Optimization Of Solid Fermentation Of Fibrinolytic Enzyme Producted By Rhizopus Chinensis And Cloning And Expression In E.coli BL21

Thrombosis intimidates mankind's life and health seriously, and fibrinolytic therapy is available. Current fibrinolytic enzymes available for clinical use are mostly plasminogen activators. Despite their widespread use, all these agents have undesired side effects and are very expensive. Therefore the search for novel fibrinolytic enzymes from various sources are being continued.The dissertation focuses on the breeding of Rhizopus chinensis producing a novel fibrinolytic enzyme. On the base of lab's small-sized fermentation, solid fermentation technologies in the 1t biology reactor were optimized and rude extraction of fibrinolytic enzyme was carried out.The result on solid fermentation of Rhizopus chinensis indicated: the appropriate conditions were beginning moisture content 0.75L/kg substance, fermentation humidity 70%, initial ventilation 640L/min, 560L/min after 30h,28℃, fermentation cycle 60h, Productivity of the fibrinolytic enzyme went up peak value 706.3U/g by above conditions. The rude enzyme was purified with ultrafiltration concentration and ammonium sulfate fractionation. The specific activity was 134.84U/mg, 70.1% of the fibrinolytic enzyme was recovered with 27.91-fold purification. The rude enzyme was examined by SDS-PAGE.The expression of novel fibrinolytic enzyme in E.coli was investigated. According the N-terminal amino acid sequence of fibrinolytic enzyme protein, generate primers were designed. RNA of Rhizopus chinensis was extracted using TRIZOL reagent. The cDNA encoding fibrinolytic enzyme was cloned by RT-PCR, the cDNA was inserted into plasmid pUCm-T and transformed into E.coli JM109, the sequence rcfe encoding novel fibrinolytic enzyme was obtained. rcfe gene was cloned into plasmid pET22b(+) and transformed into E.coli BL21(DE3), resulting the vector pET22b(+)-rcfe. PCR and SDS-PAGE results indicated the fibrinolytic enzyme gene from Rhizopus chinensis has been successfully transformed and expressed in E.coli BL21(DE3). The recombinant protein was proved no fibrinolytic enzyme activity.