Purification and characterization of an endopeptidase from Lactococcus lactis subsp. cremoris SK11

Abstract

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 this enzyme in the early stages of casein breakdown by the lactococcal proteolytic s ys tem .

The proteolytic systems of lactic acid bacteria

“…Similar enzymes as the ones described above have been purified from other strains ofL. lactis (Muset et al, 1989;Baankreis 1992;Pritchard et al, 1994).…”

Bioprospecting for Bioactive Peptide Production by Lactic Acid Bacteria Isolated from Fermented Dairy Food

“…Endopeptidases are probably the first enzymes involved in the processing of peptides transported inside the cells. PepG, PepE, and its paralog PepE2 are cysteine-peptidases, whereas PepF and PepO have been classified as metallopeptidases [248][249][250][251][252]. At least in L. helveticus, two paralogs of PepO, named PepO2 and PepO3, with different specificity have been characterized [250,253].…”

Cloning and Expression of an Oligopeptidase, PepO, with Novel Specificity from Lactobacillus rhamnosus HN001 (DR20)

“…The peptide ␣ s1 -casein f(1-17) was extracted from an 8-week-old Cheddar cheese by the hot water extraction method of Kuchroo and Fox (18) and purified by the RP-HPLC method of Exterkate and Alting (12). The method used for the hydrolysis of all substrates was essentially identical to that described by Pritchard et al (34). Enzyme activity was deduced from the decreasing concentrations of substrate as detected by peptide absorbance at 214 nm after separation of substrate and products by RP-HPLC.…”