| Project/Area Number |
16560684
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Biofunction/Bioprocess
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| Research Institution | University of Hyogo |
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Principal Investigator |
TAKEO Masahiro University of Hyogo, Graduate School of Engineering, Associate professor, 大学院・工学研究科, 准教授 (40236443)
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| Co-Investigator(Kenkyū-buntansha) |
NEGORO Seiji University of Hyogo, Graduate School of Engineering, Professor, 大学院・工学研究科, 教授 (90156159)
KITAMURA Chitoshi University of Hyogo, Graduate School of Engineering, Assistant professor, 大学院・工学研究科, 准教授 (60295748)
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| Project Period (FY) |
2004 – 2006
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| Project Status |
Completed (Fiscal Year 2006)
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| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2006: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 2005: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2004: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Keywords | alkylphenol / nonylphenol / biodegradation / error-prone PCR / degradation genes / evolutionary engineering / Pseudomonas putida / Sphingomonas sp. / Sphingomonas / 分解経路 / 環境ホルモン / ブチルフェノール / DNAシャッフリング |
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| Research Abstract |
In this study, to elucidate biodegradation mechanisms of alkylphenols (APs) including an endocrine disruptor, nonylphenol, and to construct biodegradation system for APs, the butylphenol (BP) degradation gene cluster of Pseudomonas putida MT4 was at first analyzed in detail. The substrate specificities of the key enzymes in the BP degradation pathway, BP hydroxylase and butylcatechol 2,3-dioxygenase, were investigated at cell and enzyme levels. It was found that the former was able to oxidize a wide range of APs including 4-n-APs with an alkyl chain length of up to 7 carbons (C7) into the corresponding alkylcatechols, while the latter also oxidized 4-n-alkylcatechols with a long alkyl chain (【less than or equal】 C9). This result indicates that both enzymes have broad substrate specificity. Another strain, Sphingomonas sp. NP5 was isolated as a nonylphenol (NP)-degradaing bacterium, which was capable of degrading many kinds of NP isomers with a various branching at the alkyl chain. In the degradation of two NP isomers, 4-(1-ethylheptyl)phenol and 4-(1-ethyl-l-methylhexyl)phenol, were degraded efficiently by NP5. 3-Nonanol was detected as a metabolite from 4-(1-ethylheptyl)phenol, which was further oxidized into 3-nonanone, indicating that the alkyl chain of NP was released as a secondary alcohol and further converted into a ketone. This is a novel NP degradation pathway. These results show that it is possible to construct biodegradation system for a wide range of 4-APs with an alkyl chain of up to C9 using these strains. To expand the oxidation range of the BP hydroxylase of MT4, error-prone PCR was carried out using bupA4, which encodes the oxygenase large subunit of this enzyme. Several recombinant P.putida strains harboring bupA4 with some point mutations (3.2 substitutions in 1000 by on average) oxidized a mixture of NP isomers (technical grade NP) weekly. The screening of recombinant strains with high activity for NP is now continued.
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