Dyes Decolorization By Laccase From Trametes Sp.LK13

Laccase(EC1.10.3.2) is a group of multi copper-containing polyphenol oxidasesa. Laccase is able to catalyse diverse forms of chemical reaction, the broad substrate spectrum of laccases. Laccase is an very important and valuable enzyme for various biotechnological and industrial applications, such as biodegradation of lignin without polluting the environment, thorough degradation of different recalcitrant compounds, decolorization of dyes.environmental protection and bioremediation of soils and water,biological bleaching in paper industry, textile dye decolorization. The study on laccase possessed a certain oretical and practical significance. Unfortunatly, its production is always poor, which refrain its wild range of application.In this thesis, a strain of high laccase producing microorganism was isolated by the method of ABTS plate screening.The strain was identified,and the medium components were optimized by response surface method, dys was decolorized by using crude enzyme. The main results are as follows:1. Identification of strain Trametes sp. LK13According to a strain of cultural characteristic, morphologic culture, molecular biological identification, designated as Trametes sp. LK132. The optimization of medium a of Trametes sp. LK13With one factor at a time was conducted.the study indicated that the best medium carbon and nitrogen source for the produce of laccase of Trametes sp. LK13is glucose and yeast extract/peptone (1:1) respectively. Among the mineral salts, Cu2+stimulation the maximal production of laccase,while the best inducer is phenol.In the optimization of medium, the influences of eleven medium component on laccase production were frist evaluated using P-B design.The path of steepest ascent was used to attain the optimal region of the medium composition.Production of laccase using a submerged culture of Trametes sp. LK13was optimized using a central composite design of the Response Surface Methodology and response surface analysis.The optimum medium composition was glucose28g/L,yeast extract5g/L.peptone5g/L,FeSO40.4mM,VB10.2g/L,CuSO41.65mM,phenol30.54mg/L.Tween-806g/L.PEG400030.54mg/L, seed volume10%(v/v).Optimized conditions gave a laccase yield of122.85U/L, which was about13.65times of that in the GYP medium.3.The characterization of crude laccase fromTrametes sp. LK13was studiedThe optimal temperature and optimal pH of crude laccase reaction were at65℃ and pH2.5using ABTS as substrate.The laccase showed higher stability when temperature was not higher than75℃,the enzyme retained75%of its activity after10min of incubation at75℃and it kept strongly active when pH ranged from2.5to4.0. In the case of met al ions, we found that chloride ions and Ni2+, Fe2+,Ca2+were responsible for inhibition while Cu2+,Mg2+,Mn2+,Co2+enhanced laccase activity. When halides were tested, we showed the following degree of inhibition:F->Cl-Br-With ABTS,50%of the laccase activity remains for solvent concentration ranging from10%to40%depending on the solvent used while with ABTS.The stability was enhanced with veratryl alcohol and mannitol.The laccae was inhibitied by DTT and L-Cys.These excellent properties demonstrated that the laccase from Trametes sp.LK13has potentials and highly attractive candidate for the specific industrial or environmental applications.4. The decolorization of different dyes by Crude laccase from Trametes sp. LK13In this part, preliminarily explore the roles of Trametes sp. LK13laccase on decolorization anthraquinone dyes-MG.The results showed that the appropriate conditions of laccase degradating MG was70℃,pH6.0, decolorization rate was higher than95%.Met al ions of Mg2+,Mn2+obviously increased decolorization of MG by Crude laccase from Trametes sp. LK13the laccase activity,while others decreased decolorization of MG.UV-vis scan (400-800nm) of enzyme treated dyes withdrawn at3h indicated decolorization and decrease in dye concentration.Peak observed at618nm(0h) decreased without any shift inλ max upto complete decolorization of the dye (2h) incubation. Evidence of the removal of dye can be observed with absorbance at λ max being virtually zero after2h and an increase in absorbance towards UV region.The absorbance peaks, corresponding to dyes,diminished indicating that the dye had been removed. The decrease of absorbance peak of dyes indicated a rapid degradation of the dye.Similar with MG,The higher dye concentration, decolorization the more difficult.The crude laccase were able to decolorize in two hour more60%of all the dys.Among the three structurally different kinds of dyes,the bromophenol Blueafte was the most easily decolorized.Bromophenol Blueafte showed a degradation percentage of57.79%after only10min treatment with crude laccase of Trametes sp. LK13.Reactive dye of decolorize rate lower than the commonly used laboratory dyes,which may contain inhibitor. Compared with the single dye, mixed dye decolorization more easily,The reason may be that the synergy of different dyes.The results suggested that it is reasonable to use fungal laccase to decolorize the dyes redundant in textile waste water.