Purification and Characterization of Cold-Adapted Metalloprotease from Deep Sea Water Lactic Acid Bacteria Enterococcus Faecalis TN-9

This paper investigated a 3-step purification and characterization of a protease from Enterococcus faecalis TN-9, a bathypelagic lactic acid bacteria. The purification procedure includes precipitation with (NH4)2SO4, then ion-exchange chromatography with DEAE-Sephadex A-25 and DEAE Cellulofine A-500. Native PAGE analysis indicates a single protease band. The molecular weight is 30 kDa by SDS-PAGE analysis, and 69 kDa by gel chromatography analysis. It proves that the optimal temperature for protease reaction is 30 oC, and the optimal pH is 7.5-8.0. The reaction is stable while pH is 6.0-9.5 and temperature is under 45 oC. The relative activity is 6.1% at 0 oC. The enzyme is totally deactivated with heat treatment at 60 oC or over. The protease is partially inhibited by EDTA-2Na, Hg, Cu, Ni, Ag, Co and Pepstatin A. Zn shows obvious activation to the protease. Km and Vmax of purified protease acting on azocasein are 0.098 % and 72 mg/(h.mg) respectively. This protease is one of gelatinase with N-terminal sequence of VGSEVTLKNS, and shows characteristics of a cold-adapted metalloprotease.