Immobilization Of Laccase On Magnetic Fe₃O₄ Nanoparticles And The Research Of Its Application

Laccase?EC 1.10.3.2?as a multi-copper oxidase has relatively low substrate specificity and can oxidize various phenolic and non phenolic compounds.However,the industrial applications are limited due to the low stability and poor reusability of free laccase.In order to increase stability and realize continuous operations,laccase is always immobilized before application.However,the activity of laccase may decrease after immobilization.For this problem,the catalytic properties of functionalized magnetic Fe₃O₄ nanoparticles immobilized laccase were studied from two aspects of immobilization methods and carriers in this thesis.The magnetic immobilized laccase was respectively combined with the external magnetic field:alternating magnetic field and high gradient magnetic field to be used in the reactors to reduce the mass transfer limitation and inhibition of catalytic reaction caused by degradation products,which laid foundation for large-scale industrial wastewater treatment.The main results are as follows:The catalytic properties of immobilized laccase were studied from two aspects of immobilization methods and carriers.Laccase was immobilized on amine-functionalized Fe304 nanoparticles?Fe₃O₄-NH₂ NPs?by covalent binding through glutaraldehyde?GA?or Cu2+ affinity adsorption.The maximum activity recovery of Fe₃O₄-NH₂-Cu2+-Laccase was 42.74%.The maximum activity recovery of Fe₃O₄-NH₂-GA-Laccase was 31.88%.And the corresponding adsorption capacity of Fe₃O₄-NH₂-Cu2+ nanoparticles?NPs?was 1.59 times as high as Fe₃O₄-NH₂-GA NPs.Cu2+affinity adsorption was more suitable for laccase immobilization.Polyethylenimine?PEI?as a spacer-arm polymer was modified on Fe₃O₄-NH₂ NPs.The adsorption capacity of Fe₃O₄-NH₂-PEI?1200?-Cu2+ NPs was larger than Fe₃O₄-NH₂-Cu2+ NPs almost in the whole range of laccase concentration.The maximum activity recovery of Fe₃O₄-NH₂-PEI?1200?-Cu2+-Laccase?107.41%?was much higher than Fe₃O₄-NH₂-Cu2+-Laccase?42.75%?.The corresponding specific activity of Fe304-NH₂-PEI?1200?-Cu2+-Laccase was 12.97 U/mg?protein?,which was separately 1.36 times and 101.33 times as large as Fe₃O₄-NH₂-Cu2+-Laccase and free laccase product from Sigma.Km reflects the affinity of the enzymes to substrates.And smaller Km value means higher affinity between immobilized laccase and substrates.The Vmax values defining maximum reaction velocity and the ratio Vmax/Km values defining a measure of the catalytic efficiency of a laccase-substrate pair.Vmax and Vmax/Km values of Fe₃O₄-NH₂-PEI?1200?-Cu2+-Laccase are separately 1.7 times and 1.6 times as high as the Fe₃O₄-NH₂-Cu2+-Laccase.Spacer arm PEI can reduce the steric hindrance between laccase and carrier to some extent and enhance the flexibility of laccase,thus improved the maximum reaction velocity and catalytic efficiency of Fe₃O₄-NH₂-PEI?1200?-Cu2-—Laccase.The Km values of Fe₃O₄-NH₂-Cu2+-Laccase and Fe₃O₄-NH₂-PEI-Cu2+-Laccase were the same as the free laccase.Immobilized laccase had strong affinity with substrate as free laccase.The enzymatic properties of the two immobilized laccases were both improved and the immobilization realized reuse of laccase.The results showed that Fe₃O₄-NH₂-PEI?1200?-Cu2+NPs were more promising than Fe₃O₄-NH₂-Cu2+ NPs for large-scale laccase immobilization.For PEI functionalized magnetic Fe₃O₄ nanoparticles could enhance the catalytic properties of immobilized laccase,laccase was immobilized on different molecular weight PEI?Mn?1200/10000/60000?functionalized Fe₃O₄-NH₂ NPs.The immobilized laccase was separately combined with alternating magnetic field to catalyze the oxidation of catechol in the reactor to to enhance the micro vibration of magnetic immobilized laccase.The oxidation reaction rate of catechol catalyzed by Fe304-NH₂-PEI?1200?-Cu2+-laccase under an alternating magnetic field?600 Hz,10 Gs?was separately 2.10 times and 1.16 times higher than the control without any external force and with mechanical stirring at 150 rpm.This was a larger increase than Fe₃O₄-NH₂-PEI?10000/60000?-Cu2+-laccase.In addition,the reaction rate catalyzed by Fe₃O₄-NH₂-PEI?1200?-Cu2+-laccase was enhanced as the magnetic field frequency,strength and Fe₃O₄-NH₂-PEI?1200?-Cu2+-laccase or catechol concentration increased.The immobilized laccase retained 85%of its initial activity after six consecutive operations.Using an alternating magnetic field was a powerful way to intensify the reaction rate catalyzed by laccase immobilized on branched polymer chains modified magnetic nanoparticles.In order to reduce the inhibition of catalytic reaction caused by degradation products,laccase immobilized on Fe₃O₄-NH₂-PEI?1200?-Cu2+ was connected with high gradient magnetic field to degrade phenol solution continuously in magnetically fixed bed.The degradation rate of continuous treatment in the fixed bed after 18 h was 2.38 times of that in bath treatments after 6 successive operations.The immobilized enzyme reactor achieved continuous degradation of phenol and reduced the inhibition of catalytic reaction caused by the degradation products.The functionalized PEI also contributed to the efficient degradation.The degradation rate catalyzed by Fe₃O₄-NH₂-PEI?1200?-Cu2+-laccase stabilized at 72.93%after 2.5 h reaction.However,that catalyzed by laccase immobilized on Fe₃O₄-NH₂-Cu2+ NPs decreased to 62.41%.The influences of the volume fraction of nickel wires mesh,flow rate of phenol solution,phenol concentration and Fe₃O₄-NH₂-PEI?1200?-Cu2+-laccase amount in separation column were investigated to find the optimum conditions.Under the conditions,degradation rate of phenol kept over 70.30%in 48 h continuous treatment.The feasibility and advantage of this type reactor together with Fe₃O₄-NH₂-PEI?1200?-Cu2+-laccase were tested.It also provides the basis for the continuous degradation of phenolic compounds in large-scale industrial wastewater.