Production and purification of protease by fungus isolated from the Brazilian dry tropical forest (Caatinga)

The industrial demand for proteolytic enzymes is stimulating the search for new enzyme sources. Fungal enzymes are preferred over bacterial enzymes, and more effective and easy to extract. The aim of this work was the purification of proteases produced in solid state fermentation by Mucor subtilissimus UCP 1262. The crude extract was analyzed using 3 different proteolytic activities, namely collagenolytic (161.4 U/ml), keratinolytic (39.6 U/ml) and fibrinolytic (26.1 U/ml). After ammonium sulfate precipitation, the active fractions with fibrinolytic activity were dialyzed in 15 mM Tris-HCl buffer, pH 8, loaded onto DEAE-Sephadex A50 ion-exchange column and gel filtered through Superdex 75 HR10/300. The purified enzyme showed activity against a chromogenic chymotrypsin substrate, SDS-PAGE showing a molecular mass of approximately 70 kDa, and the specific activity of 25.93 U/mg. These characteristics suggest that the enzyme could be profitably and efficiently produced in large amounts for thrombolytic therapy.