Immobilization Of Oxidoreductase Based On Two-Dimensional Graphene Materials

Oxidoreductase is one of the most important biocatalysts,which can catalyze reactions with high effienciency and stereoselectivity in mind conditions,but the expensive cofactor is an indispensable part of the reactions.Formate dehydrogenase(FDH)is suitable for the regeneration of cofactor NADH.It has many advantages,namely,favorable thermodynamic equilibrium,the inertness of the substrate and reaction product(CO2)which can remove easily from the main product of the reactions.Phenylananine dehydrogenase(PheDH)can be used in the detection of phenylketonuria(PKU)as well as the production of chiral amino acid by coupling with FDH.Graphene oxide(GO)has unique planar structure with hydroxyl,epoxy,and carboxyl groups distributing on the basal and edge of the plane.GO is an ideal immobilization support for enzymes because of its large surface area and good biocompatibility.In this work,we constructed different scaffolds based on graphene oxide derivatives to immobilize oxidoreductase.The details in this study are summarized as follows:Firstly,graphene oxide was prepared by Modified Hummers Method,and was characterized by XRD,UV-Vis,FT-IR,and Fluorescene spectra.The results showed that the process of strong oxidation introduced functional groups including hydroxyl,epoxy,and carboxyl.Secondly,nickel-coordinated graphene oxide composites(GO-Ni)were prepared as a novel supporter for formate dehydrogenase immobilization.Compared with GO,the loading capacity of GO-Ni increased by 5.2 folds.The relative enzyme activity of GO-FDH and GO-Ni-FDH is 73.3%and 86.7%,respectively.The immobilized enzyme showed good thermal stability,the activity of GO-FDH and GO-Ni-FDH remained 18.2%and 40.9%after 3 h at 60?,respectively,while FDH remained 2.78%.GO-Ni-FDH exhibited a better storage stability,which showed 31.4%relative activity after 20 days' storage at 4?.After eight times usages,GO-Ni-FDH maintained 63.8%activity.Moreover,circular dichroism(CD)and SEM were used to analyze the structure and morphology of immobilized enzyme.The structure of enzyme changed in the process of immobilization,GO-Ni-FDH formed uniform crystal structure because of the multi-layer interactions between GO-Ni and FDH.Thirdly,branched polyethyleleimine(PEI)grafting graphene oxide was applied as a functional material for formate dehydrogenase immobilization.Compared to GO,the enzyme loading capacity of GO-PEI was enhanced by 2.4 folds.The relative activity of immobilized enzyme GO-PEI-FDH remained about 24.9%after 20 days storage at 4?.After eight times reaction,the residue activity was 47.4%.The mechanism of GO-PEI immolization was studied,in which electrostatic interaction plays an important role.Fourthly,CRGO was prepared by reducing GO with L-ascorbic acid(L-AA).The reduction degree of the CRGO was controlled by the reaction time.The loading capacity of CRGO-2,CRGO-4,CRGO-6 and CRGO-12 is 0.278,0.362,0.521 and 0.587 mg/mg,respectively,and the the relative activity of immobilized enzyme CRGO-2-FDH,CRGO-4-FDH,CRGO-6-FDH and CRGO-12-FDH is 88.6%,90.2%,90.3%and 90.6%,respectively.Compared with GO-FDH,CRGO-12-FDH exhibited better thermostability and reusability.The enzyme activity CRGO-12-FDH remained 20.8%after 3 h at 60?.After eight times use,CRGO-12-FDH maintained 70.4%activity.Moreover,circular dichroism(CD)and SEM were used to analyze the structure of immobilized enzyme.Finally,graphene oxide was used for phenylalanine dehydrogenase immobilization by adsorption,the optimum pH and temperature of PheDH changed in the process of immobilization.The relative enzyme acivity of GO-PheDH is 75.84%,after 8 times use,the activity remained 13.15%.Explore the possibility of PheDH immobilization based on graphene oxide materials.