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 ...

A previously identified insert expressing an endopeptidase from aLactobacillus helveticus CNRZ32 genomic library was characterized. Nucleotide sequence analysis revealed an open reading frame of 1,941 bp encoding a putative protein of 71.2 kDa which contained a zinc-protease motif. Protein homology searches revealed that this enzyme has 40% similarity with endopeptidase O (PepO) fromLactococcus lactis P8-2-47. Northern hybridization revealed that pepO is monocistronic and is expressed throughout the growth phase. CNRZ32 derivatives lacking PepO activity were constructed via gene replacement. Enzyme assays revealed that the PepO mutant had significantly reduced endopeptidase activity when compared to CNRZ32 with two of the three substrates examined. Growth studies indicated that PepO has no detectable effect on growth rate or acid production by Lactobacillus helveticusCNRZ32 in amino acid defined or skim milk medium.