Purification and characterization of a cysteine proteinase with low activation energy from Lactococcus lactis ssp lactis IAM 1198

A caseolytic proteinase with low activation energy was purified to homogeneity by conventional chromatographic techniques from Lactococcus lactis ssp. lactis IAM 1198. The molecular weight of the enzyme was estimated to be 12,000 by gel filtration and SDS-PAGE. Activity was maximal at pH 5.5 to 6.0 and 30 degrees C. The proteinase, LLP-II C-2, was almost stable from pH 5.0 to 7.0 and below 30 degrees C. The enzyme seemed to be a cysteine proteinase because it was inhibited by p-chloromercuriphenyl sulfonic acid and monoiodoacetic acid. The Michaelis constant, maximum velocity, and activation energy of LLP-II C-2 for whole casein were .071%, .018 mu g of tyrosine equivalent/min per mg, and 11,500 cal/mol, respectively. The proteinase is thought to play a major role in proteolysis during cheese ripening, which is carried out at a low temperature and for a long time, because the enzyme showed lower activation energy than those (26,000 similar to 28,000 cal/mol) of other coexisting proteinases.