Development and application of degradation system for polychlorinated dioxin

多氯二恶英降解系统的开发及应用

• 批准号: 10558103
• 负责人: ESAKI Nobuyoshi
• 金额: $ 3.14万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10558103/
• 关键词: chlorinared organic compound; fluorinated organic compound; chloropropionate; fluoroacetate; dehalogenase; クロロプロピオン酸; DL-2-ハロ酸デハロゲナーゼ; 2-ハロ酸アミド; ^<18>O取り込み実験; 質量分析; 結晶構造解析

We isolated a fluoroacetate-degrading bacterium from soil, and purified fluoroacetate dehalogenase, which catalyzes the hydrolytic dehalogenation of fluoroacetate, from this bacterium. The enzyme also acted on chloroacetate and bromoacetate, but did not catalyzed the hydrolysis of haloalkanoic acids whose carbon chain lengths are longer than three. We also carried out screening using a medium containing 2-chloropropionate as a carbon source, and isolated a bacterium degrading this substrate from lake water. The enzyme isolated from this bacterium, DL-2-haloacid dehalogenase, catalyzed the hydrolysis of both D-and L-2-chloropropionates. The enzyme acted on various haloacetates as well as 2-chloropropionamide. The gene encoding this enzyme was cloned, and its nucleotide sequence was determined. The gene product was estimated to be composed of 301 amino acid residues, and its molecular weight was calculated to be 34,049. We analyzed the reaction mechanism of DL-2-haloacid dehalogenase. When the single-turnover enzyme reaction was carried out using 2-chloropropionate as a substrate in the presence of HィイD22ィエD2ィイD118ィエD1O, ィイD118ィエD1O was found to be incorporated into the product, lactate. The enzyme was not labeled with ィイD118ィエD1O even after the multiple-turnover enzyme reaction in the presence of HィイD22ィエD2ィイD118ィエD1O. These results indicate that the water molecule activated by the enzyme directly attacks the substrate to displace the halogen atom.

从土壤中分离到一株氟乙酸降解菌，并从该菌中纯化了催化氟乙酸水解脱卤的氟乙酸脱卤酶。该酶也作用于氯乙酸和溴乙酸，但不催化碳链长度大于3的卤代烷酸的水解。我们还进行了筛选，使用含有2-氯丙酸作为碳源的培养基，并从湖水中分离出一种降解该底物的细菌。从该菌中分离的DL-2-卤酸脱卤酶催化D-和L-2-氯丙酸酯的水解。该酶作用于各种卤代乙酸酯以及2-氯丙酰胺。克隆了编码该酶的基因，并测定了其核苷酸序列。基因产物由301个氨基酸残基组成，分子量为34049。分析了DL-2-卤酸脱卤酶的反应机理。当使用2-氯丙酸酯作为底物，在H2O D22 β D2 β D118 β D1 O存在下进行单转换酶反应时，发现β D118 β D1 O掺入产物乳酸中。即使在存在H β D22 β D2 β D118 β D1 O的情况下进行多次转换酶反应后，该酶也未被β D118 β D1 O标记。这些结果表明，被酶激活的水分子直接攻击底物以置换卤素原子。

项目成果

Vincenzo Nardi-Dei et al.: "DL-2-Halo acid dehalogenase from Pseu domonas sp.113 is a new class of dehalogenase catalyzing hydrolytic dehalogenation not involving enzyme-substrate ester intermediate"The Journal of Biological Chemistry. 274(30). 20977-2098

Vincenzo Nardi-Dei 等人：“来自 Pseu domonas sp.113 的 DL-2-卤酸脱卤酶是一类新的脱卤酶，催化水解脱卤，不涉及酶-底物酯中间体”《生物化学杂志》。

Nobuyoshi Esaki et al.: "Crystal Structures of Reaction Intermediates of L-2-Haloacid Dehalogenase and Implications for the Reaction Mechanism" J.Biol.Chem.273. 15035-15044 (1998)

Nobuyoshi Esaki 等人：“L-2-卤酸脱卤酶反应中间体的晶体结构及其对反应机制的影响”J.Biol.Chem.273。

Nobuyoshi Esaki et al.: "DL-2-Haloacid dehalogenase from Pseudomonas sp. 113 is a new class of dehalogenase catalyzing hydrolytic dehalogenation not involving enzyme-subustrate ester intermediate."J. Biol. Chem.. 274(30). 20977-20981 (1999)

Nobuyoshi Esaki 等人：“来自假单胞菌 113 的 DL-2-卤酸脱卤酶是一类新型脱卤酶，催化水解脱卤，不涉及酶-底物酯中间体。”

Nobuyoshi Esaki et al.: "X-Ray Structure of a Reaction Intermediate of L-2 -Haloacid Dehaligense with L-2-Chloropropionamide"J. Biol. Chem.. 124(1). 20-22 (1998)

Nobuyoshi Esaki等人：“L-2-卤代酸脱卤化物与L-2-氯丙酰胺的反应中间体的X射线结构”J。

Nobuyoshi Esaki et al.: "Cystal Structures of Reactio Intermediates of L-2Haloacid Dehalogenase and Implications for the Reaction Mechanism"J. Biol. Chem.. 273(24). 15035-15044 (1998)

Nobuyoshi Esaki 等：“L-2卤酸脱卤酶反应中间体的晶体结构及其对反应机制的启示”J。