Applications Of Laccase In Decolorization Of Dyes And Degradation Of Hydrophobic Aromatic Compounds

In the 21st century,one of the most important problems facing human beings is environmental problem.The environmental protection has become the topics actively researched by many scientific researchers.With the rapid development of biological science and engineering,biotechnology has been displaying its great power in the environmental protection field increasingly.The use of enzyme in environmental protection presents good development prospect.Laccases are oxidoreductases belonging to the multinuclear copper-containing oxidases.They can catalyze oxidation of phenols and aromatic amine compounds.In the presence of mediators, the range of their oxidizable substrates can be expanded further.Because their widely substrate specificity,laccases have important applications in wastewater treatment, bio-bleaching,aromatic compounds conversion and environment monitor,etc.The enzymatic properties of laccase are different from its origin.In this paper, firstly,we studied the kinetics of the decolorization of textile dyes by laccase obtained from Irpex lacteus dft-1 and investigated the effects of reaction conditions,such as mediums and additives on the decolorization.Response surface methodology(RSM) was used for the evaluation of effects of various parameters and their interactions on the decolorization of Remazol Brilliant Blue R(RBBR) by laccase from Irpex lacteus dft-1.Secondly,we studied the effects of different types of surfactants(cationic, anionic and nonionic) and polyethylene glycol(PEG) on the enzymatic properties of laccase and the influence of nonionic surfactant Triton X-100 on the hydrophobic aromatic compounds bisphenol A(BPA) conversion catalyzed by laccase. Subsequently,we investigated the catalytic performance of laccase from Irpex lacteus dft-1 in different reverse micelles formed by different types of surfactants and discussed the effect of PEG400 on the BPA conversion catalyzed by laccase in AOT/ isooctane reverse micelles,which can provide theoretical basis for further research on the laccase-catalysed oxidation of aromatic compounds in the reverse micelles. 1.Decolorization of textile dyes by laccases from Irpex lacteus dft-1Different sources of laccase have different ability to degrade and decolorize the textile dyes,and the optimum conditions are also different from one another.The mediators are very expensive.In order to reduce the treatment cost,it is necessary to find laccase-catalyzed systems that can decolorize the textile dyes without mediators. Irpex lacteus dft-1 laccase is a novel laccase.In this paper,we studied the kinetics of laccase-catalyzed decolorization of three types of dyes without mediators and investigated the effects of mediums and additives on the decolorization.The results show that Irpex lacteus dft-1 laccase can decolorize the anthraquinone dye(alizarin red),azo dye(methyl red) and triarylmethane dye(bromocresol green) effectively without mediators.The decoloration efficiency and optimum pH were different for different dyes.The decoloration efficiency can be improved when the nonionic surfactant solution of Triton X-100 or Brij30 with low concentration was added to the decolorzation system due to their activation and stabilization effects on the laccase,Response surface methodology(RSM) that combines mathematical and statistical techniques is a useful tool for modeling and analyzing the interactions of multi-variable process.It has been applied to optimize and evaluate interactive effects of independent factors in numerous chemical and biochemical processes.A three-level Box-Behnken design was used to investigate the effects of major operating variables (temperature,pH,concentration of enzyme and incubation time) on the decolorization of Remazol Brilliant Blue R(RBBR) by laccase from Irpex lacteus dft-1.A quadratic model was obtained for dye decolorization through this design.The model high F-value(2279.28) and low P-values(＜0.0001) indicated that the model is significant. The results show that enzyme concentration was the principal factor and the interaction between pH and temperature was the most significant during the enzymatic process.Very high regression coefficient between the variables and the response(R²=0.9996) indicated the fitness of response surface quadratic model during the present study. 2.Effects of additives on the catalytic performance of laccase in aqueous solutionsThe enzyme shows different catalytic performance in the reverse micelles formed by different types of surfactants,which should be related to the interfacial properties of the reverse micelle systems.Polyethylene glycol(PEG) is a synthetic water-soluble polymer,which can improve the conversion of phenolic compounds catalyzed by laccase.To elucidate the difference of catalytic performance of enzyme in different reverse micelles and the effects of PEG on laccase-catalyzed system,we selected three types of surfactants(cationic CTAB,anionic AOT and nonionic Triton X-100) and PEG to investigate their effects on the enzymatic properties of laccase. The results indicated that the types of surfactants and PEG with various concentrations have different effects on the laccase activity.PEG has no effect on the laacase activity.The nonionic Triton X-100 with low concentration has activation effect on laccase activity while the cationic CTAB and anionic AOT inhibit the laccase activity.At high concentration,all the three types of surfactant inhibit the laccase activity.PEG has no effect on the optimum pH of laccase activity.CTAB and AOT make the optimum pH of laccase activity shift to lower value while Triton X-100 make it shift to higher value.Triton X-100 and PEG have no effect on the optimum temperature of laccase activity while CTAB and AOT make it decrease.In the aqueous solutions,Triton X-100 and PEG are beneficial to the thermal stability of laccase while CTAB and AOT reduce it.The laccase-catalyzed conversion of bisphenol A(BPA) in aqueous solutions was studied in the presence of nonionic surfactant Triton X-100.It was found that the conversion of BPA was increased with adding Triton X-100,especially near the critical micelle concentration(CMC) of Triton X-100.It was also found that the stability of laccase was greatly improved in the presence of TritonX-100.The endogenous fluorescence emission of laccase indicated that there existed an interaction between Triton X-100 and laccase,which was beneficial to the folding and stabilization of laccase.The binding of Triton X-100 to the laccase surface also mitigated the inactivation effect caused by the free radicals and polymerization products.Under otherwise identical conditions,a lower dosage of laccase was needed for the higher conversion of BPA in the presence of Triton X-100.3.Research on the catalytic performance of laccase from Irpex lacteus dft-1 in reverse micellesIn order to achieve higher catalytic efficiency,the enzyme activity and stability are the critical factors for enzyme engineering.There were few researches on the stability of laccase in reverse micelles,especially on the effects of various factors on the stability.The effect of various parameters,e.g.,W₀,pH,buffer composition and ionic strength,the concentration of AOT and temperature on the activity and stability of Irpex lacteus dft-1 laccase in the AOT/isooctane/buffer were investigated.It was found that the enzyme activity increased with the increase of W₀ and did not change significantly when the W₀ exceed 35,and the enzyme stability was better at lower W₀. The optimum pH for the enzyme reaction was 4.2 whereas the enzyme stability increased with the increase of pH.Both the enzyme activity and stability decreased with the increase of the concentration of AOT.The enzyme activity and stability were different in the various buffer media.The enzyme activity first increased then decreased with the increase of buffer ionic strength whereas the enzyme stability was better at lower ionic strength.The enzyme optimum reaction temperature was 50℃, but the enzyme stability was decreased with the increase of temperature.The enzyme activity of Irpex lacteus dft-1 laccase was significantly lower in the CTAB/hexanol/isooctane/buffer and Triton X-100/hexanol/isooctane/buffer reverse micelles than in the AOT/isooctane/buffer reverse micelles.The change tendency of laccase activity with W₀ was different in the three types of reverse micelles.In both CTAB and Triton X-100 reverse micelles,the optimum pH and temperature were 4.2 and 50℃,respectively,which consistent with the AOT reverse micelle system.In addition,the activity and stability of laccase were both decreased with the increase of the concentration of the surfactant.The hexanol as additives has little effect on the iaccase activity at low concentration,but the laccase activity was obviously decreased at higher concentration of hexanol.Conclusively,the laccase stability in CTAB reverse micelle was the best,followed by in Triton X-100 reverse micelle,while the decrease of laccase activity was fastest in AOT reverse micelle at the initial stages.Bisphenol A can be effectively catalytic conversion by Irpex lacteus dft-1 laacase in AOT//isooctane/buffer reverse micelle.The transformation rate was enhanced when low molecular weight polyethylene glycol(PEG 400) was introduced into the reaction system.The influence of various parameters,such as W₀,pH,ionic strength and surfactant concentration were investigated and compared with those in simple AOT reverse micelles.The results show that the transformation rate was improved under all the conditions when the PEG400 was introduced.PEG400 can soluble in the water pool of the reverse micelle and decrease the interaction between enzyme and the head group of AOT by embedding charge at the micelle interface.In addition,PEG400 can bind with laccase non-covalently.These factors improved the laccase activity and stability in AOT//isooctane/buffer reverse micelle,thus increasing the transformation rate of BPA.