Purification and Characterization of an Endopeptidase from Propionibacterium freudenreichii

A 44-kDa endopeptidase, isolated by lysozyme and sonic treatment from the cytoplasm of Propionibacterium freudenreichii ATCC 9614, was purified to homogeneity. Ion-exchange chromatography of the cytoplasmic fraction separated the enzyme from several fractions with caseinolytic activities and one fraction with proline iminopeptidase activity. The endopeptidase was subsequently purified by chromatography and by gel filtration. The enzyme was a monomer with a pI of 3.8, and the enzyme hydrolyzed bradykinin most actively between pH 6.5 and 8 and between 45 and 50°C. The specificity of the Propionibacterium endopeptidase on bradykinin, methionine enkephalin, angiotensin I, and αs1-CN (f1-23) was determined; specificity on αs1-CN (f1-23) was different from that of the 70-kDa endopeptidase (PepO) from Lactococcus spp. The activity on oxidized insulin β-chain was very low. The enzyme was inhibited more by EDTA than by 1,10-phenanthroline and was not sensitive to phosphoramidon.