Optimization Of Fermentation Conditions And Purification For Fibrinolytic Enzyme From Marine Actinomycete MY0504

Thrombosis is mainly caused by the accumulation of fibrin in the blood,plasmin can dissolve fibrin,so that it can make the blood vessels unblocked,and prevent people from thrombotic diseases.At present,the cost of thrombolytic drugs is high and the side effects are seriously.Therefore,it is a hot spot to study safe and effective thrombolytic agents.In this paper,the growth curve and enzyme production curve of the marine actinomycete strain MY0504 were determined,the fermentation time was confirmed to be 4.5 d.The optimum carbon source,nitrogen source,liquid volume,inoculation amount and inorganic salt concentration were determined by single factor test.Based on the single factor experiment,three significant factors,namely,glucose,yeast powder and temperature,were selected from the eight factors influencing the enzyme activity by Plackett-Burman design.The value of the factors approached the center point by the steepest climbing test,and then the optimal value of the three factors was obtained by the Box-Behnken response surface design.Under the optimized fermentation conditions,the activity of fermentation broth reached 2190.6 U / mL,which was 4 times of that before optimization.After we tried kinds of methods,such as salting out,ion exchange chromatography,hydrophobic chromatography and gel filtration chromatography,the purification process of fibrinolytic enzyme was determined as follows: fermentation broth centrifugal,711 resin adsorption pigment,salting out,Phenyl Sphere hydrophobic chromatography,Sephadex G-75 gel chromatography.The activity of plasmin reached 11000 U/mg,the purification multiple was 101.3 times,the recovery rate was 26.1%.The partial properties of the fibrinolytic enzyme were studied,and we found that its molecular weight is about 32 kDa and its isoelectric point is about 8.5.The fibrinolytic substance in the fermentation broth is simply fibrinolytic enzyme,does not contain plasminogen and does not contain plasminogen activator.The fibrinolytic enzyme can effectively degrade fibrinogen in the short time.It has strong and efficiency thrombolytic properties.