Synthesis And Continuous Catalytic Application Of Second Immobilized Protease-Inorganic Hybrid Nanoflowers

Protease is an important industrial enzymes,the harsh production environment will often lead to the loss of activity.Immobilized enzyme is an effective method to solve the above problems.Immobilized protease not only has higher stability,but also can be recycled,if put into industrial production can save a lot of bioenergy.Enzyme immobilization at nanometer scale is a hot research topic in recent years.Although immobilized enzymes obtained by this method have very high activity,they also have a lot of disadvantages such as high separation cost and fragile structure.Therefore,on the foundation of the nanosized immobilize protease,this paper carried out the secondary immobilization,aiming to develop an immobilization method with high catalytic activity and easy separation.In this paper,papain nanoflowers was firstly prepared by nanosized immobilization,and then papain nanoflowers were embedded in polyvinyl alcohol（PVA）hydrogel to obtain papain nanoflowers composite hydrogel（PNP）.The structure of papain nanoflowers and PVP were characterized by FT-IR,XRD and EDS.These results showed that papain was successfully immobilized into PNP.Then the pattern of PNP was observed by SEM,and the results clearly showed that the nanoflowers was embedded in the 3D network of PVA hydrogel.Simultaneously,the activity of PNP was evaluated.And the result shows the enzyme activity of PNP was 21.11 times higher than free papain.In addition,PNP has not only excellent temperature and p H stability,but also excellent recycling.After 7 times of recycling,it still retains 212.4%of the relative activity.In order to study whether the secondary immobilization method has universal applicability to other proteases,this paper applied the same method to immobilize alkaline protease obtaining alkaline protease nanoflowers composite hydrogel（NPCH）.NPCH were characterized by FT-IR,XRD and EDS,and the results showed that this method could also successfully immobilize alkaline protease.The highest enzyme activity of NPCH was only5.96 times of free enzyme,but it still retained 109.1%of relative activity after 12 cycles.In order to solve the problems of high separation cost and impaired catalytic activity of papain nanoflowers in the separation process,this paper introduced magnetic nanoparticles into the immobilized materials to synthesize papain-Cu₃（PO₄）₂·3H₂O-magnetic nanoflowers（PCMN）.On the premise of retaining the advantage of large specific surface area of the nanomaterials,the magnetic nanoparticles could be rapidly separated from the reaction solution.PCMN were characterized by FT-IR,XRD and EDS,and the results showed that the carrier was Cu₃（PO₄）₂·3H₂O and magnetic nanoparticles.The flower pattern of the nanoparticles can be clearly observed by SEM,and the magnetic nanoparticles are evenly distributed on the surface.In addition,the highest enzyme activity of PCMN was 1556.6%of free enzyme,and the highest enzyme activity recovery was 597.6%.In order to explore the application of PCMN in practical production,the experiment of hydrolyzing milk sensitization protein in PCMN was carried out,and the results showed that PCMN had a good hydrolysis effect onβ-lactoglobulin（β-Lg）.