NADH Regeneration Using Graphene-modified TiO₂ As Photocatalyst And Immobilization Of [Cp*Rh?bpy??H₂O?]²⁺ Precursor

NAD?H?is a widely existing pyridine nucleotide coenzyme,which can assist the oxidoreductase in completing the catalytic role.However,the application of the oxidordeuctase is greatly restricted because of the stoichiometric amount consumed and high cost of the coenzyme.The research on coenzyme regeneration can not only expand the application range of biocatalysis and lower the cost of biocatalytic processes,but also simplify the product separation.It is in favor of the enzyme-catalyzed reaction towards the direction of forward reaction.Therefore,it is of great significance to regenerate coenzyme.Firstly,the dispersion conditions of TiO₂ nanoparticles and the adsorption properties of NAD⁺and NADH on the TiO₂ nanoparticles were investigated.The results show that the time and speed of magnetic stirring affected the dispersion of low concentration of TiO₂ in water.The dispersion reached the stable status with the stirring speed of 400 r/min after 15 min.When the concentration of TiO₂ was higher than?g/L,the time and speed of magnetic stirring on the dispersion of TiO₂ had little effect.TiO₂ had no adsorption to NAD⁺and NADH.Secondly,RGO-TiO₂ composite photocatalyst was successfully synthesized by hydrothermal synthesis method and characterizd by XRD and FTIR.The catalytic properties and influencing factors of RGO-TiO₂ composite catalyst for the photocatalytic reduction of coenzyme NAD⁺were studied.The reaction rate and conversion rate in the system using RGO-TiO₂ composite catalyst was close to that of TiO₂,both of wihichwere obviously superior to the system using the mixture of GO and TiO₂.The ratio of RGO and TiO₂ had no obvious effect on the reaction.EDTA-Na₂-NaOH as the reaction medium,the optimum pH value was 7.0.The addition of[Cp*Rh?bpy??H₂O?]²⁺could greatly upgrade the reaction rate,shorten the time required to achieve chemical equilibrium.Under the optimum concentration of[Cp*Rh?bpy??H₂O?]²⁺,which was 0.25 mM,the conversion rate of NADH was up to94%.The activity of[Cp*Rh?bpy??H₂O?]²⁺was attenuated with the prolongation of the storage time,and the reaction rate of NADH regeneration after 43 days of storage was only 50%that of the 18 days of storage.The catalytic activity of RGO-TiO₂ and TiO₂ under the visible light is extremely low.The[Cp*Rh?bpy??H₂O?]²⁺plays an important role in the photocatalytic process,however,it is expensive and not easy to seperate.Thus,using TiO₂ as the carrier,the surface modification of TiO₂ and the immobilization of[Cp*Rh?bpy??H₂O?]²⁺precursor were carried out.The results of FTIR analysis show that 3-amino propyl triethoxy silane?APTES?is successfully grafted on the surface of TiO₂.TGA results show that when the addition amount of APTES and TiO₂ was 0.5 mL amd 0.15 g,respectively,the highest grafting rate was approximately 2%.Further,as the precursor of[Cp*Rh?bpy??H₂O?]²⁺,5-amino-1,10-phenanthroline adjacent dinitrogen was conjugated to the surface of APTES modified TiO₂ by glutaraldehyde,which provided the basis for the further immobilizationof[Cp*Rh?bpy??H₂O?]²⁺on TiO₂.