Characterization of a novel extracellular thermostable metalloprotease from Pseudomonas fluorescens migula
A novel extracellular thermostable metalloprotease was isolated from Pseudomonas fluorescens Migula, and the enzyme was estimated to have a molecular of weight of approximately 49.5kDa, pI of 4.25. The thermostability of this purified protein showed its activity even at an elevated temperature of 121°C for 15 minutes, and the LC50 value of purified protein was found to be 0.03µg/ml for both pre- and post-treatment against the pupae of Cx. quinquefasciatus. The enzyme activity was strongly inhibited by chelating agents such as EDTA and 1-10-phenanthroline, and activated by certain detergents. The metal ions Na+, Cu2+, Ca2+, Mg2+,Zn2+ and K+ enhanced the action of the enzyme, other metal ions such as Hg2+, Fe2+, Co2+, Mn2+, Ni2+, Sn+, Ba2+, and Al2+ reduced the activity of enzyme. EDTA inhibited the enzyme activity, and Zn2+, Cu2+ ions enhanced the enzyme activity, thus shows the nature of the enzyme as a metalloprotease. The enzyme was stable in the presence of detergents, surfactants, and organic solvents and hydrolysis the casein, fibrin, gelatine, elastin, and collagen. The Vmax and Km values of casein hydrolysis were 0.67µmole/min and 5.10 mM, respectively. The amino acid sequence of protease is homologous with the sequence of metalloprotease M36 and the serralysin family. Thus, the results suggest that the extracellualr enzyme represents a novel thermostable metalloprotease with fibrinolytic activity.