Purification and Biochemical Characterization of a Novel Fibrinolytic Enzyme from edible mushroom Lentinula edodes

Abstract

The crude fibrinolytic enzyme was purified to full homogeneity using fractional precipitation by ammonium sulphate, DEAE cellulose chromatography and gel filtration on Sephadex-G100. An over all of 69% fold purification with 2.5 recovery was obtained. The apparent molecular mass of the purified enzyme was estimated to be 45 kDa using SDS-PAGE. The optimum pH and temperature of the purified enzyme were 6 and 35˚C, respectively. The fibrinolytic enzyme was completely inhibited by Hg²⁺ and partially inhibited by Cu²⁺, Ni²⁺ and Al³⁺. Its activity strongly enhanced by Zn²⁺, Mg²⁺, Ca²⁺, K⁺ and Fe²⁺ in descending order. EDTA and EGTA inhibited the enzyme activity suggesting that it is a metalloprotease. The enzyme was also inhibited by the serine inhibitors PMSF and aprotinin. The pure enzyme showed strong specificity to fibrin as substrate in vitro. It was also specific to gelatin and fibrinogen but not specific to casein, elastin and egg albumin. The amidolytic activity toward synthetic substrates showed high specificity to the synthetic peptide N-Succinyl-Ala-Ala-Pro-Phe-pNA suggesting that it is a chymotrypsin-like protease. Additionally, it was discovered that the enzyme decreased fibrinogen (FIB) and prothrombin activity (PA) levels while prolonging activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT). These investigations suggest that the enzyme has a lot of promise for use as a natural remedy to cure and prevent thrombotic illnesses.