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Abstract Optimization of growth media components and the cultural conditions was carried out by using statistical factorial design L18 (21x37). 18 media were constructed with different composition and conditions. 2- In all media the fibrinolytic activity (F), proteolytic activity (P), F/P ratio, protein and specific activities were determined. 3- The highest mycelial extracellular fibrinolytic activity and high F/P ratio were obtained when P. ostreatus was cultured on medium (2) composed of (g/100ml): fibrin, 0.2; yeast extract, 1.5; glucose, 1.0; MgSO4, 0.1 and K2HPO4, 0.20; inoculums, 7.0 discs; initial pH, 6.0; Temperature, 35.0°C and incubation period 7 days. 4- Highest proteolytic with low F/P ratio was achieved on medium (6) composed of (g/100ml): fibrin, 0.3; yeast extract, 1.0; glucose, 1.0; MgSO4, 0.1 and K2HPO4, 0.20; inoculums, 5.0 discs; initial pH, 7.0; Temperature, 45.0°C and incubation period 7 days. 5- Based on the results of the factorial design experiment, extra medium was constructed to contain the optimal levels of all factors for fibrinolytic activity only. This medium was signaled as modified medium. Promising increase in fibrinolytic enzyme production exceeded that gained in medium (2) by 1.2 fold was observed with higher F/P ratio. Since this medium was selected as the optimum one for purification, characterization and molecular studies. 6- The extracellular fibrinolytic enzyme from P. ostreatus mycelium was purified to homogeneity using ammonium sulphate precipitation, DEAE-cellulose column and gel filtration on Sephadex G-100 column. 7- Three separate peaks of fibrinolytic activity were detected by DEAEcellulose, named FA, FB and FC. The composite sample of the three active peaks when applied on Sephadex G-100 showed one fraction Summary 90 peak with high fibrinolytic activity with 69% recovery and 2.5 purification fold. 8- The purified fibrinolytic enzyme (PoFR) from P. ostreatus mycelium showed a single band using SDS-PAGE. The apparent molecular mass was estimated to be 44 kDa. 9- Characterization of the pure fibrinolytic enzyme was done to include the effect of pH, temperature, metal ions, protease inhibitors, substrate specificity and amidolytic activity, in addition to Km and Vmax determination. 10- The optimum pH of the fibrinolytic enzyme was observed at pH 6. The enzyme was highly active at pH range 5-8 at 37°C for 1 h but below or above this range the enzyme activity decreased rapidly. 11- The optimum temperature of the fibrinolytic enzyme PoFR from P. ostreatus was 35°C. The enzyme activity was stable between 30-40°C. The enzyme remain active but with decreasing values up to 75°C. 12- The effect of metal ions indicated that the fibrinolytic enzyme completely inhibited by Hg2+ and partially inhibited by Cu2+, Ni2+ and Al3+. It strongly activated by Zn2+, Mg2+, Ca2+, K+ and Fe2+ in decreasing order. 13- EDTA and EGTA strongly inhibited the fibrinolytic activity of PoFR. The enzyme was also inhibited by serine protease inhibitors PMSF and aprotinin. The cysteine protease inhibitor TLCK and aspartic acid protease inhibitor pepstatin A exerted weak effect on the enzyme activity. 14- The purified fibrinolytic enzyme (PoFR) from P. ostreatus was appeared to be serine metalloprotease. 15- The fibrinolytic enzyme showed high specificity towards fibrin. This direct action toward fibrin degradation suggests it could be used as an Summary 91 effective thrombolytic agent. The enzyme was specific also to fibrinogen and gelatin but it could not hydrolyse casein, egg albumin and elastin. 16- The amidolytic activity for several synthetic substrate of PoFR showed that the strongest fibrinolytic activity was exhibited for the substrate NSuccinyl- Ala-Ala-Pro-Phe-pNA for (subtilisin and chymotrypsin). The enzyme also shows high activity for N-benzoyl-Phe-Val-Arg-pNA for (trypsin and thrombin). So the fibrinolytic purified enzyme from P. ostreatus mycelium is considering chymotrypsin or subtilisin metalloprotease. 17- The effect of different concentration of the substrate (N-Succinyl-Ala- Ala-Pro-Phe-pNA) was studied. The Km and Vmax were determined to be 0.35 mM and 21 U ml-1, respectively. 18- Molecular study and sequence analysis of the gene encoding fibrinolytic enzyme (PoFR) from P. ostreatus were investigated. The resulting cDNA showed one fragment about 845 bp. The cDNA fragment was transformed into E. coli (α-DH5) competent cell. 19- Two screening procedures were done to detect recombinant clones harboring the specific gene encoding fibrinolytic enzyme. 20- The 1st procedure achieved by PCR screening using pair of specific primers which indicated the same predicted size of cDNA (about 845 bp). 21- The 2nd procedure was done by restriction digestion where the plasmid from transformant bacteria was prepared and digested by restriction enzyme. This procedure indicated also that a DNA fragment of about 845 bp was released and separated by agarose gel electrophoresis. 22- The DNA sequence revealed that the putative mature fibrinolytic gene exhibited a high sequence homology (95%) with fibrinolytic enzyme Summary 92 isolated from P. ostreatus fruiting bodies (GenBank Accession No. AY640032.1). 23- The sequence analysis of the fibrinolytic gene showed that it contains an Open Reading Frame (ORF) of 845 bp with start code ATG and encodes 281 amino acids. The deduced amino acids indicated dissimilarities to other mushrooms and Bacillus producing fibrinolytic enzymes. It showed high homology (90%) with fibrinolytic enzyme isolated from P. ostreatus fruiting bodies (GenBank Accession No. AAU94648.1). 24- The fibrinolytic enzyme from P. ostreatus mycelium has an extended zinc-binding consensus sequence which indicated that it is from metzincin family of metalloprotease. 25- In vitro assay showed the direct action and the high specificity of PoFR toward fibrin clot. 26- In vivo assay of the thrombolytic activity of the purified enzyme (PoFR) in P. ostreatus showed that the enzyme exerted fibrinolysis in male and female mice which predicted by decrease in the coagulation marker hematocrit percentage and prolonged prothrombin time (PT) and thrombin time (TT). This indicated that PoFR has anticoagulating activity in addition to fibrin clot degradation |