BIOCHEMICAL AND GENETIC-CHARACTERIZATION OF PEPF, AN OLIGOPEPTIDASE FROM LACTOCOCCUS-LACTIS

Lactococcus lactis possesses a complex proteolytic system which is ess ential for its growth in milk. me characterized one of the peptidases of this system, oligopeptidase PepF, together with its structural gene . PepF hydrolyzed peptides containing between 7 and 17 amino acids wit h a rather wide specificity. It was purified to homogeneity. The N-ter minal sequences of PepF and of peptides resulting from tryptic digesti on of PepF were determined and used to design degenerate oligonucleoti des which served to amplify a DNA fragment internal to pepF. This frag ment was used as a probe to screen a lactococcal genomic library in Es cherichia coli and to clone the entire gene pepF. The gene coded for a 70 kDa protein and was located on a 55-kilobase lactose-protease plas mid. A motif His-Glu-X-X-His, characteristic of metallopeptidases was evidenced. Two regions of PepF were found similar, first to a stretch of 43 amino acids around the zinc-binding site of several other peptid ases, second to a stretch of 33 amino acids well conserved among creat ine and arginine kinases. Preliminary results suggest the presence of a second copy of pepF.