Preparation Of Functionalized Oxidized Graphene And Its Applications In Immobilizations Of Enzymes

Graphene is a single or few layered two dimensional sp2-hybridized carbon sheets.Based on the unique physical and chemical properties such as ultra high conductivity,excellent mechanical properties and large specific surface area,it has a wide range of applications in the field of catalysis,especially as a catalyst carrier.The graphene oxide(GO)is an oxygen-containing derivative of graphene,similar in structure to graphene,contains a large amount of oxygen-containing groups such as carboxyl,hydroxyl and epoxy groups,it provides sufficient active sites for the functionalization of materials.Is used as a precursor to prepare graphene and its composites synthesis.In this paper,GO was used as immobilized carrier material,preparation craft and surface from the GO group modification to research.NADH oxidase(NOX)and papain were used as the research object to construct a new immobilized enzyme system for nanomaterials.So as to improve the use of free enzyme in the process of separation is complex,expensive,poor stability and can not be reused and other defects.The main contents and results of this paper are as follows:(1)Functionalized graphene oxide was successfully prepared.Graphene oxide(GO)was synthesized by Hummers method,then GO was activated by ?-sulfate ethyl sulfone aniline(SESA)to obtain ABSE-GO.Rapid immobilization of NOX was carried out by covalent coupling onto the prepared functionized graphene oxide.The optimum temperature of immobilized NOX(45 ?)was 10 ? higher than that of free enzyme(35 ?);The immobilized enzyme retained about 50% of the initial enzyme activity after storage at 4 ? for 720 h,and the free enzyme was reduced to zero after 200 h,the half-life of immobilized enzyme was 4 times of that of free enzyme at 4 ?,and the storage stability was better than that of free enzyme;after repeated use of 6 times,the relative viability of the immobilized enzyme still maintained about 70%.(2)The properties of the organosilicon-functionalized graphene(APTES-GO)by the reaction of the amine groups on the ?-aminopropyltriethoxysilane(APTES)with the oxygen-containing functional groups on the oxidized graphite surface were successfully prepared by covalent bonding.The APTES silane coupling molecules were successfully modified to the surface of the graphene sheets by FT-IR and XRD.Through the XRD pattern,it can be seen that APTES-GO is monolithic and has good dispersibility.The results showed that the optimal pH of the free enzyme was 7.0 and the immobilized enzyme was 6.0;Immobilized NOX has a wider pH suitability and higher thermal stability than free enzymes;The results of kinetic parameters were as follows: the Vmax of free enzyme and immobilized enzyme were 31.53 U/mg and 22.78 U/mg,respectively,and the corresponding Km values were 91.4 ?M and 74.9 ?M,respectively.(3)Using APTES-GO immobilized papain,the optimum conditions for the immobilization process were as follows: the immobilized enzyme loading was 6.75 mg/g,the immobilization time was 45 min,the immobilization temperature was 35 ?,and the immobilization pH was 8.0.The results of kinetic parameters were as follows: the Vmax and Km values of free enzyme were 0.129 g/(L · min)and 7.1 g/L,respectively.The Vmax and Km values of immobilized enzyme were 0.115 g/(L · min)and 3.77 g/L.The half-life of the free enzyme was 432 h after stored at 4 ? for one month,and the remaining enzyme activity of immobilized enzyme was 62.71% after 720 h,so the stability of immobilized enzyme was improved by more than 1.7 times.