Abstract

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, Hg2+, Cu2+, Ni2+,
Ag2+, Co2+ and Pepstatin A. Zn2+ 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.

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