Secretory Expression And Characterization Of A Soluble Laccase From The Ganoderma Lucidum Strain7071-9in Pichia Pastoris

Laccases are multicopper-containing oxidases that catalyze the oxidation of many substrates, such as phenols and pamphyl, arylamine, hydroxy acid, steroid hormone, biochrome and their derivatives, it also plays important roles in many fields of industry, including detoxification, wine stabilization, paper processing, and enzymatic conversion of chemical intermediates. To construct the heterologous expressive system of a laccase gene GILCCI from Ganoderma lucidum is necessary for the further study of its characterization and function.Laccases are strong oxidizing enzymes that oxidize chlorinated phenols, synthetic dyes, pesticides and polycyclic aromatic hydrocarbons as well as a very wide range of other compounds with high redox potential. According to a yeast bias codon, a laccase gene GILCCI from Ganoderma lucidum was successfully synthesized through a PCR-based two-step DNA synthesis method (PTDS) and was overexpressed in Pichia pastoris with an alcohol oxidasel promoter(AOX1). The recombinant protein GILCC I was estimated to have a molecular weight of approximately58kDa. The Km values of GILCC I for2-2’-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid)(ABTS) and guaiacol were0.9665mM,1.1122mM respectively, and the Vmax of GILCC I for these substrates were3,024μM mg-1min-1and82.13μM mg-1min-1respectively. With ABTS as substrate, the enzyme had an optimal temperature of around55℃, activity over a broad range (pH2to8). The enzyme was strongly activated by K+, Na+, Cu2+and mannitol. Six amino acids increased the catalytic ability of the enzyme. The activity of laccase was obviously inhibited by Fe2+, Fe3+, sodium hydrosulphite, and sodium azide. In an attempt to understand the decolorization role of laccase GlLCC I, we choose two kind of dyes to study its decolorization, azo dye includes methyl orange(MO), triphenylmethane dyes include malachite green, crystal violet and brilliant green. Under optimal conditions, GlLCC I decolorized37.62mg L-1of azo dye methyl orange in cultural medium. Under given conditions, the decolorization rate of malachite green, crystal violet and brilliant green are87.5%,72.1%and70.6%, respectively. The high degradation ability enables GILCCI to be used in the treatment of industrial effluent containing azo dyes and triphenylmethane dyes.