Construction Of High Active And Stable Recombinant Nattokinase Based On Free Energy Computational Design

Natto is a traditional Japanese fermented soybean food with a history of more than a thousand years,Nattokinase,a type of alkaline serine protease,is a strong fibrinolytic serine protease produced by Bacillus subtilis natto during the fermentation of soybeans,and is considered to be a new generation of thrombolytic drugs because of its ability to dissolve blood clots.The American Natural Science Association has evaluated the safety of nattokinase and has confirmed that nattokinase can be investigated as a novel thrombolytic drug in clinical studies.However,in industrial production,this protease is sensitive to external conditions such as temperature and acid-base.Basically,aiming to achieve high expression may face a massive loss of nattokinase activity that can result from high temperature.Therefore,how to retain and enhance the activity of nattokinase in a high temperature environment is a current research hotpot.Nowdays,there is a trend to modify the properties of enzymes with the help of computational simulations.This thesis aims to explore the mutation sites of nattokinase by computational design software,construct a mutant nattokinase recombinant expression system,obtain the target protein,and improve its heat resistance.In this study,we used ABACUS and Rosetta＿ddg protein simulation softwares to screen a large number of nattokinase mutation sites,and finally screened six mutation sites with high stability by molecular dynamics simulation: T71 L,S89V,E156 F,E195W,Q206 L,Y256P.The recombinant plasmid containing wild-type nattokinase,PGEX-6P-NK,was constructed as a template,and six mutant nattokinase containing mutant sites were constructed by overlap extension PCR method,and after sequencing to verify the successful mutations,they were transformed into E.coli BL21 expression vector.The supernatant and precipitate of wild-type and mutant nattokinase were obtained by inducing protein expression in the strain,collecting the protein,and after cell crushing,and the mutant nattokinase E156 F,Q206L and Y256 P were verified to have thrombolytic activity by fibrin plate method.To verify the heat resistance of the above three mutants,the enzyme activity was measured after treatment at 65℃ for 30 min,and the results showed that the heat loss rate was 84.24% for the wild type and less for Q206 L,with a heat loss rate of 21.4%.In order to verify the maximum heat resistance temperature,the results after incubation at different temperatures showed that the highest heat resistance was achieved by Y256 P at 70℃,which was 5℃ higher than that of the wild type.the heat resistance of E156 F and Q206 L was also improved compared to the wild type,and the highest heat resistance temperature was 69℃.Therefore,this study succeeded in screening and constructing nattokinase mutants E156 F and Y256 P with improved enzyme activity and heat resistance,and mutant Q206 L with improved heat resistance compared with wild-type nattokinase,although the enzyme activity was not improved.In summary,the above research results remedied the defects of nattokinase in industrial production,which is intolerant to high temperature and has too low activity,and provide a solid foundation for further mass production of novel nattokinase drugs.