Preparation And Application Of Immobilized Enzymes Based On Zeolitic Imidazole Frameworks Materials

Supports are one of the major factors affecting the properties of immobilized enzymes,and the regulation and preparation of supports with better performance are of great importance to improve the catalytic performance of immobilized enzymes.Zeolitic imidazole frameworks-8（ZIF-8）,a member of the metal-organic frameworks（MOFs）series,composed of 2-methylimidazole（2-Me Im）and zinc ions(Zn²⁺)through coordination bonds,possesses high porosity,large specific surface area and and mild preparation conditions.Therefore,it has been widely used in the immobilization of enzymes,and the prepared immobilized enzymes have shown good catalytic performance,which have been well applied in the fields of biomedicine,bioassay and environmental protection.However,with the continuous research,some defects of ZIF-8 were gradually exposed,such as small pore size,strong hydrophobicity,and precursors affecting enzyme activity,etc.These defects affected the catalytic activity and stability of immobilized enzymes to some extent.Meanwhile,it was difficult to obtain immobilized enzymes with superior performance using ZIF-8 as the support due to the lack of understanding of the structure-function relationship between structure and catalytic performance of immobilized enzymes.In this study,ZIF-8 immobilized enzyme with excellent performance was prepared based on pore size modification engineering,competitive ligand hybridization strategies,and investigated its catalytic performance and structure-function relationship,which were used for phenol removal and glucose detection in aqueous solutions,the specific content was as follows:（1）Aiming at the defects of small pore size（mostly micropores,less than 2 nm）and high mass transfer resistance of ZIF-8,based on the mechanism of hard template pore formation,this chapter prepared magnetic ZIF-8（m NMZIF-8）with 3D ordered macropores by using 3D ordered polystyrene（PS）microspheres as a template and combining with magnetic nanoparticles,which was used as a support for the immobilization of enzyme by co-precipitation cross-linking method.Compared with conventional ZIF-8,the pore size of macroporous ZIF-8 reached 69 nm;after immobilization of catalase（CAT）,the recovery activity and immobilization efficiency of the immobilized enzyme（CAT/m NMZIF-8）increased 5-fold and 34-fold than conventional ZIF-8 adsorbed CAT（CAT/ZIF-8）,respectively;protein loading was 325 mg/g,showing better catalytic activity and immobilized enzyme capacity;Meanwhile,it was found that due to the presence of large pores,the Km value of CAT/m NMZIF-8 decreased by 20%and Vmax/Km increased by2-fold compared with CAT/ZIF-8,indicating that the enlargement of the support pore structure not only weakened the mass transfer resistance during the reaction and accelerated the diffusion rate of the substrate,but also increased the loading of the enzyme protein,which in turn enhanced the catalytic efficiency of the enzyme.Under the effect of magnetic field,CAT/m NMZIF-8 maintained 90%of its initial activity after 6 times of repeated use,while CAT/ZIF-8 had largely lost its enzymatic activity,demonstrating a good reusability.（2）The pore size of ZIF-8 was modified by using a soft template.ZIF-8（HZIF-8）with hierarchical pore core-shell structure was prepared based on Zn SO₄·7H₂O colloid in methanol as a soft template and ZIF-8 precursors.Compared with conventional ZIF-8,HZIF-8 possessed a mesoporous structure with a pore size of 11 nm;Cytochrome C（CytC）was encapsulated in HZIF-8（CytC@HZIF-8）using a co-precipitation method,compared with the conventional ZIF-8 immobilized enzyme（CytC@ZIF-8）,CytC@HZIF-8 showed a 3-fold increase recovery activity and exhibited higher catalytic activity;Moreover,the Km value of CytC@HZIF-8 decreased by 67%and the Vmax/Km value increased by4.4-fold than CytC@ZIF-8,indicating that the presence of mesopores accelerated the diffusion rate of substrate and product,enhanced the affinity of substrate and enzyme,and thus improved the catalytic efficiency of enzyme;After seven repetitions,CytC@HZIF-8retained 70%of its initial activity,while only 10%of CytC@ZIF-8 remained.An immobilized multienzyme detector（CytC@ZIF-8/GOx）was constructed for glucose detection by adsorbing glucose oxidase（GOx）to the external shell layer of CytC@HZIF-8,which exhibited a low limit of detection of 0.8μM,as well as a rapid response time for glucose of 10 min.（3）In view of the strong hydrophobicity of ZIF-8 and the large effect of protonation of the precursor 2-Me Im on the enzyme activity,the pore size modification engineering and competitive ligand hybridization strategy were combined to use Zn SO₄·7H₂O colloid as a soft template.3-methyl-1H-1,2,4-triazole（3-MTZ）and 5-methyltetrazole（5-MTA）with better hydrophilicity were selected to replace part of 2-Me Im,and new hybridized ligand-hierarchical pore HHMOFs with better hydrophilicity than HZIF-8 were prepared.Compared to HZIF-8,HHMOFs showed a 20°reduction in contact angle,enhanced hydrophilicity,which facilitated to maintain the molecular conformation of the enzyme.And also,the pore size of HHMOFs reached 41 nm,which was larger than that of HZIF-8.;Horseradish peroxidase（HRP）was immobilized by adsorption using HHMOFs as supports,and the resulted immobilized enzyme（HHMOFs/HRP）showed a 5.8 and 4.5-fold increase in recovery activity as compared to ZIF-8/HRP and HZIF-8/HRP,respectively,resulting in a significant increase in catalytic activity;The measurement of CD specturm showed that the contents ofα-helix,β-fold andβ-turn in the HRP secondary structure immobilized on HHMOFs/HRP were closer to the natural free HRP.however,the contents ofα-helix,β-fold andβ-turn in the HRP secondary structure immobilized on ZIF-8/HRP and HZIF-8/HRP were significantly changed compared with the natural free HRP,indicating that HHMOFs have better ability to stabilize the HRP secondary structure than ZIF-8 and HZIF-8.And due to the reduction of 2-Me Im ratio in HHMOFs,the protonation of 2-Me Im was weakened,which reduced the microenvironmental p H of HHMOFs from 8.8 to 6.8,which was closer to the optimal catalytic p H of HRP,thus improving the catalytic efficiency;After30 days of continuous storage,HHMOFs/HRP still maintained 100%of their initial activity,while ZIF-8/HRP was essentially inactive.The application of HHMOFs/HRP to the phenol removal reaction resulted in 88%phenol removal after 40 min,which was 2.9 and 1.6 times higher than HRP and ZIF-8/HRP,respectively,under the conditions of the same enzyme activity.（4）CAT,GOx and lipase（lipase）with different substrates were selected as the target,and HZIF-8 and HHMOFs were used as supports for their adsorption immobilization.The results showed that the activity of HHMOFs/CAT was comparable to that of HZIF-8/CAT,and the activity of HHMOFs/GOx and HHMOFs/lipase showed 1.83 and 2.3-fold higher activities than HZIF-8/GOx and HZIF-8/lipase,respectively,indicating that the improved HHMOFs showed better immobilization enzyme effects and were an excellent enzyme immobilization support;The catalytic performance of HZIF-8/GOx and HHMOFs/GOx was also evaluated,and the results showed that the large mesoporous structure and improved hydrophilicity of HHMOFs could not only improve the affinity between enzyme and substrate and maintain the natural conformation of enzyme,but also the weakened protonation of 2-Me Im could provide a good catalytic microenvironment for enzyme,thus improving the catalytic efficiency of enzyme.;An immobilized multi-enzyme detector（HHMOFs/GOx HRP）was constructed for glucose detection by co-adsorption of GOx and HRP using HHMOFs,which exhibited a low limit of detection of 0.54μM,as well as a response time of only 3 min for glucose.