Isolation and characterization of proline iminopeptidase from Propionibacterium freudenreichii ATCC 9614

A dimeric, 90 kDa subunit intracellular proline iminopeptidase from Propion ibacterium freudenreichii ATCC 9614 was purified to homogeneity by chromato graphy on hydroxyapatite, Sephacryl 200, Phenyl Superose and Mono Q. The en zyme was specific on Pro-p-nitroanilide and Pro-X dipeptides. It hydrolyzed 2 fragments of hormone oligopeptides with an N-terminal proline : bradykin in, f2-7 and substance P, f4-11. A number of oligopeptides containing 5-11 amino acids residues and proline at the penultimate position from N-terminu s or other internal position were not hydrolyzed. The enzyme was most activ e at pH 7-7.5 and at 37-40 degrees C but it retained 9% of maximal activity at pH 5.5 and >12% of maximal activity at 10 or 60 degrees C. The enzyme w as inhibited strongly by the serine protease inhibitor 3,4-dichloroisocouma rin, and stimulated markedly by 1 mol/l of NaCl. The results indicate that the enzyme may lead to the accumulation of proline from dipeptides and olig opeptides during the ripening of cheese.