Purification And Characterization Of Laccase From Cerrena Unicolor

Abstract:Laccases(EC 1.10.3.2) are blue multi copper-containing phenol oxidases.The ability of these fungi to degrade a variety of chemical different dyes is correlated with their ligninolytic enzymes.They are widely applied in industry.In this work,the culture conditions for laccase production by Cerrena unicolor was optimized using a one factor as well as a L₉(3⁴) orthogonal design.The result showed that the optimal culture medium components for laccase production by Cerrena unicolor were as follows:sucrose 10 g/L,peptone 1 g/L,CuSO₄.5H₂O 0.2 mM,0.5 mM veratryl alcohol as the best inducer and 5.0 as the optimal pH value.The Cerrena unicolor produced more laccase under the culture conditions of 28℃with a rotating rate of 160 r/min.At the same time,in our work,the medium was used to inoculate Cerrena unicolor,and the liquid culture was harversted after 18 d.Then the laccase was purified from the white rot fungus Cerrena unicolor to evaluta its application in dry decolorization.The purification methods,including ultrafiltration,anion-exchange chromatography and gel filtration,were used in the purification process.The purification fold was 3.12 and recovery of total laccase activity was 26.61%.SDS-PAGE analysis showed the purified laccase to be a monomeric protein of 63.77 kDa.The optimum pH for the oxidation of 2,2'-azinobis-(3-ethylbenzthiaoline-6-sufonic acid) (ABTS) was 2.2 and the optimum temperature was 50℃.The K_m values of the purified laccase for the substrate ABTS,syringaldazine and 2,6-dimethoxyphenol were 0.217,0.306 and 0.199 mM,respectively.The activity of the purified enzyme was strongly inhibited by sodium azide and partially inhibited by cysteine,dithiothreitol at the concentration of 1 mM.However, EDTA only demonstrated a slight inhibitory effect even at a concentration of 50 mM.Chemical different dyes were used in the dye decolorization test by the purified laccase.The result indicated that all the dyes could be decolorized by the purified enzyme in the absence of redox mediators.The anthraquinone dye Remazol Brilliant Blue R(RBBR) was the most suitable substrate for the laccase from Cerrena unicolor.About 90%of RBBR was decolorized in the first 10 min,and the maximal decolorization percentage reached as high as 95.7%after 3 h. Azocarmin B and crystal violet were less decolorized than RBBR.The result demonstrated the potential application of the laccase from Cerrena unicolor in dye-containing wastewater treatment.