Synthesis And Performance Of Enzyme/Covalent-Organic Framework Composites

Covalent organic frameworks（COFs）are a new class of porous crystalline nanomaterials with highly ordered pore distribution,high specific surface area,good thermal/chemical stability,and highly designable structure,based on which they have excellent potential for immobilization of enzymes.Given that most COFs are synthesized by solvothermal methods,the main methods for enzyme immobilization are impregnation and covalent modification,the former of which requires the size of the enzyme to match the pore size of COFs to facilitate enzyme immobilization inside the pores of COFs;the latter of which is prone to enzyme inactivation.In addition,COFs are composed of light elements with low density,which makes them difficult to be separated from the reaction system after use.How to effectively solve the above problems has become a new challenge in the field of COFs immobilized enzymes.In this paper,we started from ionic COFs and realized the rapid adsorption of different biomolecules on the surface of COFs by electrostatic induction;followed by the introduction of Fe₃O₄nanoparticles to prepare magnetic COFs with core-shell structure,which showed excellent magnetic recovery ability and could be used for biodiesel production after loading lipase;finally,we realized the cascade reaction between nanoenzymes and natural enzymes by the peroxidase-like property of Fe₃O₄ nanoparticles.The thesis research is divided into three parts:（1）Different biocomposites（BSA/EB-COFs and BCL/EB-COFs）were induced by the strong electrostatic interaction generated by the positive and negative charges based on the positive electrical properties of EB-COFs and the negative electrical properties of biocatalytic systems（e.g.,bovine serum protein（BSA）and lipase（BCL））without considering whether the pore sizes of biomolecules and COFs match.The adsorption of BSA and BCL was confirmed by Confocal laser scanning microscope（CLSM）and other means,with loadings of 0.1 mg/mg and 0.88 mg/mg,respectively.In p-nitrophenyl palmitate hydrolysis experiments,BCL/EB-COFs exhibited 95%of the original catalytic activity relative to free BCL.In addition,EB-COFs can effectively protect lipase under certain acidic or high temperature environments.Excluding the effect of material recovery loss,the BCL/EB-COFs still had 75%of the initial activity after 10 cycles.Interestingly,elution of almost all lipases on EB-COFs could be achieved with Triton X-100 phosphate solution,and the eluted enzyme activity was not affected,and the materials still showed excellent stability with substantial reloading capacity after 8repeated adsorption/desorption cycles.In addition,the result that aminated bovine serum protein（A-BSA）could not be successfully adsorbed onto the surface of COFs further confirmed the strong contribution of electrostatic interaction to surface adsorption.（2）The Fe₃O₄@EB-COFs core-shell structure nanomaterials were developed and synthesized,and the BCL/Fe₃O₄@EB-COFs biocomposites were formed and used for biodiesel production under electrostatic induction by virtue of the positive charge on the surface of the materials.The core-shell structure formed by Fe₃O₄ core and EB-COFs shell layer was confirmed by SEM,TEM and other characterization methods.The adsorption of BCL was confirmed by means of CLSM e with a maximum loading of 0.24 mg/mg.In the p-nitrop Henyl palmitate hydrolysis experiments,BCL/Fe₃O₄@EB-COFs still exhibited upwards of 90%activity retention and good stability under acidic or high temperature environments.No significant degradation of the catalytic performance was found after 10 repetitions of BCL/Fe3O4@EB-COFs under the external magnet.In addition,high yields were obtained for the catalytic production of biodiesel when BCL/Fe₃O₄@EB-COFs were used as ester exchange catalysts.Therefore,it is reasonable to believe that the excellent magnetic recovery ability of magnetic COFs with high lipase loading capacity shines in the actual production of biodiesel.（3）Glucose oxidase-magnetic covalent organic framework biocomposites（GOx/Fe₃O₄@EB-COFs）were prepared based on the high stability,peroxidase-like activity and positive surface charge of Fe₃O₄@EB-COFs core-shell nanomaterials,induced by electrostatic interaction,to achieve colorimetric determination of glucose concentration by one-pot method.The maximum loading of GOx was achieved by optimizing the immobilization conditions to approximately 0.045 mg/mg.Under acidic conditions,GOx/Fe₃O₄@EB-COFs showed an excellent cascade reaction between catalytic glucose oxidation and catalytic H₂O₂ oxidation.The kinetic study showed that the reaction followed the Lineweaver-Burk equation,indicating that the cascade reaction between the constructed nano-enzyme and the artificial enzyme still showed biocatalytic characteristics.The results of the cascade reaction with different temperature and p H environments showed excellent stability of GOx/Fe₃O₄@EB-COFs.In the presence of Fe₃O₄ magnetic nanoparticles,GOx/Fe₃O₄@EB-COFs showed excellent recoverability and high sensitivity,high selectivity and wide linear range for glucose colorimetric assay.Therefore,the multi-enzyme catalytic system constructed by combining the high stability of nano-enzyme with the high selectivity of natural enzymes is important for the realization of biomimetic catalysis.