Construction Of Dual-immobilized Enzyme System Based On Protein Nanocapsules And Its Application Study

Industrial biotechnology is consistently searching for a method to improve enzyme stability and make them recyclable and reusable.Enzyme immobilization is an effective approach to improve the stability and reusability of enzymes in solution.To construct a robust immobilized enzyme,searching for a suitable supporting matrix and choosing a suitable immobilization method are the most important steps.Based on the above two considerations,a new enzyme immobilization method based on protein nanocapsules,namely dual-immobilized method,was proposed in present study.The in situ radical polymerization technique was applied to construct a polymer shell around the enzyme surface to form the protein nanocapsule.Then,protein nanocapsules assembled on the surface of graphene oxide(GO)to fabricate the dual-immobilized enzyme.Here,native organophosphorus hydrolase(OPH)and OPH nanocapsules(nOPHIO)were immobilized on GO to form the traditional immobilized OPH(OPH@GO)and dual-immobilized OPH(nOPH10@GO),respectively.We systemically studied the enzymatic properties,catalytic performance and assembly mechanism of the immobilized enzymes,and then a biosensor for organophosphorus pesticides(OPs)detection was developed based on the dual-immobilized enzyme.The protein nanocapsules were synthesized through two steps:enzymes grafting with N-acryloxysuccinimide(NAS)and nanocapsulation.The relative activities were used as optimization criterions,and the proportions of different compotents were optimized through single factor experiment.The relative activity of resulted nOPHs was 97.3%.TEM,AFM,SDS-PAGE and DLS were adopted to characterize the nOPHIO.Compared with native OPH,the zeta size of nOPHIO increased,and the zeta potential and electric property of nOPHIO changed,and the nOPHIO exhibited different electrophoresis phenomenon.Therefore,combining the above results,it is reasonable to conclude that the proposed enzyme nanocapsules are successfully constructed with a "core-shell" structure.GO was prepared according to the modified Hummers method.Native OPH and nOPHIO self-assembled on the surface of GO through noncovalent interactions to fabricate the OPH@GO and nOPH10@GO.AFM,CLSM,DLS and CD were adopted to characterize the morphgraphy and structure of immobilized enzyme and confirm the successful fabrication of dual-immobilized enzyme system.Further studies found that the loading efficiency of nOPHIO on GO was higer than that of native OPH.The nOPHIO was immobilized on GO mainly because of the electrostatic attraction and hydrogen bonding,whereas the hydrophobic interaction was the driving force for the immobilization of OPH on rGOsThe enzymatic parameters,catalytic performance and stability of immobilized enzymes were studied.Compared with native OPH,the enzymatic acticity of OPH@GO decreased,while Michaelis constant(Km)and catalytic constant(kcat)increased,and kcat/Km decreased The all above countparts of nOPH10@GO increased.The thermal stabilities followed the order nOPH10@GO>OPH@GO>OPH.The pH ranges of native OPH,OPH@GO and nOPH10@GO maintaining above 90%relative activity were about 7.6-8.7,7.2-9.0,and 6.5-9.2,respectively.Incubating in 20%volume fraction of methanol,the nOPH10@GO maintained about 60%of its original activity,nearly 3 times more than that of native OPH(20.3%)and OPH@GO(22.0%).The nOPH10@GO maintained 75%of its original activity after five freeze-thaw cycles and almost all activity throughout 30 days' storage,and it kept more than 90%of its original activity after 10 times reuse.All these properties were better than native OPH and OPH@GOThe biosensors(OPH@GO/GCE and nOPH10@GO/GCE)for OPs detection were fabricated based on OPH@GO and nOPH10@GO.The peak currents were used as optimization criterions,and the optimized amount of immobilized enzyme and Nafion was 6 uL.SEM found that the surfaces of OPH@GO/GCE and nOPH10@GO/GCE were coarse with some tunnels.EIS of electrods increased after modified with immobilized enzymes Further studies found the reactions on OPH@GO/GCE and nOPH10@GO/GCE surface was diffusion-controlled process.The responding current,pH adaptation,accuracy,repeatability and storage stability of nOPH10@GO/GCE were better that that of OPH@GO/GCE,which was related to the catalytic performance and stability of immobilized enzymes.