Glutathione/Enzyme/ZIF-67 Preparation Of Hybrid Nanocatalyst And Study On Its Catalytic Properties

The immobilization of enzymes is t he main method to improve th e catalytic performance of enzymes,and the selection of carriers with excellent performance is crucial to the succes sful immobilization of enzymes.As one of nanomaterials,metal-organic frameworks(MOFs)have attracted extensi ve attention in the field of immobilized en zymes due to their tunable porous crystal structure and large specific surface area.As one of the MOF s series materials,ZIF-67 is a candidate carrier for enzyme imm obilization due to its mild synthesis conditions and simple preparation process.However,due to the hydrophobicity of ZIF-67 itself and the strong alkaline environment formed by the ligand 2-methylimidazole,the activity of the im mobilized enzyme was seriously affected.In this study,catalase(CAT)was encapsulated in hybrid ZIF-67 using glutathione(GSH)and 2-methylimidazole as co-ligands.A highly active hybrid nano immobilized enzyme(CAT@GZIF-67)was prepared.Synthesis mechanism and the catalytic performance of CAT@GZIF-67 were investigated and elucidated.In addition,with CAT@GZIF-67 as the core,a mild and efficient method for preparing hollow nanocore-shell structure materials for embedding immobilized enzymes was devel oped,a hollow nano-silicon core-shell structure immobilized enzyme(CAT@HSN)was prepared,and its catalytic perfo rmance was studied.The main research contents are as follows:(1)Preparation and synthesis mechanism of CAT@GZIF-67:Using glutathione and 2-methylimidazole as dual ligands of ZIF-67,a highly active CAT@GZIF-67 was prepared.The optimal preparation conditions of CAT@GZIF-67 were obtained:the concentration of 2-methylimidazole was 160m M,the concentration of cobalt nitrate was 40 m M,the co ncentrati on of glutathione was 0.67 mg/m L,and the concentration of enzy me was 30 mg/m L,under optimal conditions,the recovery activity of CAT@GZIF-67 is the highest,which can reach 80%.Fourier transform infrared s pectroscopy(FTIR)and X-ray photoelect ron spectroscopy(XPS)an alysis showed that the abundant sulfhydryl groups(-SH)on GSH can enrich m etal ions(Co2+)through coordination and accelerate nucleation process of metal ions and ligands around the enzyme protein,thus promoting the rapid formation of CAT@GZIF-67.At the same time,as a bioaffinity ligand,GSH is beneficial to weaken the stron g alkaline envi ronment of imidazole and improve the biocompatibility of ZIF-67to enzyme,and thus increases the immobilized enzyme activity.(2)The catalytic performance of CAT@GZ IF-67 was investigated:the results showed that the optimal catalytic tempe rature of CAT@GZIF-67,traditional ZIF-67 immobilized enzyme(CAT@ZIF-67)and free CAT were all30°C.However,CAT@GZIF-67 exhibited a broad catalytic temperature range compared with CAT@ZIF-67 and free CAT,and exhibited highe r temperature tolerance.After being treated at 55℃for 75 min,the CAT@GZIF-67 could still maintain 62.4%of its initial activity.However,the CAT@ZIF-67 could only retain 25.9%of its initial activity.Compared with CAT@GZIF-67 and CAT@ZIF-67,the Michaelis constant(K_m)of free C AT was the lowest,indicating that the immobilization increased the mass transfer resistance between the enzyme and the substrate.However,the K _m of CAT@GZIF-67 is lower than that of CAT@ZIF-67,indicating that CAT@GZIF-67 has a higher substrate affinity than CAT@ZIF-67.Moreover,compared with free CAT and CAT@ZIF-67,the CAT@GZIF-67 exhibited better organic solvent t olerance,storage stability,and reusability.After 7 consecutive uses,the CAT@GZIF-67still maintained 70%of its initial activity,but the CAT@ZIF-67 was only 40%of its initial activity.(3)Preparation of CAT@HSN and its catalytic performance:A laye r of mesoporous silica shell was grown on the surface of CAT@GZIF-67 particles by biomimetic silicification technology,and the inner ZIF-67 was removed by neutral phosphate bu ffer at room temperature to prepare the hollow nano-silicon core-shell structure immobilized enzyme(CAT@HSN).The opti mal preparation conditions of CAT@HSN were as follows:concentrations of APTES,TEOS and Pluronic F-127 were at 5 mg/m L,14 mg/m L and 12.5 mg/m L respectively;and the synthesis time was 2 h.Under optimal conditions,the recovery activity of CAT@HSN was 79.8%.The catalytic performance of CAT@HSN showed that the optimal catalytic temperature of CAT@HSN was 40℃,and was higher10℃than that of free CAT;the optimum catalytic p H of free CAT was 7.0,while that of CAT@HSN was 8.Meanwhile,CAT@HSN exhibited high temperature tolerance and p H tolerance.After being treated at 55℃for 75 min,the CAT@HSN could still retain 80%of its initial activity,while free CAT could only retain 62%of its initial activity;after being treated under different p H conditions(p H 5-9)for 1 h,the residual activity of CAT@HSN was higher than that of free CAT.Moreover,CAT@HSN showed better organic solvent tolerance,storage stability and reusability.After 10 days of continuous storage,the activity of free CAT was almost undetectable,while CAT@HSN still retained 64%of its initial activity,and after repeated use for s even times,the CAT@HSN could retain 73.4%of its initial activity.The above results showed that the hollow silicon core-shell structure immobilized enzyme could not only retain the high activity of CAT,but also play a role in stabilizing the enzyme.