| Project/Area Number |
11460048
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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 |
応用微生物学・応用生物化学
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| Research Institution | KYUSHU UNIVERSITY |
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Principal Investigator |
FURUKAWA Kensuke Faculty of Agriculture, Kyushu University, Prof., 農学研究院, 教授 (90221556)
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| Co-Investigator(Kenkyū-buntansha) |
GOTO Masatoshi Faculty of Agriculture, Kyushu University, Assistant Prof., 農学研究院, 助手 (90274521)
YOSHINO Sadazo Faculty of Agriculture, Kyushu University, Associated Prof., 農学研究院, 助教授 (80117291)
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| Project Period (FY) |
1999 – 2001
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| Project Status |
Completed (Fiscal Year 2001)
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| Budget Amount *help |
¥12,000,000 (Direct Cost: ¥12,000,000)
Fiscal Year 2001: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 2000: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 1999: ¥8,800,000 (Direct Cost: ¥8,800,000)
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| Keywords | oxygenase / hybrid enzyme / DNA shuffling / substrate specificity / degradative genes / alkyl benzene / molecular evolutionary engineering / ダイオキシン / ジオキシゲナーゼ / Pseudomonas / PCB / 遺伝子シャフリング / 芳香族炭化水素代謝 / ジベンゾフラン |
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| Research Abstract |
Oxygenases are involved in the direct introduction of molecular oxygen into the substrates, which consisted with terminal dioxygenase (comprised with a large and a small subunits) and electron transfer system from NADH. We first revealed that the large subunit is crucially involved in the substrate specificity of oxygenase. Then we performed DNA shuffling between the two large subunits of biphenyl dioxygenases derived from Pseudomonas pseudoalcaligenes KF707 and Burkhorderia cepacia LB400. The evolved bphA1 genes were expressed in Escherichia coli along with other bph genes. Some evolved biphenyl dioxygenases exhibited enhanced transformation abilities for various polychlorinated biphenyls by introducing molecular oxygen at the 2,3- and/or 3,4 positions depending on PCB congeners. Some other evolved enzymes acquired new and novel degradation capacities for benzene, toluene and some alkylbenzenes. The mutation using random hexanucleotide oligomers allow us to get another interesting mutant oxygenase which can degrade dibenzofuran and dibenzo-p-dioxin by both lateral dioxygenation and angular dioxygenation.
The amino acid sequences of above mutant dioxygenases were determined and the relationships between structure and function were discussed.
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