Least hemolytic, 12.6 kDa, plasmin-like fibrinolytic protease from marine Penicillium steckii KU1.

Abstract

A novel fibrinolytic enzyme, from the marine fungus Penicillium steckii KU1, was purified to electrophoretic homogeneity. The fibrinolytic protease was purified to 13.56 times with a specific activity of 57.64 U/mg and final yield of 13.93 %. It was found to be a monomeric protein of 12.6 kDa, having optimum activity at 30 °C and pH 8.0. It is a plasmin-like enzyme, showing resemblance to ATP-dependent zinc metalloprotease with isoelectric point (pI) 8.0. Its activity is enhanced by Zn²⁺, and inhibited by ethylenediaminetetraacetic acid (EDTA), Co²⁺ and Fe²⁺. The enzyme interaction with substrate azocasein was endothermic and with inhibitor EDTA exothermic. The K_m, V_max, K_cat and catalytic efficiency of the enzyme for azocasein were determined to be 142.71 μg mL^-1, 285.71 μg min^-1 mL^-1, 6.35 S^-1 and 4.45 × 10^-2 S^-1 μg^-1 mL respectively. It hydrolyzed all three chains of fibrinogen within 9 h, and dissolved fibrin completely within 24 h. 2 mg/mL enzyme could dissolve blood clot completely within 30 min, with negligible hemolysis (2.60 %). Lowering the immunogenicity by the application of natural or engineered small proteins is a strategy to enhance the safety and efficacy of thrombolytic therapy. Hence, the present 12.6 kDa, plasmin-like fibrinolytic enzyme appears worthy of further investigations towards a thrombolytic therapeutic.