The proteolytic system of Bifidobacterium animalis subsp. lactis was analyzed, and an intracellular endopeptidase (PepO) was identified and characterized. This work reports the first complete cloning, purification, and characterization of a proteolytic enzyme in Bifidobacterium spp. Aminopeptidase activities (general aminopeptidases, proline iminopeptidase, X-prolyl dipeptidylaminopeptidase) found in cell extracts of B. animalis subsp. lactis were higher for cells that had been grown in a milk-based medium than for those grown in MRS. A high specific proline iminopeptidase activity was observed in B. animalis subsp. lactis. Whole cells and cell wall-bound protein fractions showed no caseinolytic activity; however, the combined action of intracellular proteolytic enzymes could hydrolyze casein fractions rapidly. The endopeptidase activity of B. animalis subsp. lactis was examined in more detail, and the gene encoding an endopeptidase O in B. animalis subsp. lactis was cloned and overexpressed in Escherichia coli. The deduced amino acid sequence for B. animalis subsp. lactis PepO indicated that it is a member of the M13 peptidase family of zinc metallopeptidases and displays 67.4% sequence homology with the predicted PepO protein from Bifidobacterium longum. The recombinant enzyme was shown to be a 74-kDa monomer. Activity of B. animalis subsp. lactis PepO was found with oligopeptide substrates of at least 5 amino acid residues, such as met-enkephalin, and with larger substrates, such as the 23-amino-acid peptide ␣ s1 -casein(f1-23). The predominant peptide bond cleaved by B. animalis subsp. lactis PepO was on the N-terminal side of phenylalanine residues. The enzyme also showed a post-proline secondary cleavage site.Bifidobacteria are gram-positive anaerobic bacteria commonly found in the human intestinal tracts of mammals. Many bifidobacterium-containing dairy products have been developed due to their reported health-promoting effects. These organisms are employed to increase the beneficial properties of fermented milks, infant formulas, cheese, and ice cream (10,28,31,43). One of the strains commonly used in the industry is Bifidobacterium animalis subsp. lactis, which is particularly suitable due to its technological properties such as tolerance to oxygen, acid resistance, and ability to grow in milk-based media (20,30,33).Information in the literature regarding the metabolism of bifidobacteria focuses mainly on their glycolytic capabilities, since these organisms have been reported to grow well on oligosaccharide-based substrates (21, 49). Analysis of the genome sequence of Bifidobacterium longum NCC2705 revealed a large number of predicted proteins specialized for oligosaccharide metabolism (45). Genetic and biochemical characterization of Bifidobacterium glycosyl hydrolases has identified several enzymes that utilize nondigestible oligosaccharides as substrates (19,21,22,26). On the other hand, very little is known about the proteolytic enzyme systems of Bifidobacterium spp. However, analysis of the B. long...
