Isolation and characterization of a novel gene for degradation of environmental pollutants from environmental genetic source

从环境遗传源中分离和表征用于降解环境污染物的新基因

• 批准号: 13660078
• 负责人: NAGATA Yuji
• 金额: $ 2.3万
• 依托单位: Tohoku University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13660078/
• 关键词: Environmantal DNA; Environmental pollutant; Uncultured bacteria; Dehydrochlorinase; Dehalogenase; Naphthalene; BHC; Haloalkane; 芳香族化合物酸化酵素; ハロアルカンデハロゲナーゼ; 機能相補; 土壌ライブラリー; 環境汚染物質川

It is difficult to isolate a novel gene or enzyme for degradation of environmental pollutants by using the conventional enrichment-culture method. We directly isolated and purified long chain DNA from soil environment, and constructed soil DNA library using cosmid vector. The resultant soil DNA library was introduced into the host strain that could not grow on naphthalene because of its lack of one of genes for naphthalene assimilation. The clones which grew on naphthalene were isolated. The gene that complemented the naphthalene assimilation ability of the strain did not show homology to knwon genes for naphthalene degradation. Furthermore, we constructed strains which could not grow on BHC or haloalkanes becuse of their lack of one of genes for assimilation of BHC or haloalkanes. By using these strains, it is theoretically possible to isolate novel genes for BHC dehydrochlorinase or haloalkane dehalogenase. Introduction of the soil DNA library into these stratns are in progress.

利用传统的富集培养方法很难分离出降解环境污染物的新基因或酶。我们直接从土壤环境中分离纯化了长链DNA，并利用粘粒载体构建了土壤DNA文库。将获得的土壤DNA文库导入到不能在萘上生长的宿主菌株中，因为其缺少萘同化基因之一。分离出在萘上生长的克隆。补充萘同化能力的基因与已知的萘降解基因没有同源性。此外，我们构建了不能在六六六或卤代烷上生长的菌株，因为它们缺乏六六六或卤代烷同化基因之一。通过使用这些菌株，理论上有可能分离BHC脱氯化氢酶或卤代烷脱卤酶的新基因。将土壤DNA文库引入这些地层的工作正在进行中。

项目成果

永田裕二: "有機塩素系農薬による汚染浄化のための環境バイオテクノロジー"J.Environ.Biotechnol.. 2. 25-38 (2002)

Yuji Nagata：“清除有机氯农药造成的污染的环境生物技术”J.Environ.Biotechnol.. 2. 25-38 (2002)

Bohac,M. et al.: "Halide-stabilizing residues of haloalkane dehalogenases studied by quantum mechanic calculations and site-directed mutagenesis"Biochemistry. 41. 14272-14280 (2002)

博哈克，M.

Oakley, A. et al.: "Exploring the structure and activity of haloalkane dehalogenase from Sphingomonas paucimobilis UT26 : evidence for product-and watermediated inhibition"Biochemistry. 41. 4847-4855 (2002)

Oakley, A. 等人：“探索少动鞘氨醇单胞菌 UT26 的卤代烷脱卤酶的结构和活性：产物和水介导抑制的证据”生物化学。

Miyauchi, K.et al.: "Cloning and characterization of linR, involved in regulation of downstream pathway for gamma-HCH degradation in Sphingomonas paucimobilis UT26"Appl.Environ.Microbiol.. 68. 1803-1807 (2002)

Miyauchi, K.等人：“linR 的克隆和表征，参与少动鞘氨醇单胞菌 UT26 中γ-六氯环己烷降解下游途径的调节”Appl.Environ.Microbiol.. 68. 1803-1807 (2002)

Lee, H.-S.et al.: "Employment of the human estrogen receptor β ligand-binding domain and co-activator SRC1 nuclear receptor-binding domain for the construction of a yeast two-hybrid detection system for endocrine disrupters"J. Biochem.. 131. 399-405 (2002

Lee, H.-S. 等人：“利用人雌激素受体 β 配体结合域和共激活剂 SRC1 核受体结合域构建酵母二杂交检测系统来检测内分泌干扰物”。生物化学.. 131. 399-405 (2002