| Laccase (p-diphenol:oxygen oxidoreductase; EC1.10.3.2) is a group of copper-containing polyphenol oxidases, widely distributed in higher plants and fungi. It’s an important component of the extracellular ligninolytic enzymes secreted by white-rot fungi. In white-rot fungi, laccase is associated with many biological functions, including lignin degradation, removal of potentially toxic phenols, fruiting body development, pigment synthesis, sporulation and fugal virulence. Because of its wide range of substrates and unique biodegradability, laccase has great application potential in paper pulping, biomass energy, food processing, lignin degradation, dye decolorization, as well as degradation of industrial effluents. Therefore, acquisition of laccase gene and studying its biological functions and expression regulation are of great theory and application value, to enrich laccase gene resource, reveal the function of laccase multi-gene family, and improve laccase expression. As core with laccase of white-rot fungi, this dissertation performed a serial of research about laccase genes’cloning and analysis, heterologous expression of laccase, characterization of the recombinant laccase and the expression regulation of laccase multi-gene family. The main results are described as following:Based on a newly isolated white-rot fungus Ganoderma sp. En3and Pleurotus ostreatus BP2, with degenerate PCR, thermal asymmetric interlaced PCR (TAIL-PCR), Self-Formed Adaptor PCR (SEFA-PCR), conserve primer PCR and RT-PCR, the laccase gene lac-En3-1and its corresponding full-length cDNA were cloned from strain En3. the11members of laccase multi-gene family and their5’-flanking promoter regions were cloned from strain BP2. We further analyzed the promoter region of the laccase multi-genes from Pleurotus ostreatus BP2. Several of cis-acting elements were predicted based on the conserve sequences, such as xenobiotic-responsive elements (XREs), metal-responsive elements (MREs), CreA-binding sites, NIT2-binding sites etc.The laccase gene of Lac-EN3-1from Ganoderma sp. En3was heterologously expressed in pichia pastoris, in which the laccase function was verified. The recombinant laccase rLac-EN3-1was purified and studied. rLAC-EN3-1exhibited strong stability under alkaline conditions. rLAC-EN3-1also showed strong resistance to some enzyme inhibitors such as SDS and EDTA, as well as a very strong ability to tolerate many metal ions and organic solvents widely used in the industry. In addition, rLAC-EN3-1could efficiently decolorize and detoxicate the synthetic dye.we studied the expression pattern of laccase multi-gene family under different inducers such as metal irons, aromatic compounds and alcohols etc. Cu2+showed the most prominent stimulating effect on all of the laccase genes’ transcription. While Fe2+, Mn2+and Cd2+etc. only increased some laccase genes transcription. Seven aromatic compounds were found to significant increased the transcription level of laccase multi-genes in Pleurotus ostreatus BP2. But the effects varied from different aromatic compounds. The synergistic stimulating effect of metal ions and aromatic compounds was observed. Different alcohols and other inducers also showed promoting effect on the laccase multi-genes transcription of Pleurotus ostreatus BP2.Time course analysis of the effect of different aromatic compounds on Pleurotus ostreatus BP2laccase gene transcription was further performed. The promoting effect of cinnamic acid,3,4,5-Trimethoxycinnamic acid,3,4-dimethoxycinnamic acid, coumaric acid, ferulaic acid and vanillic acid on laccase genes transcription was most distinctly on day8. While the promoting effect of4-methoxycinnamic acid and3-hydroxy-4-methoxycinnamic acid on laccase gene transcription was most obviously on day10.In conclusion, in this study, a novel laccase gene Lac-EN3-1from Ganoderma sp. En3was cloned. The11members of laccase multi-genes and the correspending promoter regions in Pleurotus ostreatus BP2were cloned. The laccase gene of Lac-EN3-1from Ganoderma sp. En3was heterologously expressed in pichia pastoris successfully. The recombinant laccase rLac-EN3-1exhibited distinctive characters such as strong resistance to many metal ions and organic solvents, good performance in the degradation and detoxification of dyes. At last, the expression pattern of Pleurotus ostreatus BP2laccase multi-gene family’s transcription under different inducers was studied. Metal ions, aromatic compounds, alcohols etc. showed prominent stimulating effect on the expression of laccase multi-gene family... |
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