Biochemical and molecular characterization of fibrinolytic enzyme purified from Pleurotus ostreatus

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 44 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. The PoFR-cDNA encoding gene was cloned in E. coli (α-DH5) and its nucleotide 
sequence was determined (GenBank accession no. AB551656). The PoFR-cDNA was found to 
consist of 845 bp in an Open Reading Frame (ORF) encoding 281 amino acids. The sequence 
showed high degree of homology with the fibrinolytic enzyme gene from P. ostreatus fruiting 
bodies (AY640032.1). In vivo assay using thrombus induced mice showed that the enzyme exerted 
thrombolysis in the mice blood marked by decrease in hematocrit percentage and prolonged 
prothrombin time (PT) and thrombin time (TT).