The development of a new method for specific cleavage of lignin beta-ether linkage by genetic engineering.
| Project/Area Number |
04454088
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
林産学
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| Research Institution | Tokyo University of Agriculture & Technology |
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Principal Investigator |
KATAYAMA Yoshihiro Tokyo University of Agriculture & Technology, Center for Cooperative Research, Associate Professor., 共同研究開発センター, 助教授 (10214339)
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| Co-Investigator(Kenkyū-buntansha) |
KAWAI Shinya Tokyo University of Agriculture & Technology, Faculty of Agriculture, Assistant, 農学部, 助手 (90202027)
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| Project Period (FY) |
1992 – 1993
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| Project Status |
Completed (Fiscal Year 1993)
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| Budget Amount *help |
¥6,600,000 (Direct Cost: ¥6,600,000)
Fiscal Year 1993: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 1992: ¥5,200,000 (Direct Cost: ¥5,200,000)
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| Keywords | Lignin biodegradation / beta-Arylether linkage / beta-Etherase / Calpha-Dehydrogenase / DNA sequence / Gene function / Glutathione-S-transferase / Glutahione / β-エーテラーゼ / β-エーテル結合 / 遣伝子機能 / 塩基配列 / 酵素機能領域 / NAD-結合部位 |
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| Research Abstract |
Lignin is the most abundant aromatic material in the biosphere. It is a polymer constructed with phenylpropanoid units. In the structure, beta-arylether linkage is the most aboundant (approximately 50 %). Cleavage of beta-arylether is the most important process in lignin biodegradation. We already isolated Pseudomonas paucimobilis which was able to degrade beta-aryl eher linkage. And we deteced beta-etherase acivity in the cellular membrane fraction.
In this research program, we try to establish a specific modification process constructed with gene functions of lignin degradable P.paucimobilis by genetic engineering. At first, we isolated the beta-etherase gene which contains an open reading frame of 843 bp. This gene was expressed in Escherichia coli, and the enzyme had the same properies as the P.paucimobilis enzyme. The substrate specificity of beta-etherase is a beta-aryl ether that contains a carbonyl group at the Calpha-position. In P.paucimobilis, Calpha-dehydrogenase catalyzes the oxidation of alcohol group at the Calpha-position of beta-arylether compound. Then, we isolated the Calpha-dehydrogenase gene. This gene contains an open reading frame of 915 bp and located in 1 kbp upstream of the beta-etherase gene. This gene was wxpressed in E.coli, and the enzyme had the same properies as the P.paucimobilis enzme.
We idenified another beta-etherase gene, which lies between two genes described above. The beta-etherase activity of the new gene expresed in E.coli was more than 200 times as high as that of P.paucimoblis. These two beta-etherase genes are homologus to gultathion-S-transferase, and upon addtion of glutahione a remarkable acceleration of beta-etherase activity was observed in the E.coli carrying the beta-etherase gene.
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Report
(3 results)
Research Products
(6 results)
