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

• Published in: International journal of biological macromolecules 2024-12, Vol.283 (Pt 3), p.137854, Article 137854
• Main Authors: Kunhiraman, Swapna, Haridas, Madhathilkovilakathu, Basheer, Soorej M., Chellappan, Sreeja, Abdulhameed, Sabu
• Format: Article
• Language: English
• Subjects: Exothermic reaction; Fibrinolytic enzyme; Hemolysis; Metalloprotease; Plasmin-like enzyme
• ISSN: 0141-8130; 1879-0003; 1879-0003
• DOI: 10.1016/j.ijbiomac.2024.137854

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 Km, Vmax, Kcat 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. •Fibrinolytic protease from the marine fungus Penicillium steckii KU 1 was purified to homogeneity.•The enzyme is a metalloprotease, 12.6 kDa, pI 8.0, optimum activity pH and temperature 8.0 and 30 °C respectively.•Zn2+ is enhancer and Co2+ and Fe2+ were inhibitors of the enzyme.•Mode of fibrin degradation is reported and 2 mg/mL is sufficient to dissolve blood clot within 30 min.•It showed negligible rate of hemolysis (2.60 %).