Novel Scaffold For Nitrile Hydratase Immobilization And Research Of The Catalytic Performance

Enzymes are nature's sustainable catalysts.They are biocompatible,biodegradable and are derived from renewable resources.Enzymatic processes are conducted under mild conditions in water,with high rates and selectivities.However,the characteristics of operational stability and difficult to recycle impede the industrial applications of enzyme.These defects can be conquered by immobilized enzyme technology.Enzyme carrier plays a large role in this technology.Two new carriers prepared in this paper to work out a solution to large mass transfer resistance that traditional immobilized enzyme possessed.Structured interlock-microcapsule and layered cobalt ferrite porous microspheres take nitrile hydratase?NHase?as enzyme mimics.Catalytic performance and stability of immobilized enzyme have been studied in depth.Furthermore,the immobilized NHase was used for catalytic production of nicotinamide.The main contents are as follows:Structured interlock microcapsule was prepared using crosslinked polystyrene as template and tetraethyl orthosilicate as silicon precursor.The SIMC is composed of vast regularly shaped microcapsules which have unique particle size of 400 nm.Meanwhile,some macropores with 50 nm can be observed on the wall.The Brunauer–Emmett–Teller?BET?surface area of the SIMC is about 374.6 m²/g.The pore size of SIMC is 8.707 nm using the Barrett-Joyner-Halenda?BJH?model.Subsequently,immobilization of nitrile hydratase was achieved by biomimetic adhesion of dopamine?Dopa?.Through the optimization of the immobilization process,the optimal activity of immobilized NHase?about 62.2 U/g?was obtained when the concentration of Dopa was 0.5 mg/mL,the modification time was about0.5 h,and the immobilization time was 1 h.Nitrile hydratase?NHase?was immobilized on SIMC realized the easy separation of the enzyme and improve properties of enzyme such as pH tolerance and heat stability.This work demonstrated that the enzyme-immobilized SIMC presented high catalytic performance and significantly improved stability.NHase was immobilized on SIMC via glutaraldehyde?GA?to covalent bonding instead of Dopa.Through the optimization of the immobilization process,the optimal activity of immobilized NHase?about 287.6 U/g?was obtained when the concentration of GA was 100mg/mL,the activation time was about 1.5 h,and the immobilization time was 4 h.The activity of immobilized NHase was improved and this kind of fixed mode also endowed the immobilized NHase better pH and thermal stability.When the immobilized NHase was used in the preparation of nicotinamide,the yield of nicotinamide could be maintained at 32.67%after reused 7 times.Furthermore,the immobilized NHase showed good catalytic activity in the continuous reaction of packed bed.The hierarchical CoFe₂O₄ porous microspheres?HCPM?were prepared by hydrothermal co precipitation method,which presented a kind of morphology of dandelion pollen grains.The results of SEM show that the diameter of HCPM was about 2 nm.The morphology of HCPM was similar to that of the dandelion pollen grains,which showed a highly porous structure with a pore diameter of about 200 nm.The saturation magnetization of HCPM was56.82 emu/g.When it was applied to immobilized NHase,better stability were observed.The kinetic experiments showed that the Km value of the immobilized NHase was 5.887 mM,which was only about 1.796 times than that of the free enzyme.There was a significant decrease compared with the traditional immobilized enzyme.The results demonstrated that HCPM had a certain role in promoting the nucleophilic attack of enzyme molecules.