Rhizopus The Plasminogen Liquid Fermentation, Purification And Characterization

Thrombosis intimidates mankind's life and health seriously; the morbidity of it has increased. 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 searches for new fibrinolytic enzymes from various sources are being continued.The dissertation focused on optimization of liquid fermentation condition of Rhizopus chinensis YY-15 for production a fibrinolytic enzyme, purification of the fibrinolytic enzyme, and partial characteristics of the enzyme.The liquid fermentation condition of Rhizopus chinensis YY-15 for producing the fibrinolytic enzyme was optimized. The effects of carbon sources, nitrogen sources, ratio of carbon to nitrogen sources, and initial pH of culture medium on the enzyme productivity were investigated by single factor experiment. Fermentation time and inoculum size were optimized by orthogonal experiment. The results showed that the suitable carbon and nitrogen sources were wheat bran and soybean meals. Optimized composition of culture medium was wheat bran of 3% (w/v) and soybean meals of 5% (w/v) , and suitable initial pH of culture medium was 5.0. Optimized fermentation time and inoculum size were 72 h and 6%, respectively. Produced the fibrinolytic enzyme reached 98.31 U/ml (urokinase unit) at the optimized condition.Purification process of the fibrinolytic enzyme form Rhizopus chinensis YY-15 was explored and established. The fibrinolytic enzyme was purified by using fractional salting-out with (NH₄)₂SO₄ of 40%-70% , Phenyl Sepharose Fast Flow hydrophobic interaction chromatography, DEAE-Sepharose FF ion exchange chromatography and Sephacryl S-100 gel filtration chromatography. The purified enzyme was homogeneous, as examined by high resolution Resource Phe hydrophobic interaction chromatography. The purification protocol resulted in a 90.5-fold purification of the enzyme, with a final yield of 46.5%.Partial characteristics of the purified fibrinolytic enzyme from Rhizopus chinensis YY-15 were investigated. The molecular weight was 20.3kDa determined by gel filtration chromatography. The isoelectric point estimated by isoelectric focusing electrophoresis was 8.2. The specific activity of the purified enzyme was 2416.86 U/mg. The enzyme had an optimal temperature of 50℃. The...