The Study On Fermentation Technology、Characterization And Cloning Of The Fibrinolytic Enzyme From Bacillus Amyloliquefaciens

Fibrinolytic enzyme was isolated from fermentation broth of Bacillus amyloliquefaciens,which not only can dissolve fibrin directly, but also can activate plasminogen into plasmin todissolve fibrin, so it is a promising thrombolytic agents.In this paper we optimized the producing fibrinolytic enzyme fermentation condition ofBacillus amyloliquefaciens; then the enzymatic properties of the enzyme were studied, layingthe foundation for future application; Finally the cloning and expression of the fibrinolyticenzyme gene were studied, hoping to further enhance the fibrinolytic activity.In this paper, we use the agarose fibrin plate method of plasmin to measure the activity ofthe enzyme, this method is simple, intuitive and can simultaneously test multiple samples. Soit’s the most widely method.During the process of optimizing the producing fermentation condition of Bacillusamyloliquefaciens, we use the single factor experiment, PB experiment design experiment andfinally climbing central composite experiment and the effect of surface analysis. Finally, wegot the best fermentation condition: bacteriological peptone concentration2.203%, dextrinconcentration2.644%,NaCl1%, NaH₂HPO₄0.6%, NaH₂PO₄0.2%, peptone bacteriologicalconcentration2.25%, dextrin concentration2.65%, pH7; The best fermentation condition:fermentation time45h, fermentation temperature37℃, shaking speed of225r/min. On thiscondition,the fibrinolytic activity is96.346IU/mL.By the research on enzymatic properties of Bacillus amyloliquefaciens producingfibrinolytic enzyme, we found the optimum plasmin temperature is37℃, the optimum pH isalkaline, and it is suitable for short-term storage at low temperature. The enzyme is a serineprotease and maybe belongs to metal enzyme. Its existing formation is plasmin, not only candirectly disslove fibrin and can active plasminogen to degrade fibrin indirectly.This fibrinolytic enzyme gene was cloned and expressed. According to the subtilisin DFEprecursor gene DQ132806.1, primers were designed. After the cloning and transformation, wesuccessfully realize the expression of the gene in Bacillus subtilis WB800, the enzyme activityof the fermentation liquid reached48.694IU/mL, which is lower than our expectations, so weneed further study.