Study On Cloning, Expression And Evolution Of Douchi Fibrinolytic Enzyme

Thrombosis is one of this most prevalent, deadly and costly diseases. Recently, thrombolytic agents and health-care foodstuff with function of thromboclasis have developed rapidly. As a new type of thrombolytic agent, fibrinolytic enzyme of microbial origin has potential applications in both theory and clinical practice, and has positive effect in the field of medicine. A bacterial strain with fibrinolytic activity was isolated from douchi in our lab, which can produce enzyme with high fibrinolytic activity, and was identified as Bacillus subtilis LD-8547.In this study, the fibrinolytic enzyme gene (fe) was cloned, and the expressing of the fibrinolytic enzyme gene and the activity analysis of the enzyme were performed. SDS-PAGE analysis and enzymatic activity determination showed that E. coli BL21/pET28a-fe can produce a protein with molecular weight of 60 kDa, and this protein can be converted into a protein with molecular weight of 28 kDa, and the proteins showed fibrinolytic activity.Directed evolution is a new powerful method for improving virtually all properties of a protein for which an appropriate screening method can be devised in the absence of any knowledge of structure and functional mechanism in advance. In order to improve the activity of recombinant fibrinolytic enzyme, we modified the fibrinolytic enzyme gene using error-prone PCR and staggered extention process (StEP). Four recombinants with higher fibrinolytic activity were screened, and the kinetic analysis of the mutated fibrinolytic enzyme were performed. In order to test the evolution result, the douchi fibrinolytic enzyme was fermented and purified, and the purified enzyme was applied to kinetic analysis. The results showed that the michaelis constant(Km) of the mutant mut1 was reduced, which indicate that the affinity of this mutant was improved, and the catalytic constants(Kcat) of the four mutants were increased, which indicate that the catalytic efficiency of the mutants were all improved, and the specificity constants of mutant mut2, and mutant mut3 were increased, which indicate that the efficiency of the two mutants both improved.