Study On The Laccase Production By Ganoderma Lucidum And Its Application In Decolorization Of Dyes

Laccase is a type of multi copper-containing polyphenol oxidoreductases that catalyze the oxidation of a variety of phenolic compounds. It has a wide range of substrates, including phenols, aromatic amines, carboxylic acids, biological pigments, metal organic compounds and non-phenolic substances. It has various potential applications in areas such as food processing, paper industry, environmental protection, biological testing, organic synthesis and energy development.A strain of Ganoderma lucidum which can produce a high yield of constitutive laccase was screened. In this thesis, the liquid fermentation and solid-state fermentation of G. lucidum laccase, the enzymology characteristics and the application of laccase in dye decolorization were investigated. The major results were as follows:Using single factor experiments and orthogonal design, the suitable liquid-state fermentation parameters of sucrose 35g / L, wheat bran 8g / L, ammonium sulfate 6g / L, casein 8g / L,CuSO4·5H2O 0.02g/L, KH2PO4 3g/L, MgSO4·7H2O 1.5g/L, VB1 10mg/L, VC 500mg/L; The optimal culture conditions: initial pH 5.0, culture temperature 32℃, inoculum 3.75% (cultured for 5 days of seed solution), rotating speed 120 r/min. The activity of laccase reached the highest of 2126U/mL after 3 days. The pH value of fermentation medium decreased from 5.0 to 4.0, when laccase activity reached the maximum, and then it remained stable.The laccase producted by G. lucidum was studied with solid-state fermentation. The sugar cane bagasse and wheat bran were found to be good substrates and the appropriate ratio of bagasse to wheat bran appropriate was 1.5:1. The optimum solid-state fermentation medium contained: glucose 0.5%, ammonium sulfate 5%, CuSO4·5H2O 0.01%, moisture content of substrate 73%. The optimal culture conditions were as follows: initial pH4.0, inoculum dosage 20%, culture temperature 28℃, high laccase activity (23803U/g) was obtained by solid-state fermentation after 8 days. According to the above conditions, solid-state fermentation of G. lucidum was carried out in polypropylene plastic bag to enlarge culturing, the time of the highest activity was still 8 days, while laccase activity was only 16087U/g. The characterization of laccase from G. lucidum was studied and the result showed that the optimal temperature and optimal pH of laccase reaction were at 60℃and pH 3.0. The laccase showed higher stability when temperature was not higher than 50℃, and it kept strongly active when pH ranged from 2.5 to 4.0. The laccase was activized by Cu2+, Mn2+ and Mg2+ while its activity was obviously inhibited by Cr3+, Hg2+, Fe2+ and inhibitor. The Km and Vmax of laccase were 0.13mmol/L and 234nmol/(mL·min) respectively with guaiacol as substrate under optimal conditions.The decolorization of different structure dyes including anthraquinone, azo and triarylmethane dyes by laccase from G. lucidum was studied. The oxidized substrates by laccase can be extended in the presence of small molecular mediators. The optimal decolorization conditions of brilliant blue KN-R were obtained as follows: dye concentration 400mg/L, pH 4.0, temperature 30℃, laccase 30U/mL and ABTS 10μmol/L. the decolorization rate reached about 91% in 60 minutes. The optimal decolorization conditions of methyl orange were as follows: dye concentration 50mg/L, pH 3.5, temperature 30℃, laccase 30U/mL and ABTS 10μmol/L. The decolorization rate reached about 78% in 90 minutes. The optimal decolorization conditions of crystal violet were as follows: dye concentration 50mg/L, pH3.0, temperature 30℃, laccase 30U/mL and ABTS 10μmol/L. With these conditions the decolorization rate reached about 85% in 180 minutes. The effect of inorganic ion on decolorization was investigated, the results showed that decolorization was significantly promoted by Cu2+, Mn2+, Mg2+ while it was inhibited by Zn2+ and Fe2+. The absorption spectrum of dyes decreased significantly after laccase/ABTS mediator system treatment, which did not appear other absorption spectrum in the visible range. Laccase decolorization reaction rate and substrate concentration relationship was consistent with Michaelis-Menten kinetics. The Km of brilliant blue KN-R, methyl orange and crystal violet reacted with laccase/ABTS mediator system were 3556mg/L, 100mg/L, 132mg/L and Vmax were 91.7mg/L·min, 2.4mg/L·min, 1.67mg/L·min.