PURIFICATION AND CHARACTERIZATION OF AN ENDOPEPTIDASE FROM PSEUDOMONAS FLUORESCENS ATCC 948

Intracellular, ca 55 kDa monomeric endopeptidase (PsPepO) from Pseudomonas fluorescens ATCC 948 was purified to homogeneity by chromatography on Q-Sepharose, Sephacryl 200, Phenyl-Superose and Mono Q. It was strongly inhibited by the metalloproteinase inhibitor, 1,10 phenanthroline and by dithiothreitol, but less strongly by EDTA; it was stimulated by Co²⁺. Activity was optimal at pH 7.0 and 40–45C, with considerable activity at pH 5.0 and 12C. The enzyme was relatively heat-stable with a D_80Cvalue of 1.2 min. It did not hydrolyze α_s1-, β-or κ-casein (CN) and peptides with less than 5 amino acids but readily hydrolyzed α_s1-CNfl-23, α_s1-CN f157-164, α_s1-CN f165-199 and various β-CN fragments and peptide hormones. On α_s1-CN fl-23, α_s1-CN f165-199, insulin B-chain and bradykinin, it mainly catalyzed the hydrolysis of peptide bonds in which a hydrophobic amino acid (most commonly Phe or Leu) residue occupied the P'₁position. β-CN f193- or 194-209, which are the source of bitter peptides in cheese ripening, were hydrolyzed slowly or not at all. β-CN fragments from the sequence 58-70 were degraded or did not inhibit the endopeptidase activity as well as β-CN f193-209. The characteristics of the endopeptidase of Ps. fluorescens ATCC 948 were compared with those of lactococcal endopeptidases.