Construction Of Immobilized Double Enzymes Catalytic System And Study On The Degradation Performance Of Polycyclic Aromatic Hydrocarbons In Water Solution

Polycyclic aromatic hydrocarbons(PAHs)are persistent and refractory organic pollutants.As the number of benzene rings in the structure increases,their toxicity becomes stronger and stronger.Especially in recent years,the content of PAHs in the freshwater environment in some areas has reached several milligrams or even tens of milligrams per Rise,seriously exceeding the upper limit of safety.PAHs in the water will gradually enter the human body through the accumulation of biological chains,which will not only cause teratogenic,carcinogenic,mutagenic,and even endanger life and safety.At present,traditional physical treatment methods(adsorption),chemical treatment methods(chemical oxidation)and biological treatment methods(strain degradation)still cannot effectively remove PAHs.Studies have shown that the laccase extracted from sumac tree or white rot fungi has potential application prospects in the treatment of PAHs in water without causing secondary pollution.However,the natural free enzyme is unstable,which limits its practical application.Many studies have shown that immobilization is an effective method to improve stability.This study tried to construct double enzymes catalytic system immobilized on hollow nanofibers to degrade four PAHs of naphthalene,anthracene,pyrene and benzo[a]pyrene in water.Firstly,the polyvinylidene fluoride(PVDF)hollow nanofiber membrane was prepared by coaxial electrospinning as a carrier for enzyme immobilization;Subsequently,the model protein bovine serum albumin(BSA)was fixed in-situ in the cavity of the PVDF nanofibers under different conditions to determine the best fixation condition,and the typical glucose oxidase(GOx)/horseradish peroxidase(GOx)/horseradish peroxidase(HRP)two enzyme andβ-galactosidase(β-Gal)/GOx/HRP three-enzyme cascade catalytic system were immobilized,to explore the feasibility and activity of the immobilized multi-enzyme catalytic cascade reaction of this method;Finally,laccase and HRP were immobilized in the cavity of PVDF nanofibers in the same way,and the four PAHs of naphthalene,anthracene,pyrene and benzo[a]pyrene were degraded by the immobilized Laccase/HRP double-enzyme catalytic system.The following conclusions were obtained:(1)Preparation of PVDF hollow nanofiber membrane immobilized carrier by coaxial electrospinning method.Optimized the solvent ratio of the outer polymer spinning solution of the coaxial electrospinning and the electrospinning parameters such as temperature,drum receiver revolutions,drying method and other influencing factors to prepare PVDF hollow nanofiber membrane carrier with uniform morphology and holes on the surface.Analyzing the pore structure of the fiber surface,the results show that the pores on the surface of the PVDF nano fiber are larger than 5nm,which is ensured to the sufficent contact of PAHs and enzymes;According to the needs of immobilized enzymes to degrade PAHs in water,the hydrophilicity and hydrophobicity,mechanical properties of PVDF nanofiber membrane carrier and the adsorption properties of naphthalene,anthracene,pyrene,and benzo[a]pyrene in water were tested.The experimental results showed that have outstanding adsorption performance of mechanical properties and the four kinds of PAHs.In the single-system PAHs adsorption reaction,the adsorption rate of pyrene is as high as 100% within 3 hours.(2)In-situ encapsulating of multiple enzymes in PVDF hollow nanofibers and multi-enzyme catalyzed cascade reactions.Study the immobilization of model protein BSA in hollow PVDF nano fiber membrane carrier under different conditions.When the dopamine(DA)in the inner core spinning solution is 2mg/m L,it in-situ polymerized to produce polydopamine(PDA),and BSA was immobilized on PVDF nano in the inner cavity of the PVDF nano fiber.and the fixation effect is the best,the amount of fixed BSA is0.01195mg/mg;The GOx/HRP double-enzyme and β-Gal/GOx/HRP three-enzyme cascade catalytic system were immobilized in the inner cavity of nanofibers in situ by using PDA.Experiments confirmed that enzymes were randomly co-immobilized in the inner cavity of PVDF nanofibers The GOx/HRP double enzyme and β-Gal/GOx/HRP triple enzyme have the catalytic properties of cascade reaction;And due to the protective effect of the outer shell of PVDF nanofibers for the immobilized multi-enzymes in the inner cavity and the micro-nano confinement effect formed by the inner cavity as the cascade catalytic reaction site,the activity of the multi-enzyme catalytic cascade reaction is improved.It provides a theoretical basis for the experiment of the immobilized Laccase/HRP double enzyme catalytic system to degrade four PAHs in water.(3)The immobilized Laccase/HRP double-enzyme catalytic system degrades four PAHs in water.Using the same method to fix Laccase and HRP in situ in the inner cavity of PVDF nanofibers,using 2,2’-diazo-bis-3-ethylbenzothiazolin-6-sulfonic acid(ABTS)as the substrate the activity and stability of Laccase/HRP-PDA@PVDF were tested.After immobilization,the total activity of Laccase/HRP kept 80% compared with the free enzymes,and the activity still kept the original 80% after storage in pH 7.4 buffer for 7 days.After the ABTS oxidation experiment is operated 10 times,the activity still retains the initial 40%.The experimental results show that the storage stability and operation stability of Laccase/HRP are greatly improved after immobilization.Laccase/HRP-PDA@PVDF,free Laccase,free HRP and free Laccase/HRP were used to degrade four PAHs in water.After 8 hours of reaction,no residual PAHs were detected in the system.Analyze the degradation performance of Laccase/HRP-PDA@PVDF,free Laccase,free HRP and free Laccase/HRP by gas-phase mass spectrometry(GC-MS)and liquid-phase mass spectrometry(LC-MS),The experimental results show that Laccase/HRP-PDA@PVDF can achieves better degradation performance than free Laccase/HRP.Furthermore,benzo[a]pyrene was used as an example to analyze the degradation products.The results showed that Laccase and HRP co-fixed in the inner cavity of the nanofiber may possess a synergistic effect,and some of the intermediate products were degraded to produce oxalic acid;And compared with free enzymes,the by-products like linear alkanes,etc.generated in the reaction are adsorbed on the PVDF nano fiber membrane carrier,reducing the secondary pollution.