Construction And Application Of Anew Zirconium Based Coordination Polymer Immobilized Enzyme

Metal coordination polymer(CPs)are regarded as a promising enzyme immobilization material.In this paper,2-methylimidazole(2MIm)was used as an organic ligand to self-assemble metal organic coordination polymer(Zr-2MIm)with zirconium ion in water phase,and its application was explored.The main results are as follows:1.In this paper,the method of in-situ self-assembly of CPs by zirconium ion and 2-methylimidazole in aqueous phase was studied.It was found that high buffer concentration and neutral buffer were beneficial to the formation of the coordination polymer.Curcumin reductase(CurA)was selected as the model enzyme for in situ self-assembly,and the characterization experiment proved that the enzyme was successfully encapsulated in the composite.It was found that CurA@Zr-2MIm had a high affinity for the substrate.After incubation at high temperature and strong acid for 1 hour,the activity of immobilized enzyme remained good acticity.In addition,CurA@Zr-2MIm had long-term storage stability at room temperature,and maintained nearly 70%initial activity in 11 days.2.It was found that the metal site Zr4+of Zr-2MIm was unsaturated,which was favorable for the coordination with hexahistidine-tagged enzyme and the specific adsorption of the enzyme.Based on this property,an enzyme catalyzed reactor with clear enzyme distribution was constructed.Firstly,G6PD was successfully encapsulated for NADPH regeneration by in situ self-assembly.The encapsulated G6PD has excellent storage stability,good tolerance to different pH and temperature,and excellent catalytic performance.Then,the composite materials capture the α,β-unsaturated ketone reductase with his-tag on its surface through coordination interaction,thus obtaining a multi enzyme reactor.The activity of bioreactor remained high at high temperature,but the activity of free enzymes was lost.In addition,the bioreactor showed high stability and repeatability.3.To solve the problem of difficult recovery of immobilized enzyme,a magnetic nano carrier(Fe3O4@Zr-2MIm)was formed by coating a layer of Zr-2MIm polymer on the surface of Fe3O4 nanoparticles.We successfully expressed and purified two proteins with his-tags by means of genetic engineering.The magnetic immobilized enzyme was prepared by adsorption of his-enzyme on Zr-2MIm.The immobilization conditions were explored.It was found that the highest immobilization rate was at 25℃for 30 minutes.It is worth noting that the activity of immobilized enzyme is 1.8 times higher than that of free enzyme.The immobilized enzyme also has good stability and shows good tolerance to different pH and temperature.The residual activity of the magnetic immobilized enzyme at room temperature for 11 days was about 60%of its initial activity.In addition,the immobilized enzyme still had more than 50%activity after 6 cycles,which proved that the immobilized enzyme could be recovered effectively.