| Project/Area Number |
16380115
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
林産科学・木質工学
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| Research Institution | KYOTO UNIVERSITY |
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Principal Investigator |
WATANABE Takashi KYOTO UNIVERSITY, Res.Inst.Sustainable Humanosphere, Professor, 生存圏研究所, 教授 (80201200)
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| Co-Investigator(Kenkyū-buntansha) |
NAKAMURA Yoshitoshi Kanazawa University, Graduate School of Natural Science & Technology, Assoc.Prof., 大学院・自然科学研究科, 助教授 (20172455)
HONDA Yoichi KYOTO UNIVERSITY, Res.Inst.Sustainable Humanosphere, Assc.Prof., 生存圏研究所, 助教授 (70252517)
WATANABE Takahito KYOTO UNIVERSITY, Res.Inst.Sustainable Humanosphere, Assist.Prof., 生存圏研究所, 助手 (30362403)
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| Project Period (FY) |
2004 – 2005
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| Project Status |
Completed (Fiscal Year 2005)
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| Budget Amount *help |
¥15,300,000 (Direct Cost: ¥15,300,000)
Fiscal Year 2005: ¥5,100,000 (Direct Cost: ¥5,100,000)
Fiscal Year 2004: ¥10,200,000 (Direct Cost: ¥10,200,000)
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| Keywords | laccase / ethanol fermentation / biomass / white rot fungi / fermentation inhibitor / 発酵阻害 / 異種発現 / リグニン |
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| Research Abstract |
To obtain high yield of the ethanol production from wood biomass, it is necessary to minimize the influences of fermentation inhibitors produced in the pretreatment process. A white rot fungus Trametes versicolor RC3 secreted laccase isozymes and they abolished the inhibitory effect of the compounds and promoted the fermentation efficiency by Pichia stipitis. To elucidate the removal mechanism of fermentation inhibitors by T.versicolor RC3 laccase, we conducted gene cloning and heterologous expression of a RC3 laccase isozyme and analyses of the reaction products of the fermentation inhibitors treated by T.versicolor RC3 laccase fractions. We purified a laccase isozyme and the N-terminal amino acid sequence was determined. Using degenerated primers for the gene encoding this isozyme, a DNA fragment was amplified by PCR and cloned. After cloning of the flunking sequences, we constructed an expression plasmid which has P.ostreatus sdi1 promoter followed by the coding sequence of the cloned laccase gene from T.versicolor RC3. The expression plasmid was introduced in P.ostreatus and extracellular laccases were characterized. On the other hand, 5-HMF, vanillin, syringaldehyde and the SF was treated with the acidic or alkaline fractions of laccase from culture filtrate of T.versicolor RC3, and the resulting compounds were analyzed by GC-MS. The determined nucleotide sequence demonstrated that the cloned gene encodes a new laccase isozyme, and the recombinant gene was expressed in P.ostreatus. We also found that the acidic fraction was significantly stable against incubation with SF compared to any other laccases tried, suggesting possible contribution of this fraction in the high removing activity of fermentation inhibitors by RC3 laccases.
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