Mutagenesis And Optimization Of Submerged Culture Conditions Of Cordyceps Militaris For High Yield Of Fibrinolytic Enzyme

Thrombosis is one of the most serious diseases, which has a higher disability rate and mortality. Need of exploitation for thrombolysis drug is urgent. Microbial is an important source of thrombolytic drug. Cordyceps militaris is one of the medicinal mushrooms. A kind of enzyme with stronger fibrinolytic activity has been found, by our partner our laboratory, in the fermentation culture fluids of Cordyceps militaris, which has a potential for treating thrombus. The fermentation conditions for the fibrinolytic enzyme production with Cordyceps militaris were optimized in our laboratory, which has made the fibrinolytic enzyme productivity be increased.In the dissertation, based on optimized condition, a Cordyceps militaris strain with high fibrinolytic enzyme productivity was obtained through protoplast mutagenesis. The fermentation conditions for the fibrinolytic enzyme production with Cordyceps militaris were further investigated, and the fermentation kinetics were also studied. And the process of the fibrinolytic enzyme production was scaled-up successfully from 250mL flask to 10L and 100L fermentor.The productivity of fibrinolytic enzyme for eight Cordyceps militaris strains, which obtained from different area of China,were compared. C.LSG-1 was selected as mycelidium for protoplast mutagenesis. The lethal dose of UV irradiation on Cordyceps militaris protoplast was 45s. Then 4 positive mutants were screened from 400 mutants, the productivity of fibrinolytic enzyme in liquid medium of the mutants were 15% higher than that of orignial strains.The fermentation conditions were investigated. Inoculums size of liquid spawn, age of liquid spawn, pH control mode, the time of supplementary component of nutrition, the volume of supplementary component of nutrition, the concentration of supplementary component of nutrition , the element of supplementary component of nutrition were studied. The results showed that a better inoculum size was 0.5%(v/v)liquid spawn of 3-5 day. Controlling the pH of culture medium was not suitable for the production of fibrinolytic enzyme. Fed batch cultivation was better for mycelia production, but not better for the production of fibrinolytic enzyme.The fermentation kinetics of Cordyceps militaris submerged culture for fibrinolytic enzyme production was investigated. The kinetics equations for biomass growth, production of fibrinolytic enzyme and consumption of glucose were established as follow: dx/dt=0.0918(1-X/30.9043)X;dp/dt = 0.1379dx/dt+0.0126X;-ds/dt=0.621dx/dt+0.0193dp/dt。The process of fibrinolytic enzyme production with Cordyceps militaris was scaled-up from 250mL flask to fermentors of 10L and 100L according to KLa equal- principle. The fermentation of 10L fermentor under the conditions of agitation rate of 100r/min and aeration rate of 600L/h got a better result for the fibrinolytic enzyme production. The area of the fibrinolytic enzyme active reached 214.28mm~2, equal to 286.21U/mL of urokinase, which was 3.2 times more than the results of flask. The YP/x was 5.5 times more than the results of flask. The fermentation of 100L fermentor under the conditions of agitation rate of 250r/min and aeration rate of 1m~3/h got a better result for the fibrinolytic enzyme production. The area of the fibrinolytic enzyme active reached 154.30mm~2, equal to 87.65U/mL of urokinase, which was equal to the results of flask. The YP/x was 1.32 times more than the results of flask.