| With the fossil fuels are being consumed more and more, Most of attentions are paid to the using of renewable energy. Biomass material is the only energy which can be large-scale regenerated on earth, which is renewable energy,it can unceasingly provide energy to the human. And plant photosynthesis fixed much of the energy as the plant cell wall of lignocellulose substance, therefore cellulose,hemicellulose and lignin are the most extensive renewable biological energy sources in the world.In the plant fiber materials, hemicellulose, cellulose, lignin and protein chemical have a close connection by the chemical bond, hemicellulose and lignin connect with ester bond or ether bond. The ferulic acid connect the hemicellulose with ester bond, and connect the lignin with the ether bond, then form the structure of the bridge league of’hemicellulose-ester-ferulic acid-ether-lignin’.Feruloyl esterase (FAE) is one type of hemicellulose degrading enzymes. FAE hydrolyzed the ester bond between the arabinose substitutions and ferulic acid, which is involved in crosslinking xylan to lignin, and destroyed three-dimensional network structures of plant cell walls. Feruloyl esterase has been widely used in the bio-ethanol production, pulping industry, animal feed production and the biological synthesis process. But now, the production capacity of the strains which can product the feruloyl esterase can not meet the requirements of industrial application, in this paper, we cloned the faeA gene from Penicillium decumbens114-2,which is the wild strain that can secrete cellulose enzyme, then restructed the strains through genetic engineering method. In this thesis:1The faeA gene of Penicillium decumbens expression in E. coliThe work constrcted recommbinatant plasmid pET-faeA and transformed it into E.coli BL21for expressing FaeA protein. We obtained the positive on LB plates with ampicillin. The resulting recombinant strain was named as pET/FaeA. pET/FaeA could produce FaeA when induced by IPTG, the expressed protein has been evaluated by SDS-PAGE and the molecular mass was about30kDa, but this protein was expressed as insoluble, inactive inclusion bodies. Through the optimization experiments, the strain pET/FaeA still cannot express an active soluble protein.2The faeA gene of Penicillinm decumbens114-2expression in Penicillium decumbens M12The work constrcted recommbinatant plasmid ANip-faeA and transforms it into Penicillium decumbens MI2for expressing FaeA protein. We obtained the positive strain on screening plates without uridine. The resulting recombinant strain was named as M/FaeA. Through the PCR identification, we have the resulting recombinant strain.3The degradation of feruloyl esterase in fiber materialsAsperigillus flavue was screened from the soil, this fungus has a good ability to produce Feruloyl esterase. The crude enzymes was fermented for4days,We get a pure xylanase and the mix enzymes including xylanase and feruloyl esterase. Through the concentration, ammonium sulfate precipitation, dialysis, ion exchange chromatography. The single sugars contents and morphology of wheat straw were analyzed by HPLC and infrared spectroscopy after hot water-pretreated wheat straw was treated with the pure xylanase and the mix enzymes. It was shown that the sugar contents were increased in the saccharification liquid, especially for the xylose content, the arabinose concentration also has weak increase; and absorption of ester bond was decreased, which means the FAE produced by the strain4-3-α can break ester bond and promotes degradation of arabinoxylan. |
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