Purification and characterization of an endopeptidase from Lactococcus lactis

1 Department of Chemistry and Biochemistry, Massey University, Palmerston North, New Zealand * New Zealand Dairy Research Institute, Palmerston North, New Zealand An endopeptidase has been purified from Lactococcus lactis subsp. crernoris SK11. The enzyme is a 70 kDa monomer, strongly inhibited by the metalloproteinase inhibitors 1,l O-phenanthroline and phosphoramidon but relatively insensitive to EDTA. It is not significantly inhibited by the thiol enzyme inhibitor plchloromercuribenzoate nor by the serine protease inhibitor phenylmethylsulphonyl f luoride. The action of the endopeptidase in catalysing the hydrolysis of several peptide hormones has been studied and the hydrolysis products identified by sequence analysis. The enzyme catalyses hydrolysis of peptide bonds in which a hydrophobic amino acid (most commonly a Phe or Leu) residue occupies the position immediately C-terminal to the hydrolysed bond. It thus has a specificity very similar to that of thermolysin. T w o of the oligopeptides produced during the early stages of & casein digestion by the lactococcal cell-wall proteinases were hydrolysed by the endopeptidase, the others were resistant to hydrolysis. Cell fractionation studies have shown that the distribution of endopeptidase activity between the different cell fractions is the same as that of the intracellular marker enzyme fructose bisphosphate aldolase, and thus indicate a cytoplasmic location for the enzyme. These observations argue against a role for th is enzyme in the early stages of casein breakdown by the lactococcal proteolytic s ys tem .