Purification and characterization of a fibrinolytic enzyme from the food-grade fungus, Neurospora sitophila

Abstract

A fibrinolytic protease was purified from the culture supernatant of a GRAS fungus, Neurospora sitophila. The enzyme displayed a molecular mass of 34 kDa, as estimated by SDS-PAGE and gel filtration chromatography. The isoelectric point (pI) of the enzyme was 9.3 ± 0.2 as determined by iso-electric focusing (IEF). It was maximally active at pH 7.6 and 41 °C and displayed remarkable stability in a wide pH range (4–11) and up to 52 °C. The enzyme activity was inhibited by phenylmethane sulfonyl fluoride (PMSF) and ethylenediamine tetracetic acid (EDTA), indicating that it is a metal-dependent serine protease. It was found to be a direct acting plasmin like protein which efficiently cleaved the α-chain of fibrin(ogen), followed by β-chain and γ-chain. Three internal peptide sequences LASTANSGVLSGLLAGTVGGK; AYTSKSSVPSSVGLAR; LLDTGLNTAHSDFNR were determined by Q-TOF2. These results indicate no sequence similarities with other fibrinolytic enzymes suggesting it to be a novel enzyme. This fibrinolytic enzyme may be developed as a safe potential candidate for oral administration as a functional food additive or as a drug for prevention and/or treatment of thrombolytic diseases.