Fermentation Conditions Optimization And Characterization Of Laccase Produced By Mycena Purpureofusca And Its Application On Dye Decolorization

Laccase is a multicopper oxidases (MCO), which is widely used in wastewatertreatment, paper industry, textile industry etc. In this dissertation, the fermentationconditions, enzymology characters and application on dye decolorization of laccasewere studied. The research contents were as below:(1) Strain selection and optimizaiton of fermentation conditionsMycena purpureofusca was screened from six basidiomycetes fungi, and it waswell characterized in submerge fermentation for high production of laccase and verylow mycelium generation. Optimization of liquid fermentation medium for laccaseproduction by M. purpureofusca was carried out with two statistical methodsincluding Plackett-Burman (P-B) and Box-Behnken (B-B) designs. Three variables,i.e. sucrose, MgSO₄and CuSO₄, were found to affect laccase production significantlyby P-B screening. B-B design with three-factors at three levers was performed toexplain the combined effects of the three medium constituents. The optimum mediumconsisted of sucrose (4.26g/L), yeast powder (15g/L), MgSO₄(4.83g/L), KH₂PO₄(2.7g/L), CuSO₄(5.625mg/L), Vitamin B₁(0.1g/L). The laccase production wasincreased by1.87fold (277.5U/L) using this optimized medium.(2) Purification and characterization of laccaseThe extracellular laccase produced by Mycena purpureofusca was purified andcharacterized biochemically and biologically. The purified laccase showed a relativemolecular mass of61.7kDa according to SDS-PAGE experiment. The optimum pHwas2.2and the optimum temperature was50°C. The enzymatic activity was stable atneural pH and temperatures between10and30°C. The values of kinetic parametersKm and Vmax for purified laccase were0.296(mM) and0.0645(mM/min),respectively. Among the metal ions used, Fe³⁺, Mn²⁺, Cu²⁺, Ag⁺,Ca²⁺, Ba²⁺and Zn²⁺2+were found to have a slightly stimulating effect on the enzyme activity at theconcentrations of0.05mM. The enzyme activity can be enhanced by18.7%and 130.5%when Ag⁺was added to the medium at the concentration of0.05and0.5respectively. On the contrary, Fe²⁺strongly inhibited enzyme activity up to98%at aconcentration of0.05and0.5mM.(3) The application of crude laccase on dye decolorizationThe extracellular crude enzyme from M. purpureofusca showed good activity insynthetic dye colour removal. The decolourization of RBBR, an anthraquinonic dyeextensively used in the textile industry was further studied. Experimental resultshowed that RBBR decolorizing efficiency during first1h was greater than in otherdecolorizing process; The optimal temperature and pH were relatively40℃and3;The decolorizaion decreased when the concentration of substrate was increased.Increasing the laccase concentration could lead to a greater decolorization if substratewas saturated; A Box-Behnken design was used to evaluate the effects of threeparameters on the RBBR decolourization yield. The results clearly indicated that theeffect of the incubation time and enzyme concentration is significant. High efficiencydecolourization of RBBR by crude laccase was gained by low laccase concentrationwithout redox mediators. The selected optimal conditions allowed97.03%of RBBRdecolourization versus94.21%for the predicted value.