Enzyme Immobilization On Hierarchical Porous Metal Organic Frameworks For Efficient Biocatalysis

Biocatalysis is an efficient way to synthesize products with high purity.Candida rugosa lipase(CRL)has become one of the most widely used biocatalysts because it can be used in various catalytic reactions such as hydrolysis,esterification,resolution and transesterification.However,in actual industrial applications,lipase has various problems,such as high production cost,poor stability,difficulty in recovery and reusability.In order to solve the above problems,the immobilization of lipase on a carrier is considered to be an effective method to enhance its catalytic stability,facilitate recovery and increase its reusability,and significantly reduce costs.Metal Organic Frameworks(MOFs) is a three-dimensional porous material composed of metal ions or metal cluster secondary building units as nodes,organic linking groups as ligands,and self-assembly through coordination.Because MOFs have the advantages of high porosity,large specific surface area,good biocompatibility,high stability and adjustable structure,they can be widely used in gas storage and separation,catalysis,drug delivery,supercapacitors,and enzyme immobilization.At present,MOFs materials have been used as carriers for enzyme immobilization.Covalent crosslinking and physical adsorption are the common methods to immobilize lipase on the carriers.Covalent cross-linking method needs to functionalize MOFs first,and then cross-linking with enzyme.The method is more complex,and the porosity of MOFs after functionalization is reduced,which ultimately affects the enzyme microenvironment and enzyme loading capacity;while physical adsorption can successfully load lipase through simple soaking,however,most MOFs only have microporous structure(microporous pore size<2 nm).The size of lipase is 4.5 nm×3 nm×3 nm,so it is difficult to enter into the channels of MOFs and can only be adsorbed on the surface of MOFs.The high porosity of MOFs and the micro environment of the channels have not been effectively utilized.Therefore,this project plans to add regulators and control the reaction temperature at the same time,or introduce mesoporous(2 nm<mesoporous pore size<50 nm)in microporous MOFs by template method,so as to maintain the high porosity of MOFs,expand the pore size and improve the enzyme loading capacity and efficiency.HKUST-1 is a classic MOF with Cu²⁺as the metal and trimellitic acid as the ligand.This topic uses monocarboxylic acid as a regulator to induce HKUST-1 to produce a hierarchical porous structure with both micropores and mesopores(2 nm<mesopore pore diameter<50nm)under solvothermal conditions,and use it for fat Enzyme immobilization research.Experiments have shown that,compared with the ordinary uninduced microporous HKUST-1,the maximum enzyme capacity of the graded porous HKUST-1 is 122.0 mg·g^-1,which is about 2.57 times that of the microporous HKUST-1.The graded porous HKUST-1 can significantly increase the load of lipase.In addition,the p H and temperature stability of the lipase immobilized on the graded porous HKUST-1 is significantly enhanced than that of the free lipase.In order to further explore the application of hierarchical porous MOFs in immobilized enzymes,ZIF-8 with Zn²⁺as the metal and 2-methylimidazole as the ligand was selected,and the hydrogel was used as a template to prepare ZIF-8 with a hierarchical porous structure.The loading capacity of graded porous ZIF-8 on lipase is about 2.68 times that of microporous ZIF-8,and the stability of enzyme activity immobilized on graded porous ZIF-8 has also been significantly improved.The above results indicate that hierarchical porous MOFs are an ideal platform for immobilizing macromolecular enzymes.In the course of the experiment,it was found that due to the high dispersibility of MOFs,it can only be collected by high-speed centrifugation during the recycling process,which will have a certain impact on the enzyme activity and reduce the enzyme activity.Therefore,the introduction of magnetic Fe₃O₄ nanoparticles into MOFs,and the rapid collection of MOFs-enzyme complexes through the addition of magnets,can quickly separate the immobilized enzyme from the reactant solution and achieve the purpose of recycling.The Fe₃O₄nanoparticles with carboxylic acid groups were synthesized by hydrothermal method.Through the coordination of Zn²⁺and carboxylic acid,ZIF-8 crystals can be grown on them.During the synthesis process,by adding cetyltrimethylammonium bromide as a template and histidine as an auxiliary agent,a magnetic ZIF-8 with a hierarchical porous structure was synthesized.Its loading capacity for lipase is about 2.51 times that of microporous magnetic ZIF-8.In addition,the synthesized graded porous magnetic ZIF-8 has good magnetic responsiveness and can quickly recycle the immobilized enzyme through the magnet.After 10 cycles of reaction,the enzyme activity of the immobilized enzyme can reach 65%of the remaining activity.