Immobilization Of Laccase On Monolithic Silica-based Macroporous/Mesoporous Composite And Its Application In Dye Degradation

Laccase has been proved to have the special catalytic ability of highly efficient degradation of dyes,and friendly to environment.However,the free laccase is not easy to be recovered from the reaction system and can not be reused,which limits the application of laccase in the industrial field.Immobilized enzyme technology is considered as the most effective improvement method,which takes into account the characteristics of enzyme and realizes the reuse of enzyme,and also improves the stability of enzyme,so that enzyme can be applied in modern industry.In this work,a monolithic macroporous/mesoporous SiO₂ with three-dimensional?3D?continuous pass-through macropore structure was prepared and modified for the immobilization of laccase.Using azophloxine as a simulated water pollutant,the catalytic performances of two kinds of immobilized laccases(Lac-PDA/SiO₂ and Lac-Cu²⁺-PDA/SiO₂)for the degradation of dye were compared.Meanwhile,a packed bed laccase reactor was designed and fabricated for continuous decolorization of azophloxine.Specific contents are as follows:?1?A monolithic macroporous/mesoporous SiO₂ with three-dimensional?3D?continuous pass-through macropore structure was prepared.Through the in situ oxidative polymerization of dopamine?DA?on the surface of the pore wall of the macroporous/mesoporous SiO₂,a monolithic composite material modified by polydopamine?PDA?was obtained.Samples were analyzed by SEM,BET,FTIR and TG.The monolithic composite material?PDA/SiO₂?was used to immobilize commercial Novozymes laccase and the effects of pH,immobilizing time and temperature on the immobilization were investigated.Using azophloxine as a simulated water pollutant,the catalytic performance of the immobilized laccase?Lac-PDA/SiO₂?for the degradation of azophloxine was studied.The results show that the activity of Lac-PDA/SiO₂reaches to 348.9 U/g under the optimized conditions.Up to 99.9%of azophloxine was decolored under azophloxine concentration 10 mg/L,pH of 7.0,temperature of 30?and degradation time of8h.The immobilized laccase exhibited good decolorization reusability.?2?The composites with PDA chelated Cu²⁺(Cu²⁺-PDA/SiO₂)were prepared by using monolithic macroporous/mesoporous SiO₂ as the substrate and Cu?CH₃COO?₂/H₂O₂ as the oxidants to initiate free radical polymerization of dopamine.The PDA film with Cu²⁺was rapidly deposited on the surface of the monolithic macroporous/mesoporous SiO₂.Samples were analyzed by SEM,BET,FTIR,EDX and XPS.Laccase was immobilized using Cu²⁺-PDA/SiO₂ as the carrier by covalent binding and ion affinity methods,and the conditions of laccase immobilization were optimized.The immobilized laccase(Lac-Cu²⁺-PDA/SiO₂)was applied to the decolorization of azophloxine,and its decolorization performance was compared with that of Lac-PDA/SiO₂.The results show that the activity of Lac-Cu²⁺-PDA/SiO₂ reaches to 463.3 U/g under the optimized conditions;its thermal stability,pH stability and operating stability far exceed those of Lac-PDA/SiO₂ and its enzyme activity and enzymatic properties are improved further.Up to 99.9%of azophloxine was decolored under pH of 7.0,azophloxine concentration 10 mg/L,temperature of30?and degradation time of 2 h.Lac-Cu²⁺-PDA/SiO₂ also exhibited good reusability.?3?A packed bed reactor filled with the immobilized laccase was designed and fabricated for continuous decolorization of dye.The conditions of decolorizing azophloxine using the packed bed laccase reactor were optimized and the reusability was tested.The results show that the packed bed laccase reactor has the high decolorization rate and good reusability under the flow of 1/3 L/h,pH of 7.0,azophloxine concentration 10 mg/L,temperature of 30?.