Co-immobilization Of NADH Oxidase And Dehydrogenase Onto Epoxy-functionalized Nanoparticles For In Situ Regeneration Of NAD

Oxidoreductase is a class of important biocatalyst,which usually requires coenzyme to complete the electron transfer in enzyme catalyzed reaction.Its' industrial application is limited because the coenzyme is very costly.Regeneration of the cofactor could effectively lower the cost.NAD is the most widely used cofactor of oxidoreductase.The regeneration of NAD using NADH oxidase has the advantages of no by-product and high efficiency.In this work,the cofactor regeneration system was designed and examined with enzymatic production of 1,3-dihydroxyacetone?DHA?via immobilized NADH oxidase?No_x?coupled with glycerol dehydrogenase?GDH?.The reuse of immobilized enzyme is very easy due to the magnetism of magnetic support,which is beneficial for an efficient regeneration of cofactor.In the present work,preparation of magnetic nano-carrier,enzyme immobilization,optimization of immobilization process,properties of immobilized enzyme,and applications of immobilized enzyme were performed.In Chapter 2,magnetic Fe₃O₄@SiO2-Epoxy nanoparticles were prepared and used as a carrier for enzyme immobilization.Firstly,Fe₃O₄ magnetic nanoparticles were synthesized by chemical co-precipitation method.Then,the magnetic nanoparticles were coated with SiO2 by sol-gel method.Finally,the surface of silica nanoparticles was modified by ?-ether oxygen trimethoxysilane?GPS?.Thus,epoxy-functionalized magnetic nanoparticles were obtained.The characterization of nanoparticles was performed by transmission electron microscopy?TEM?and Fourier transform infrared spectrometer?FTIR?.It confirmed that nanoparticles had been successfully prepared and the diameter of nannoparticle was about 16-24 nm.The prepared nanoparticles were used for covalent immobilization of Alcohol dehydrogenase?ADH?.The optimal immobilization conditions were obtained as follows: enzyme/support 5.27 mg/g,pH 8.0,temperature 30 oC and buffer concentration 0.05 M.Under optimal conditions,a high immobilization yield of 90% was obtained.After immobilization,the enzyme displayed improved stability and good reusability.The immobilized ADH retained more than 80% initial activity after six cycles.Finally,the immobilized ADH was applied to catalyze the reduction of phenylglyoxylic acid to?R?-mandelic acid,and the yield of 64% was obtained.In Chapter 3,NADH oxidase was immobilized onto the surface of epoxy-functionalized nanoparticles by covalent attachment.The resulted immobilized enzyme had a good stability and was easy to recycle.The immobilization conditions of No_x were optimized.The optimal conditions were obtained as follows: enzyme/support 6.47 mg/g,immobilization time 2 h,pH 7.0 and temperature 30 oC.After optimization,a high immobilization yield of 92% was obtained.It was found that the optimal temperature of immobilized No_x was 45 oC.The activity of immobilized No_x was much higher than that of free one under acidic conditions.The immobilized No_x had better affinity to substrate than the free enzyme.It reserved almost 83% initial activity after nine cycles,indicating that the immobilized enzyme has good reusability.In Chapter 4,we applied the immobilized No_x coupled with GDH for efficient conversion of glycerol to DHA with an in situ cofactor regeneration system.The coupled reaction system was also investigated in view of various operational factors.The optimal conditions for DHA production were obtained as follows: GDH/No_x ratio?w/w?2:1,temperature 25 oC,pH 7.5 and glycerol concentration 100 mM.Under optimal conditions,a high DHA concentration can reach up to 3.5 mM,which was about 4 times higher than that in the absence of No_x.The results suggested that the enzyme-coupled cofactor regeneration system could be utilized for efficient enzymatic production of DHA from the inexpensive substrate.