| 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 new fibrinolytic enzymes from various sources are being continued.The dissertation focuses on the breeding of Rhizopus chinensis 12# producing a fibrinolytic enzyme, optimization of solid-state and liquid fermentation condition, purification of the fibrinolytic enzyme, optimization of preparative chromatography process, and characteristics of the fibrinolytic enzyme.Rhizopus chinesis 12# producing the fibrinolytic enzyme was mutagenized with UV combining LiCl and 74 nystatin-resistant mutants were obtained. All mutants were further screened through repeated solid-substrate fermentations and then 4 plus-mutants with highly genetic stability,Q1,Q18,Q26and Q39, were acquired. The productivity of the fibrinolytic enzyme of plus-mutants increased by 32.9%, 21.5%, 22.3% and 18.0% respectively compared with that of original strain.The solid-substrate fermentation condition of plus-mutant Q1 for producing the fibrinolytic enzyme was optimized. The effects of carbon source, different nitrogen sources, ratio of carbon to nitrogen sources, beginning pH and moisture content of culture , inorganic salts on the fibrinolytic enzyme productivity were investigated by single factor experiment, uniform experiment and response surface methodology. 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, the suitable weight ratio of wheat bran and soybean meals was 1:2, and other components of the culture medium were distilled water 0.75ml, MnSO4-H2O 0.0025g and (NH4)2SO4 0.0142g / g solid substrate (DW). Optimized fermentation time and inoculum size were 72 h and 107 spores /g solid substrate respectively. Each gram solid substrate yielded 791.81 units the fibrinolytic enzyme (urokinase unit) at the optimized condition. The fibrinolytic activity produced at optimized condition was 3.26 times of original condition.Medium composition of liquid cultivation of plus-mutant Q1 for producing the enzyme was optimized, and mechanism of producing the fibrinolytic enzyme was investigated. Two kinds of available mediums were determined by single factor experiment, orthogonal experiment, uniform experiment and Plackett-Burman Experiment. The mediums composition were wheat bran 5%, soybean meals 6%, NaNO3 0.2%, NH4C1 0.1%, tryptone 2.5%, pH4.3 and wheat bran 5.6%, soybean meals 5.3% pretreated with 0.1N HC1, (NH4)2HPO4 0.4%, NaNO30.5%, pH6.1. The activities of the fibrinolytic enzyme produced from those two mediums were 182U/ml and 140U/ml respectively. The fibrinolytic enzyme from Rhizopus chinensis 12# was induced by tryptone, and hydrolyzate of soybean meals showed a similar inducing effect in the liquid fermentation.Purification process of the fibrinolytic enzyme form Rhizopus chinensis 12# was explored and established. The fibrinolytic enzyme was purified using fractionalsalting-out with (NH4)2SO4, Octyl-Sepharose FF hydrophobic interaction chromatography, Q-Sepharose FF ion exchange chromatography and Sephacryl S-100 gel filtration chromatography. The purified enzyme was homogeneous , as examined by SDS-PAGE and PAGE.Taking into account selectivity, resolution, throughput and scaleability of purifying unit modes, preparative purification chromatography process of the fibrinolytic enzyme from Rhizopus chinensis 12# was optimized and established. The decolor procedure of the crude enzyme was also explored and established through using anion exchange resin. The fibrinolytic enzyme was preparative purified applying a special decolor procedure of anion exchange resin, fractional salting-out with (NH4)2SO4, Octyl-Sepharose FF hydrophobic interaction chromatography, Phenyl-sepharose HP hydrophobic interact...
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