Immobiization Of Laccase From Cerrena Unicolor And Applications In Degradation Of Environmental Pollutants

Laccase(EC 1.10.3.2)is a class of copper-containing polyphenol oxidases.Laccase has broad substrate specificity and application potentials in various industries including wastewater treatment.In this study,white-rot fungus Cerrena unicolor HYB07 laccase was immobilized.The best conditions for preparing immobilized laccase,enzymatic properties and degradation of environmental pollutants with free laccase/immobilized laccase were studied.The results are as follows:1.Cerrena unicolor HYB07 laccase was immobilized by three methods:cross-linked enzyme aggregates(CLEAs),sodium alginate embedding and chitosan-glutaraldehyde crosslinking.Preparation conditions of cross-linked enzyme aggregates were optimized.With ammonium sulfate or acetone as the optimal precipitant and glutaraldehyde as the cross-linking agent,activity recovery rates of 68.08%and 59.51%were obtained,respectively.With ammonium sulfate and chitosan as the precipitant and cross-linking agent,the activity recovery rate was 40.37%.Amino-functionalized magnetic nanoparticles bonded to CLEAs,activity recovery rate of magnetic CLEAs was 46.76%.In addition,Cerrena HYB07 laccase was immobilized with carriers,sodium alginate for embedding or chitosan for crosslinking,with activity recovery rates of 29.37%and 50.50%,respectively.2.The enzymatic properties of immobilized laccase were studied,Thermal stability of immobilized laccases were improved significantly.Immobilized laccases had lower substrate affinity towards ABTS and could be reused 5 times in decolorization of Remazol Reactive Brilliant Blue,with the relative activity remained above 50%.CLEA salso demonstrated improved tolerance of metal ions,organic solvents and NaCl.Li+,Hg2+ and Ni2+ were detrimental to laccase activity,with free laccase being more sensitive than CLEAs.With various organic solvents,CLEAs displayed higher tolerance than free laccase,with relative activity above 70%.NaCl was a mixed-type inhibitor of both free and immobilized laccase,CLEAs had higher inhibition constants KI and KTIS,The values of KI and KIS of NaCl inhibition of CLEAs(8.3 and 29 mM,respectively)and free laccase(1.6 and 11.5 mM,respectively).CLEAs also displayed greater velocity and efficiency in Ramazol Brilliant Blue Reactive decolorization in the presence of NaCl compared with free laccase.3.Free laccase catalyzed decolorization of Malachite Green and Remazol Reactive Brilliant Blue,response surface methodology was adopted for optimization of decolorization conditions,and decolorization ratios were above 90%.Liquid chromatography-mass spectrophotometer was used to identify Malachite Green degradation products.Degradation pathways were deduced based on the identified products.Destaining of Coomassie Brilliant Blue R-250-stained polyacrylamidegels with fungal laccase/mediator system was studied,a clear gel background was obtained in 2 h at 37? with 2 ?m mediator(ABTS)and 15 U/mL laccase.In addition,various polycyclic aromatic hydrocarbons,phthalate esters,environmental hormones and antibiotics were degraded with immobilized laccase.Phenanthrene,diethyl-stilbestrol,dimethyl phthalate,tetracycline were degraded by immobilized laccase most efficiently,with the decolorization ratios above 80%.Furthermore,liquid chromatography-mass spectrophotometer was used to identify degradation products of tetracycline.In this research,the optimal preparation conditions and enzymatic properties of immobilized laccases,as well as degradation conditions and mechanisms of environmental pollutants by free/immobilized laccases were studied.The research findings served to facilitate industrial applications of Cerrena laccase.