Partial purification and characterization of intracellular proteinases from Lactococcus lactis subsp lactis MG1363

Abstract

Summary - Three caseinolytic proteinases (P1, P2, P3) were isolated from the cytoplasm of plasmid-free Lactococcus lactis subsp lactis MG1363 by sequential chromatography on OEAE-cellulose, Sephacryl 200, CM-cellulose, Phenyl Sepharose or hydroxyapatite and MonoQ ion exchanger. Minor caseinolytic fractions were separated during successive purification steps and the final preparations, although free from aminopeptidase activities, showed impurities by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SOS-PAGE), indicating autolysis or a very complex caseinolytic system. Proteinase P1 was a dimer with molecular mass (Ms) of ca 66 kOa by SOS-PAGE and 124 kOa by gel filtration and it was most sensitive to EOTA. Proteinase P2 (Ms ca 68 kOa by SOS-PAGE and 64 kOa by gel filtration) and P3 (Ms ca 52 kOa SOS-PAGE and 47 kOa by gel filtration) were strongly inhibited by phenylmethylsulfonyl fluoride. Proteinase P1 was most active at pH 7.0 and 35 oC, while proteinases P2 and P3 had optima at pH 7.5 and 45 C. Urea-PAGE of digests of Œ- or f3-casein showed dilferences in the peptide pattem released by the three proteinases alter 6 or 20 h incubation. The peptides released by P1, P2 and P3 were also dilferent from those produced by a crude preparation of the cell envelope-associated proteinase from L lactis subsp lactis 712, the parental strain of L lactis subsp lactis MG1363. Reverse-phase fast protein Iiquid chromatography also showed dilferences between peptides released from Œs,-casein by P1, P2 and P3.