Screening,Expression And Immobilization Of Diguaylate Cyclase

3’,5’-cycl ic diguanylate(c-di-GMP)is an important second messenger,which plays a crucial role in regulating some biological processes,like the formation of biofilms,the production of virulence factors,as well as acclimatization.c-di-GMP has powerful immune enhancement effect not only in systemic immune adjuvant,but also when it is used as mucosal adjuvant.Hence,c-di-GMP is a vaccine adjuvant with very high development potential and it also has an important application prospect in clinical.In vitro enzymatic synthesis,Diguanylate cyclase(DGC)is the catalyst of c-di-GMP.This project obtained DGC with practically usage through selection,heterologous expression and immobilization;and we also did characterization on its enzymatic property;at the same time,its catalytic reaction condition had also been optimized.Below is the detailed study:Firstly,choose 6 DGC from different sources whose amino acid sequence consistency is between 30%～90%to code DGC enzyme store;perform selection through soluble expression level and enzyme activity of recombined DGC enzyme,then best DGC enzyme from Pseudothermotoga hypogea DSM 11164=NBRC 106472 can be obtained,of which the gene can realize more than 90%soluble expression level of recombinant protein in E.coli BL21(DE3).Specific enzyme activity is 39.6 U/g.Furthermore,optimized the expression condition of DGC in escherichia coli,which was from Pseudothermotoga hypogea.The optimal expression condition was:Logarithmic prophase induction(OD600 was around 0.6);added IPTG to obtain final concentration of 0.05mM;induced for 5-8 hours under 28℃.Did characterization on enzymatic property of DGC:The optimum reaction temperature was 90℃,the optimum pH was 7.0,the activation capacity of Mg2+ was the strongest among the metal ions,and the optimum concentration was 10 mM.Among the anions,chloride ion had the best catalytic effect,and the enzyme had good thermal stability under 70℃.The kinetic parameters of DGC:Km and Vm were 0.0880 mM and 0.0263 mM/min respectively.The catalytic reaction of DGC was studied,and the optimal process conditions were as follows:substrate concentration was 5 mM,adding enzyme amount was 25 U/L,reaction time was 60 min,and the substrate conversion rate under the optimal conditions could reach more than 80%.Lastly,different curing methods were tried to immobilize DGC enzyme.The results showed that the non-covalent directional immobilization method of agar-IDA-Ni chelating carrier was the most suitable method,and the immobilization conditions and enzymatic properties of immobilized enzyme were studied.The optimal immobilization conditions were as follows:the immobilized pH was 8.0,the reaction time was 1h,adding enzyme amount was 2 mg/g(carrier),the optimal immobilized enzyme activity was 24.4 mU/g,and the enzyme activity recovery rate reached up to 40.6%.The optimum reaction temperature of the immobilized DGC enzyme was 80℃,the optimum reaction pH was 7.0,and it had good operation stability and storage stability.It laid a foundation for the large-scale preparation of c-di-GMP.