Purification and characterization of a 33 kDa subunit oligopeptidase from Propionibacterium freudenreichii ATCC 9614

A non-caseinolytic 33kDa subunit, tetrameric intracellular oligopeptidase from Propionibacterium freudenreichii ATCC 9614 was purified to homogeneity by chromatography on Fast Q Sepharose, hydroxyapatite and Mono Q. Like oligopeptidase F from Lactococcus and the 30kDa subunit olipopeptidase from Lactobacillus, the oligopeptidase from Propionibacterium did not hydrolyze the pentapeptide, methionine enkephalin, and had very low activity on peptides containing 17 or more amino acid residues. It also had very low activity on αs1-casein f90-95, β-casomorphin-7 and β-casein f58-72. The Propionibacterium oligopeptidase had a different specificity on bradykinin and substance P than the oligopeptidase F from Lactococcus and the oligopeptidase from Lactobacillus; it released N-terminal Arg from these two peptides, but did not hydrolyze Arg-, Leu-, Pro- and Gly-Pro-p-nitroanilides. The N-terminal sequence of the enzyme was X-X-Ile-Pro-Ile-Thr-Pro. The Propionibacterium oligopeptidase was strongly inhibited by o-phenanthroline and partially by p-chloromercuribenzoate. The enzyme was not inhibited by β-casein f58-72. It was most active at pH 6.7–7.5 and at 40–50°C, but it retained c. 20% of maximal activity at 7°C and pH 5.5, indicating that it may be active in cheese during ripening.