Purification and Characterization of an Extracellular Proteinase Having Milk-Clotting Activity from Enterococcus faecalis TUA2495L

Abstract

Lactic acid bacteria (157 stock cultures) were screened for their ability to produce extracellular proteinase with milk-clotting activity. A strain identified as Enterococcus faecalis TUA2495L showed the highest ratio of milk-clotting activity (MCA) to proteinase activity (PA). The molecular weight of the purified enzyme from the strain was estimated to be 34–36 kDa by gel filtration and by SDS-PAGE. Its isoelectric point was about 5.4, and the K_m value on casein (Hammarsten) was 0.61% (w/v). The optimum temperature was 70°C for MCA and 50°C for PA. The MCA increased with a decrease in the pH from 7.8 to 5.8 but the PA was optimum at pH 8.0–9.0. Both MCA and PA were stable within the pH range of 5.5–10.0. The enzyme was inhibited by heavy metal ions (Fe², Cd², Ni², Cu² and Al³), SDS and EDTA. Reactivation of the enzyme activity with Co², Mn² or Zn² indicates the importance of these metals in the catalytic function of the enzyme. The enzyme was especially active on κ-casein, and SDS-PAGE analysis showed that the degradation patterns of κ-casein by Ec. faecalis enzyme and Mucor miehei milk-clotting enzyme were almost the same.