Expression of Six Peptidases from Lactobacillus helveticus in Lactococcus lactis

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...

Section: Discussionmentioning

confidence: 99%

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

Sridhar

Hughes

Welker

et al. 2005

“…This revealed that both proteolytic and biosynthetic enzymes could be expressed through the same mRNA (35). Recently, six L. helveticus peptidase-encoding genes, including pepR, were expressed in Lactococcus lactis under the control of their own promoters (16). pepD and pepR were the only two peptidases for which no expression in Lactococcus lactis was obtained.…”

Section: Discussionmentioning

confidence: 99%

The pepR Gene of Lactobacillus sakei Is Positively Regulated by Anaerobiosis at the Transcriptional Level

Champomier‐Vergès¹,

Marceau²,

Méra³

et al. 2002

Lactobacillus sakei is a lactic acid bacterium belonging to the natural flora of meat products. It constitutes the main flora of vacuum-packed meat and is largely used in western Europe as a starter for the manufacturing of fermented sausages. This species is able to grow both under aerobiosis and anaerobiosis. In many technological processes involving it, oxygen is scarce. The aim of this study was to identify the major proteins affected by growth under anaerobiosis. Using two-dimensional electrophoresis, we showed that one spot was 10-fold overexpressed when cells were grown under anaerobiosis. By N-terminal sequencing it was identified as a peptidase (PepR), and the pepR gene was cloned. Northern analysis revealed that pepR was expressed as a single 1.27-kb transcript induced under anaerobiosis. A mutant was constructed by single crossover in the pepR gene, and its growth and survival were not affected by anaerobiosis.

“…The absence of an outer membrane in these gram-positive microorganisms makes them particularly attractive for the display or secretion of biologically active molecules such as antigens, enzymes, or cytokines (4,19,30,31). In addition to being considered nonpathogenic, LAB show a remarkable ability to survive passage along the oral route, as documented by their active metabolism in most of the compartments of the gastrointestinal tract (3).…”

Section: Discussionmentioning

confidence: 99%

Lactobacillus bulgaricus Proteinase Expressed in Lactococcus lactis Is a Powerful Carrier for Cell Wall-Associated and Secreted Bovine β-Lactoglobulin Fusion Proteins

Bernasconi

Germond

Delley

et al. 2002

Lactic acid bacteria have a good potential as agents for the delivery of heterologous proteins to the gastrointestinal mucosa and thus for the reequilibration of inappropriate immune responses to food antigens. Bovine ␤-lactoglobulin (BLG) is considered a major allergen in cow's milk allergy. We have designed recombinant Lactococcus lactis expressing either full-length BLG or BLG-derived octapeptide T6 (IDALNENK) as fusions with Lactobacillus bulgaricus extracellular proteinase (PrtB). In addition to constructs encoding fulllength PrtB for the targeting of heterologous proteins to the cell surface, we generated vectors aiming at the release into the medium of truncated PrtB derivatives lacking 100 (PrtB٢, PrtB٢-BLG, and PrtB٢-T6) or 807 (PrtB⌬) C-terminal amino acids. Expression of recombinant products was confirmed using either anti-PrtB, anti-BLG, or anti-peptide T6 antiserum. All forms of the full-length and truncated recombinant products were efficiently translocated, irrespective of the presence of eucaryotic BLG sequences in the fusion proteins. L. lactis expressing PrtB٢-BLG yielded up to 170 g per 10 9 CFU in the culture supernatant and 9 g per 10 9 CFU at the bacterial cell surface within 14 h. Therefore, protein fusions relying on the use of PrtB gene products are adequate for concomitant cell surface display and secretion by recombinant L. lactis and thus may ensure maximal bioavailability of the eucaryotic antigen in the gut-associated lymphoid tissue.An inappropriate immunological response to bovine milk proteins, including ␤-lactoglobulin (BLG), has been put forward as a key factor in cow's milk allergy (34). Studies have shown that a reequilibration of this response toward a state of specific oral tolerance can be achieved upon oral administration of partially hydrolyzed cow's milk formula or BLG peptides obtained by tryptic hydrolysis (8,22). Interestingly, the degradation of cow's milk proteins by lactic acid bacteria (LAB) used in food fermentation was also found to generate tolerogenic peptides, with a suppression of the specific lymphoproliferative response (32). In addition, certain LAB strains are more and more widely used as a vehicle for the delivery of heterologous proteins to the mucosal immune system (13,20). Therefore, it can be hypothesized that the introduction of LAB capable of expressing full-length or BLG fragments may prove them to be a useful tool to aid in host protection against allergic sensitization to this dietary antigen.Lactococcus lactis is a gram-positive, nonpathogenic bacterium that is widely used in industrial food fermentation. A noncolonizing bacterium, L. lactis has been shown to be quite resistant to gastric acidity when administered together with food, remaining metabolically active all the way through the digestive tract (3). Its use as an antigen delivery vehicle for mucosal immunization has been reported (1,4,26,31). Induction of oral tolerance might require a large amount of target antigen, in contrast to immune modulators such as cytokines (30,31). Therefo...