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PURIFICATION AND CHARACTERIZATION OF AN ENDOPEPTIDASE FROM PSEUDOMONAS FLUORESCENS ATCC 948
Author(s) -
GOBBETTI MARCO,
SMACCHI EMANUELE,
STEPANIAK LESZEK,
CREA FRANCESCA,
FOX PATRICK F.
Publication year - 1998
Publication title -
journal of food biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.507
H-Index - 47
eISSN - 1745-4514
pISSN - 0145-8884
DOI - 10.1111/j.1745-4514.1998.tb00228.x
Subject(s) - endopeptidase , pseudomonas fluorescens , chemistry , hydrolysis , biochemistry , dithiothreitol , peptide , sepharose , casein , enzyme , chromatography , bacteria , biology , genetics
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 Co 2+ . 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 80C value 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' 1 position. 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 .