Identification and Characterization of Lactobacillus helveticus PepO2, an Endopeptidase with Post-Proline Specificity

Abstract

A post-proline endopeptidase (PepO2) was detected in cell extracts from a genomic library of Lactobacillus helveticus CNRZ32 by using the synthetic substrate N-acetyl-␤-casein-(f203-209)--nitroanilide in a coupled reaction with aminopeptidase N. Isolates with activity for this substrate contained plasmids with visually indistinguishable restriction profiles. Nucleotide sequence analysis revealed a 1,947-bp open reading frame, designated pepO2, encoding a putative 71.4-kDa protein. Analysis of the predicted peptide sequence revealed that L. helveticus PepO2 contained the zinc-dependent metalloprotease motif HEXXH and exhibited levels of amino acid sequence similarity of 72, 61, 59, and 53% to L. helveticus PepO, Lactococcus lactis PepO2, L. lactis PepO, and Lactobacillus rhamnosus PepO, respectively. Northern hybridization results indicated that the transcript containing pepO2 was monocistronic. Despite the high degrees of amino acid similarity to PepO proteins from other lactic acid bacteria, the specificity of the L. helveticus PepO2 for post-proline bonds distinguishes it from other PepO-type endopeptidases characterized to date. The specificity for post-proline bonds also suggests that this enzyme may play a central role in the hydrolysis of casein-derived bitter peptides, such as ␤-casein(f193-209).The proteolytic systems of dairy lactic acid bacteria (LAB) have received extensive research attention due to their importance in the physiology of these organisms and in cheese flavor development. LAB are fastidious microorganisms with multiple amino acid auxotrophies (18). During growth in milk, LAB rely on their proteolytic systems to obtain essential amino acids from caseins (CNs), the most abundant proteins in milk (9, 17). Additionally, proteolytic enzymes from LAB produce flavor compounds and precursors that are essential for cheese flavor development (9, 23).The proteolytic systems of LAB can be functionally divided into three components: (i) cell envelope-associated proteinases which hydrolyze CNs to oligopeptides; (ii) peptide transport systems, of which the oligopeptide transport system is the most important in milk and cheese; and (iii) numerous intracellular peptidases (9, 17). The intracellular peptidases of LAB include both endopeptidases and aminopeptidases. Endopeptidases, due to their ability to hydrolyze peptide bonds within a peptide, are of particular interest in targeting peptides for rapid hydrolysis. In Lactococcus lactis, the best-characterized LAB, the endopeptidases that have been identified include PepO, PepO2, PepF1, and PepF2. All of these enzymes are metalloproteases, and PepO, PepF1, and PepF2 are encoded in operons (9, 17). The physiological roles of these endopeptidases remain unclear; however, PepF appears to be important for protein turnover during nitrogen starvation (24). To date, one metalloendopeptidase, designated PepO (8), and a thiol-dependent endopeptidase, designated PepE (13), have been characterized from Lactobacillus helveticus.The ability of L. helveticus CNRZ32 to ...

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

“…Ligation mixtures were transformed into E. coli ABLE C, and then representative transformants with each plasmid were identified via direct colony PCR using the respective gene-specific primers. Endopeptidase activity was confirmed with chromogenic substrates as described by Chen et al (5).…”

“…Cells were broken by vortexing with 300 mg of glass beads for 3 min using a Turbomix attachment to a Vortex Genie 2 (Scientific Industries, NY). CFEs from E. coli expressing CNRZ32 pepE, pepE2, pepF, and pepO3 transcriptional fusion genes were assayed for endopeptidase activity against chromogenic substrates as described by Chen et al (5). CFE protein concentrations were determined using the protein assay kit I from Bio-Rad with bovine serum albumin as the protein standard.…”

“…While numerous enzymes of the proteolytic system of L. helveticus CNRZ32 have been identified (8), our understanding of the specific enzymes responsible for this strain's ability to reduce bitterness in cheese is incomplete. As part of this endeavor, previously characterized L. helveticus CNRZ32 aminopeptidase PepN and endopeptidase PepO2 were shown to hydrolyze model bitter peptides, ␤-casein (␤-CN) (f193-209) and ␣ S1 -casein (␣ S1 -CN) (f1-9) (5,8). However, the hydrolysis specificities of previously characterized CNRZ32 endopeptidases PepE, PepO, and PepO2 toward these peptides under cheese-ripening conditions (pH 5.1, 4% NaCl, and 10°C) and in the presence of other cheese-derived peptides is largely unknown.…”

The Use of Different Proteases to Hydrolyze Gliadins

“…In addition, other specific endopeptidases with cleavage activity for Pro-residues of different substrates have been reported, e.g. endopeptidase from Lactobacillus helveticus CNRZ32 (Chen et al, 2003), metalloendoproteinase from Penicillium citricum (Doi et al, 2004) and prolyl-endoprotease from Aspergillus niger (Stepniak et al, 2006). The latter one is promising tool for celiac disease therapy.…”

Hydrolysis of Casein-Derived Peptides α S1 -Casein(f1-9) and β-Casein(f193-209) by Lactobacillus helveticus Peptidase Deletion Mutants Indicates the Presence of a Previously Undetected Endopeptidase

“…Hydrolysis samples were separated and collected by using RP-HPLC as described in the accompanying paper (5). The masses of RP-HPLC-separated peptide fractions were determined by using a Perkin-Elmer API 365 triple quadrupole electrospray ionization mass spectrometer or a Bruker Reflex II instrument for matrix-assisted laser desorption ionizationtime of flight mass spectrometry.…”