Study On The Immobilization Of Laccase Based On Biomimetic Mineralization And Its Application In Dye Wastewater Treatment

Malachite green,as a cationic triphenylmethane dye,is widely used in leather,cosmetic and textile industry.However,it is difficult to be removed and highly toxic to humans because of its high toxicity,high residue and high carcinogenicity.Malachite green wastewater is a type of refractory industrial dye wastewater.Laccase has an important value in the aspects of sewage treatment and decolorization of dyes due to its extensive substrate specificity,but the free laccase is susceptible to the external environment.Poor stability,easily inactivation and hard recovery restrict the application of free laccase.So it is imperative to develop effective immobilization technology to solve these problems.Biomimetic mineralization provides a favourable immobilization environment for enzyme with its simple operation,mild condition and rapid reaction.It makes the application of biomimetic nanomaterials in enzyme immobilization a hot topic research.Therefore,based on the mechanism of biomimetic mineralization,titania and zirconia nanoparticles were prepared,respectively,and used for laccase immobilization in this paper.Moreover,the decolorization of malachite green by immobilized laccase was studied.Titania and zirconia nanoparticles were synthesized by using protamine sulfate and lysozyme as inducers,respectively.The two materials were characterized by field emission scanning electron microscope(FSEM),transmission electron microscopy(TEM),fourier transform infrared spectroscopy(FTIR),X-ray diffraction(XRD),X-ray photoelectron spectroscopy(XPS),thermo gravimetric analyzer(TGA),and N2 adsorption-desorption.Results showed that protamine and lysozyme did not only play a catalytic role in the biomimetic process but also act as templates embedded in titania and zirconia,respectively.Finally,organic-inorganic composite of irregular spherical nanoparticles were formed.According to the mechanism of biomimetic mineralization,laccases were immobilized in titania and zirconia,respectively.The immobilization conditions and enzymatic properties of the immobilized laccase were also studied.It was fo und that the recovery rate of laccase immobilized in titania was 46.6%when protamine concentration was 5 mg/mL,Ti-BALDH concentration was 30 mmol/L,and laccase concentration was 0.1 U/mL.On the other hand,when K2ZrF6 concentration and lysozymeconcentration was 0.1 mmol/L and 5 mg/mL,respectively,the recovery rate of laccase immobilized in zirconia was 60%.In addition,the heat resistance and pH stability of immobilized laccase were significantly improved compared to free laccase in the aspect of enzymatic properties.The activity of immobilized laccase showed almost no loss after the storage of 30d,and the immobilized laccase also showed good repeatability.Finally,the decolorization of malachite green by immobilized laccase was studied.Wherein,the decoloring conditions were optimized by the response surface method.The degradation of malachite green was analyzed by the UV-Vis spectra(UV-Vis)and the gas chromatography-mass spectrometry(GC-MS).Results showed that the degradation pathway started with malachite green hydroxylation and quinone structure disappearance.It was also indicated that the degradation was mainly through a series of amino demethylation and deamination effect,and then the entire dye structure was decomposed into a single benzene ring.In summary,it provided a new way for the degradation of malachite green.