Heterologous Expression And Application Of Laccase Genes From Cerrena Sp

Laccases are a family of multi-copper oxidases containing four coppers. Type I copper transfers the electrons from polyphenol substrates to the trinuclear cluster, which is composed of two type II and one type III coppers, oxygen reacts with the electrons and protons from trinuclear cluster to form water without any other byproducts. Thus, laccase is considered as an eco-friendly polyphenol oxidase. Meanwhile, because of the lower substrate specifity, the aid of redox mediator will broaden the substrates range and hence the laccase is widely used in various industrial areas, especially in dye decolorization.In this study, several laccases genes (including promoters) were cloned from a previously isolated laccase-producing fungi strain Cerrena sp. HYB07Three laccase genes were heterologous expressed in Pichia pastoris, and efforts were made to improve expression efficiency with fermentation medium selection, codon optimization and signal peptide substitution. The expressed recombinant laccases were characterized and applied in decolorization. The results are as follows:1. Via degenerate PCR, TAIL-PCR and Site-Finding PCR,6 new laccase genes were cloned. All the HYB07 laccases, including these 6 new and 4 known laccase genes, displayed characteristics of fungal laccases. The upstream promoter sequences contained many c/s-acting elements. Based on bioinformatic analyses of the obtained laccase sequences including introns, amino acid compositions, sequence identities and phylogeny, it was predicted that Lac3, Lac6 and Lac8 might have distinctive biochemical properties.2. Seven pPICZα-Lαc vectors were cloned, and pPICZα-Lαc3,-Lac 6 and -Lac8 were transformed into Phchia pasloris X33 for heterologous expression. By means of medium optimization, codon optimization and native signal-peptide substitution, rLac6 showed the highest yield of 6.33 U·mL-1 after 16-d cultivation in YP medium at 28℃. The rLac6 was then partially purified, and the kinetic parameters were studied.3. HYB07 laccases had optimal pH at 3.0-3.5, optimal temperature between 30-35℃, stabilized pH≥4 and temperature≤50℃, in addition, the laccases showed strong resistance to 7 water miscibile organic solvents as well as inhibitors EDTA, kojic acid and surfactant Triton X-1.00. Cu2+、Al3+ and Mn2+ stimulated laccase activity. The purified Lac6 displayed higher resistance to metal ions and inhibitors.4. The fermentation broth of HYB07 could decolorizetion textile effluents with 80.3% and 58.8%efficiencies, respectively. The HYB07 laccases could also efficiently decolorize azo, tri-phenylmethane, indigotine and anthraquinone dyestuffs. In addition, the recombinant laccase can be used for decolorization directly without any purification procedures. Therefore, HYB07 and its laccases have potential in industrial applications, such as bioremediation.