A dual-function chymotrypsin-like serine protease with plasminogen activation and fibrinolytic activities from the GRAS fungus, Neurospora sitophila

Abstract In this study, we have isolated and characterized a fibrinolytic enzyme from the GRAS (Generally Recognized as Safe) fungus, Neurospora sitophila . The enzyme was purified by fractional ammonium sulfate precipitation, hydrophobic interaction, ion exchange and gel filtration chromatography to 45.2 fold with a specific activity of 415.6 U/mg protein. The native molecular mass of the enzyme was 49 kDa, while the denatured molecular mass was 30 kDa and 17.5 kDa, indicating that the enzyme was a hetero-dimer. It was optimally active at 50 °C and pH 7.4 and stable at human physiological temperature and pH. It was found to be a chymotrypsin-like serine protease which cleaved the synthetic chromogenic substrate, N -Succinyl-Ala-Ala-Pro-Phe-pNA for which the apparent K m and V max values were 0.24 mM and 4.17 × 10 −5  mM/s, respectively. The enzyme hydrolyzed all the chains of fibrinogen by cleaving α chain first, followed by β chain and then γ chain. Moreover, the enzyme possessed dual function of direct fibrinolysis as well as plasminogen activation. Due to its attractive biochemical and fibrinolytic properties and being from a GRAS fungus, the fibrinolytic enzyme has application as a safe and efficient thrombolytic drug.