Construction Of Carbon-based Immobilized Enzymeand Its Interaction With Phenols

Although the total discharges of volatile phenols from industrial sources aredecreasing year by year,the pollution of volatile phenols in inland waters of China is still severe,which not only affects the stability of the aquatic system,but also poses a serious threat to the healthy development of human society.Therefore,it is necessary to develop a technology that can effectively remove or neutralize phenol and its derivatives.The oxidoreductase stands out because it has the characteristics of microorganisms that can degrade phenolic compounds into non-toxic products and can overcome the inhibitory effect of pollutants,and the rise of enzyme immobilization technology has made it attract attention in environmental pollution control.However,the conformational changes of immobilized enzymes and their internal correlation mechanisms with enzyme activity and environmental pollutants are still unclear,which to some extent hinders the improvement of the catalytic efficiency of immobilized enzymes an d their application in the removal of refractory organic pollutants.This study will select the carbon nanomaterial as the representing carrier for immobilizing Horseradish peroxidase(HRP)and laccase,andthe immobilized enzyme will be applied to the removal of phenolic recalcitrant organics.Scanning transmission X-ray microscopy,Dynamic light scattering,high-resolution electron microscope,Raman spectrometry,Fourier infrared spectrometry,circular dichroic spectrometrywill be used to study the spatial distribution,activity and conformational change of the immob ilized enzymes,and their interaction mechanism with phenolic pollutants.The effect of surfactants on the degradation of phenolic pollutants by carb on-based oxidoreductase will also be explored.The activity and conformational changes of carbon-based oxidoreductase and the mechanism of interaction between carbon-based oxidoreductase and phenolic pollutants will be elucidated.The research results of this study will provide theoretical evidence and technical support for the effective construction of carbon-based immobilized oxidoreductases and the control of the adverse effects of environmental refractory organic pollution on the ecological environment.This paper mainly includes three parts:The first part foucuses on the optimization of oxidoreductase immobi lization on carbon nanomaterials.HRP and laccase were immobilized on carbon nanomaterialsrespectively.The effects of immobilization conditions suc h as enzyme concentration,p H,immobilization time and temperature were studied in detail,and the immobilized oxidoreductase with high load and enzyme activity was prepared.The immobilization conditions vary with the type,size and surface charge of the enzyme.HRP has the optimal catalytic activity at p H 6,while the optimal immobilization p H of laccase is 4.And the optimal temperature is also different,laccase has the highest catalytic activity at 35 °C,while that of HRP is 25 °C.Therefore,the physical and chemical properties of the carrier and enzyme should be considered comprehensively when optimizing the immobilization conditions.Under the optimal immobilization conditions,the loadings of HRP and laccase immobilized on three-dimensional ordered mesoporous carbon(3DOm C)prepared by physical methods are higher,which can be as high as 4568 mg/g carrier a nd 3206 mg/g carrier,respectively,the activities of the corresponding immobilized enzymes are 92.03% and 68.64%,respectively,which are mainly due to the excellent adsorption performance and unique pore structure of 3DOm C.Although the load and enzyme activity of immobilized HRP and laccase by covalent binding method are lower,no enzyme leakage has occurred.This study provides a theoretical basis for the construction of high-performance immobilized oxidoreductase.The second part is the study of the c atalytic activity and conformation of the carbon-based immobilized oxidoreductase.On the basis of the first part,the effects of immobilization conditions,immobilization methods,physicochemical properties of the carrier,and enzyme types on the catalyti c activity of immobilized enzymes were comparatively studied.The spatial distribution and conformational changes of enzyme on the carrier were discussed by using the characterization techniques including Scanning transmission X-ray microscopy,Fourier transform infrared spectroscopy and circular dichroism spectroscopy.The introduction of carboxyl hydrophilic functional groups can improve the hydrophilicity and Zeta potentialof carbon nanocarrier materials whilereducingits hydraulic diameter,thus effectively enhancing the catalytic performance of immobilized HRP.Although the grafting of polyethyleneimine(PEI)increases the loading of HRP,the catalytic activity decrease s due to the increase of the diffusion resistance of the substrate.HRP with smaller molecular size is more suitable for immobilization on 3DOm C with 9 nm pore size,while laccase has better catalytic performance on 3DOm C with large pore size of 15 nm.The affinity of immobilized HRP and laccase to substrate decreases,and the changes of protein secondary structure of the enzyme are reflected in the decrease of ?-helix structure,the increase of ?-turn structure,and the increase of ?-sheet structure,respectively.This study contributes to the rational design and development of high-efficiency immobilized enzyme biocatalysts.The third part is the study of the interaction mechanism between carbon-based immobilized oxidoreductase and pollutants in the environment.On the basis of the above two parts,the catalytic degradation of refractory phenolic compounds in water and sediment by immobilized HRP and laccase was investigated,and the effects of different surfactants on the degradation of phenolic compounds were investigated.The study found that HRP immobilized with magnetic 3DOm C(Fe-3DOm C-HRP)not only overcomes the problem that free HRP is easy to deactivate,but also has good thermal stability,storage stability and recycling.Fe-3DOm C-HRP has an obvious advantage in the catalytic removal of phenol,80.2% of phenol can be removed within 4 h,which is the result of the synergistic effect of 3DOm C adsorption and HRP catalytic performance.Both the anionic surfactant rhamnolipid(Rha)and non-ionic surfactant alkyl polyglycoside(APG)can improve the cata lytic activity of laccase,especially for the immobilized laccase.This is due to the fact that the interaction btweensurfactants and enzymeschanges the conformation of the enzyme,so that the active sites of enzyme that have lost freedom during the immobilization process are re-exposed,and at the same time,the affinity of the enzyme and the substrate is improved.The promotion of surfactant on the catalytic activity of laccase and the solubilization of phenol,combined with the strong enrichment performanc e of 3DOm C,the removal performance of immobilized laccase for phenol is also significantly improved,and the removal efficiency of phenol solution in the concentration range of 50-400 mg/L is above 88%.3DOm C-HRP and 3DOm C-Laccase have significant catalytic remediation effect on 2,4-dichlorophenol(2,4-DCP)in water and sediment media,especially for 3DOm C-HRP,the removal of 2,4-DCP in water can reach 81.3%,and 100 mg of 3DOm C-HRP can reduce the concentration of 2,4-DCPin sediment from 97.5 mg/Kg to 28.8 mg/Kg within 30 days.3DOm C-Laccase can be uniformly distributed in the sediment medium without serious agglomeration,and the local concentration of laccase is increased,thereby promoting the catalytic removal of 2,4-DCP in the sediment.The application of immobilized enzymes in the rem ediation of organic pollution in the sediments of rivers and lakes will not cause changes in the p H value of the river and lake environment,and can effectively increase the content of organic matter in the sediments.This study not only has significant gui ding significance for the application of immobilized enzymes to the remediation of refractory organic pollution in river and lake sediments,but also provides theoretical basis and technical support for effectively co ntrolling the adverse effects of refractory organic pollution in river and lake sediments on the ecological environment.