Functional Expression Of Ganoderma Lucidum Laccase And IT,s Application In Industrial Wastewater,s Decolorization Treatment

Laccases are copper-containing polyphenol oxidases,phenol compounds, Carboxylic acids,Aromatic amines,biochrome and Steroidal hormones can be used as their substrates. laccase is recognizedas the key enzyme in lignin degradation, and widely used in paper and pulp, treatment in forestry and agricultural residues, moreover, it has potential prospect in waste water treatment, degradation of environmental pollutants, food processing, etc. Laccasegene from Ganodermalucidum was Synthesized, integrated into the genomic DNA of Pichiapastorisand expressed heterogeneouslyleading to the production of recombinant laccase. The properties of recombinant enzymeand its ability to decolorize azo dye amino black 10 B were studied, which provided solid foundations for its production and application in industry.G.lucidum Laccase gene with P.pastoris codon bias was synthesized using modified overlap extension PCR(OE-PCR) according to the laccase amino acid sequence of Dr Chengdu’s previous work in sunyat sen university taking advantage of DNAwokrs 3.1 software to design and optimize primers. Expression vector containing target DNA was transformed and integrated into P. Pastoris GS115 through electroporation, recombinant laccase was expressed and purified using Ni spin coloumn from Qiagen company after induction with methanol. The size and purity of recombinant enzyme was determined by SDS-PAGE, and a clear band of 55 k Da was observed obviously. The studies on the enzymatic properties indicated that the enzyme has the highest activityat p H 3.6 and 30℃ using ABTS as substrate. The enzyme had high stabilityat 30℃ and kept stable in an acid p H range.Decolorization of azo dye using G lucidum Laccase was carried out according to the dye degradation function of laccase. Taking amino black 10 B as an example. Response surface methodology(RSM) was applied for optimization of amino black 10 B decolouration using immobilized ganoderma lucidum laccase. The optimum value of the six selected impact factors about decolorizing efficiency were measured through single factor experiment. A Plackett-Burman design was applied to screen potential key factors, and the steepest ascent method was adopted to approach the approximate optimal reaction system. Finally, central composite design and response surface methodogy were applied to define more precisely the optical composition of the reaction system. The data revealed ABTS concentration and Laccase amount to be the most influential parameters, and the composition of the optimal reaction system was determined to consist of p H 4.6, temperature 30℃, copper sulfate concentration 1.75 mmol/L, ABTS concentration 0.07 mmol/L, enzyme 6.23 mg, the initial amino black 10 B 100 mg/L. The decolorization ratio of amino black 10 B in the optimal reaction system was 60.02%, which was 44.46% higher than single factor method.