Optimization Of Solid Fermentation Condition And Purification Of A Fibrinolytic Enzyme From Cordyceps Militaris

The dissertation focuses on optimization of solid fermentation condition of producing fibrinolytic enzyme with rice as the main raw material by Cordyceps militaris, and the enzyme’s purification.Liquid seed culture condition, solid state culture medium components, and fermentation condition were investigated by single factor experiment and orthogonal experiment. On the basis of cordyceps militaris liquid seed growth curve optimum seed cuture time was 4 days. The mixture of rice and bran was excellent solid state fermentation material for producing fibrinolytic enzyme by cordyceps militaris. Optimized cuture medium components and condition were rice to bran ratio of 1:1, water volume of 75%(v/w), inorganic salt CaCl2 and(NH4) 2 SO4 of 0.1%(w/w) and 2%(w/w) respectively, nature initial pH, inoculation size of 25%(v/w), culture time of 5 d, and extraction time of 4 h. Under the optimized fermentation condition, the fibrinolytic activity was 167 ± 1 U/ml.The fibrinolytic enzyme produced by cordyceps militaris with solid-fermentation was sepatated and purified with ammonium sulfate precipitation, Octyl Sepharose FF hydrophobic interaction chromatography, Phenyl Sepharose HP hydrophobic interaction chromatography, and Superdex 75 gel chromatography in order. The results indicated that the fibrinolytic enzyme was purified to homogeneity(SDS-PAGE) by the established purification protocol, with 40.12 fold purification, an overall yield of 5.67% and a specific activity of 1179.93 U/mg. Relative molecular mass of the purified fibrinolytic enzyme is 32000 ± 2000 Da determined by SDS-PAGE electrophoresis.