Catalytic Activity Of Nitrile Hydratase Immobilized On Ferrous Nanoflowers

Nitriles are widely used to produce more economically valuable amides.Compared with traditional chemical methods,microbial methods（nitrile hydratase as catalyst）have the advantages of green environmental protection,high specific activity and substrate specificity,which makes them have great potential in the industrial production of amide substances.However,the activity of nitrile hydratase is easily reduced or lost under destructive environmental conditions,so it needs to be immobilized to avoid the influence of extreme environments,enhance the stability of the enzyme and realize the reuse of biological enzymes.As a carrier,organic-inorganic hybrid nanoflowers have the advantages of high specific surface area,simple operation and mild synthesis conditions,so they are widely used in the field of immobilized enzymes.In this study,nitrile hydratase derived from Rhodococcus erythropolis CCM2595 was expressed in E.coli,,and purification by affinity chromatography to obtain nitrile hydratase.Next,the carrier ferrous nanoflower Fe₃（PO₄）₂was synthesized,and a biocatalyst NHase@Fe₃（PO₄）₂was successfully prepared by immobilizing nitrile hydratase by in situ synthesis.First,Fe₃（PO₄）₂and NHase@Fe₃（PO₄）₂were characterized（scanning electron microscopy,EDS,fourier transform infrared spectroscopy,X-ray diffraction,and confocal laser scanning microscope）,and it was found that nitrile hydratase was successfully immobilized on Fe₃（PO₄）₂,and nitrile hydratase had no obvious effect on the morphology and structure of the carrier,showing a uniform flower-like structure.Next,the preparation conditions of NHase@Fe₃（PO₄）₂were optimized,and it was finally determined that 100mmol/L Fe SO₄was incubated with 1.0 mg/m L nitrile hydratase solution for 12 h.Finally,the optimal reaction conditions,substrate tolerance,storage stability and reusability of NHase@Fe₃（PO₄）₂and free nitrile hydratase were investigated using adiponitrile as the reaction substrate.The results showed that:（1）Under the destructive environment of high temperature and partial acid and partial alkali,since the carrier Fe₃（PO₄）₂played a good protective effect on nitrile hydratase,NHase@Fe₃（PO₄）₂catalyzed the production of adiponitrile.（2）It is worth mentioning that compared with the free enzyme,NHase@Fe₃（PO₄）₂has a significant substrate tolerance,when the adiponitrile concentration reaches 100 mmol/L,The free enzyme activity is almost lost,while NHase@Fe₃（PO₄）₂still maintains more than 95%catalytic activity,and still maintains 60%activity when the adiponitrile concentration is as high as 200 mmol/L;（3）NHase@Fe₃（PO₄）₂storage stability is also better than that of free nitrile hydratase.On the 5th day,only 30%of the relative activity of free nitrile hydratase remained,while NHase@Fe₃（PO₄）₂still maintained 50%of the activity,and NHase@Fe₃（PO₄）₂also exhibited good reusability,retaining 60%of the initial enzymatic activity after 4 times of reuse.In conclusion,a nano-biocatalyst NHase@Fe₃（PO₄）₂was prepared in this paper.The synthesis conditions are mild and the method is simple.This organic-inorganic hybrid nanoflower has the characteristics of high specific surface area,good biocompatibility and friendly to enzyme activity.It has a good buffering effect on nitrile hydratase and avoids the loss of catalytic activity under destructive conditions,provides new ideas for the industrial application of nitrile hydratase,and provides new possibilities for the development of immobilized enzymes.