepO3 are likely to play a key role in this strain's ability to reduce bitterness in cheese.The proteolytic enzyme system of lactic acid bacteria (LAB) includes diverse enzymes whose primary physiological functions involve housekeeping needs and acquisition of essential amino acids to support growth (9). Intracellular peptidases of LAB consist of both endopeptidases and aminopeptidases. Endopeptidases, due to their ability to hydrolyze peptide bonds within a peptide, are of particular interest, since they promote hydrolysis of peptides that are resistant to aminopeptidase activity. The proteolytic enzymes of LAB are also of practical interest because they play a major role in cheese maturation and flavor.Bitterness, a common flavor defect in Cheddar and Gouda cheeses, results from the accumulation of hydrophobic bitter peptides to concentrations higher than their taste threshold. Formation of these peptides during cheese ripening is directly related to the activity and specificity of the cell envelope proteinase and chymosin (2,14,24). Degradation of these peptides is related to the activity of peptidases derived from the starter and nonstarter bacteria present (24). Bitter peptides typically contain relatively high levels of proline (24, 36), underscoring the importance of proline-spe...

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

“…helveticus CNRZ32 has been previously reported (Chen et al, 2003;Sridhar et al, 2005). In contrast to our findings, Sridhar et al (2005) reported that PepO of Lb. helveticus CNRZ32 did not possess activity on b-CN f193-209 under cheese ripening conditions (pH 5.1, 4% NaCl, and 10 1C).…”

“…PepE did not show activity on b-CN f193-209, in agreement with Sridhar et al (2005). Table 5 lists mass-to-charge ratios (m/z) of MALDI-TOF peaks resulting from the combined action of CFEs from E. coli DH5a derivatives expressing PepE and PepN of Lb.…”

“…helveticus strains CNRZ32 and 53/7, characterization of the aminopeptidase and endopeptidases of Lb. helveticus WSU19 remains of interest (Chen & Steele, 1998;Chen, Christensen, Broadbent, & Steele, 2003;Christensen, Lin, Palva, & Steele, 1995;Dudley, Husgen, He, & Steele, 1996;Fenster, Parkin, & Steele, 1997;Fernandez, Bhowmik, & Steele, 1994;Shao, Yu¨ksel, Dudley, Parkin, & Steele, 1997;Sridhar, Hughes, Welker, Broadbent, & Steele, 2005;Vesanto, Savijoki, Rantanen, Steele, & Palva, 1995;Yu¨ksel & Steele, 1996). Lb.…”

Prolyl endopeptidases

“…Post-proline cleaving ability was detected for three enzymes (PepO2, PepO3, and PepF), PepO2 having the highest activity. Based on sequence comparison, PepO2 and PepO3 were also predicted to be metalloendopeptidases [52,53]. Recombinant L. helveticus PepO2, cloned and expressed in Escherichia coli, did not reveal activity against proline-rich, gluten-derived oligopeptides.…”

Angiotensin I-converting enzyme inhibitory activity in milk fermented by wild-type and peptidase-deletion derivatives of Lactobacillus helveticus CNRZ32

“…Even though the proteolytic system of L. helveticus (Christensen, Dudley, Pederson, & Steele, 1999) and the ability of this microorganism to hydrolyze bitter peptides (Christensen, Broadbent, & Steele, 2003;Sridhar, Hughes, Welker, Broadbent, & Steele, 2005) have been studied intensively in recent years, understanding of the specific enzymes responsible for the generation and stability of ACEinhibitory peptides is lacking. To evaluate the contributions of individual enzymes to peptide processing, it is necessary to construct recombinant L. helveticus strains differing only in the activity of known components of the proteolytic mechanism.…”