Purification and Characterization of Catechol 2,3-Dioxygenase from the Aniline Degradation Pathway of Acinetobacter sp. YAA and Its Mutant Enzyme, Which Resists Substrate Inhibition
スポンサーリンク
概要
- 論文の詳細を見る
Catechol 2,3-dioxygenase (C23O), a key enzyme in the meta-cleavage pathway of catechol metabolism, was purified from cell extract of recombinant Escherichia coli JM109 harboring the C23O gene (atdB) cloned from an aniline-degrading bacterium Acinetobacter sp. YAA. SDS–polyacrylamide gel electrophoresis and gel filtration chromatography analysis suggested that the enzyme (AtdB) has a molecular mass of 35 kDa as a monomer and forms a tetrameric structure. It showed relative meta-cleavage activities for the following catechols tested: catechol (100%), 3-methylcatechol (19%), 4-methylcatechol (57%), 4-chlorocatechol (46%), and 2,3-dihydroxybiphenyl (5%). To elevate the activity, a DNA self-shuffling experiment was carried out using the atdB gene. One mutant enzyme, named AtdBE286K, was obtained. It had one amino acid substitution, E286K, and showed 2.4-fold higher C23O activity than the wild-type enzyme at 100 μM. Kinetic analysis of these enzymes revealed that the wild-type enzyme suffered from substrate inhibition at >2 μM, while the mutant enzyme loosened substrate inhibition.
- 社団法人 日本農芸化学会の論文
- 2007-07-23
著者
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Takeo Masahiro
Department of Materials Science and Chemistry, Graduate School of Engineering, University of Hyogo
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Negoro Seiji
Department of Materials Science and Chemistry, Graduate School of Engineering, University of Hyogo
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Negoro Seiji
Department Of Materials Science And Chemistry Graduate School Of Engineering University Of Hyogo
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Negoro Seiji
Department Of Biotechnology Faculty Of Engineering Osaka University
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Takeo Masahiro
Department Of Materials Science And Chemistry Graduate School Of Engineering University Of Hyogo
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Takeo Masahiro
Department Of Applied Chemistry Himeiji Institute Of Technology
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Negoro Seiji
Graduate School Of Engineering Univ. Of Hyogo
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Nishimura Munehiro
Department of Materials Science and Chemistry, Graduate School of Engineering, University of Hyogo
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Takahashi Hana
Department of Materials Science and Chemistry, Graduate School of Engineering, University of Hyogo
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Takeo Masahiro
兵庫県立大学 工学研究科物質系工学専攻
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Negoro Seiji
兵庫県立大学 工学研究科物質系工学専攻
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SHIRAI Mizuho
Department of Materials Science and Chemistry, Graduate School of Engineering, University of Hyogo
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Takahashi Hana
Department Of Materials Science And Chemistry Graduate School Of Engineering University Of Hyogo
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Shirai Mizuho
Department Of Materials Science And Chemistry Graduate School Of Engineering University Of Hyogo
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Nishimura Munehiro
Department Of Materials Science And Chemistry Graduate School Of Engineering University Of Hyogo
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