Preparation,Characterization And Application Of Magnetic Metal Organic Frame Materials And Its Efficient Used For Glucose Oxidase Immobilization

Sodium gluconate is a deep processing product of glucose.It is widely used in food,water stabilizer,medicine and other fields.Glucose oxidase?Aspergillus niger,GOx?,which is capable of catalyzing the oxidation of glucose.However,the free GOx has a number of limitations preventing its further applications such as its easy inactivation,difficulty to recycle and separate from the reaction system.So the immobilization of GOx is used to overcome these shortcomings and will bring significantly economic benefits in industry.In recent years,metal organic framework materials?MOFs?,have attracted interest for its its high porosity,specific surface area,adjustable pore size,good thermal stability and variable functional groups compared to traditional porous materials.The Fe₃O₄ was surface-modified by Cellulose through dissolution and regeneration process.The grain size of Cellu@Fe₃O₄ was about 30 nm.Then,ZIF-8@Cellu@Fe₃O₄ was prepared by self-assembly method.The size was approximate to 170 nm.Besides,ZIF-8@Cellu@Fe₃O₄ has good thermal tolerance and superparamagnetism.The GOx was successfully embedded in ZIF-8@Cellu@Fe₃O₄ with high activity and high stability.The effects of different preparation conditions on the activity recovery of GOx-ZIF-8@Cellu@Fe₃O₄ were optimized.The results indicated that,under the optimal conditions: support/enzyme mass ratio of 10:1,immobilization temperature of 20°C,immobilization time of 1 h,immobilization pH of 7.0,the GOx was immobilized on the ZIF-8@Cellu@Fe₃O₄ with a protein loading of 94.26 mg/g and an activity recovery of124.2%.The enzymatic properties of free GOx and GOx-ZIF-8@Cellu@Fe₃O₄ were systematically studied.The optimal pH of GOx-ZIF-8@Cellu@Fe₃O₄ and free GOx were both 6.0,the optimum temperature of the free GOx was 35°C,and the immobilized GOx was40°C.Besides,the GOx-ZIF-8@Cellu@Fe₃O₄ showed superior and its thermal stability,pH stability and organic solvent tolerance comparing with its free countpart;Moreover,after 20 days storage,the GOx-ZIF-8@Cellu@Fe₃O₄ remained 88.51% relative enzyme activity recovery.The kinetic study demonstrated that the immobilized GOx supported by a lower apparent Km value?0.125 vs 0.147 mM?and significantly higher Vmax/Km value?79.288 vs19.258 min-1?than free GOx,respectively.Especially,there is a significant enhancement of Vmax value for immobilized GOx?9.911 vs 2.831 mM·min-1?.The substrate affinity of GOx-ZIF-8@Cellu@Fe₃O₄ becomes stronger under the conditions of enzyme activity center easier to contact with the substrate,with a stronger catalytic capacity.The secondary structure and enzymology properties of GOx-ZIF-8@Cellu@Fe₃O₄ were systematically investigated.The results revealed that the content of alpha helix and beta folding in secondary structure respectively increased by 1.09% and 17.15% after immobilization,leading to the increase of structural rigidity and stability of GOx-ZIF-8@Cellu@Fe₃O₄.The reaction of GOx-ZIF-8@Cellu@Fe₃O₄ catalyzes the oxidation of glucose is investigated.The optimum substrate concentration was 200 mM,the optimum temperature was 40°C,and the optimum enzyme concentration was 50 U.Under the optimum conditions,the initial reaction rate and conversion rate were 27.53 mM/h and 96.75%,respectively.After repeated use of 9 times,GOx-ZIF-8@Cellu@Fe₃O₄ retained more than 60% of its initial activity.In this study,the immobilization of GOx on ZIF-8@Cellu@Fe₃O₄ and the efficient oxidation of glucose catalyzed by the prepared GOx-ZIF-8@Cellu@Fe₃O₄ were investigated.The preparation process of the novel nanometer enzyme preparation is simple,and it can realize the rapid separation of the enzyme and the reaction medium under the action of the magnetic field.It has the characteristics of high enzyme load,high enzyme activity and high stability.This study is helpful to enrich the enzyme localization technique and deepen the understanding of the enzyme-carrier interaction.