Directed Evolution of Aromatic Dioxygenases for Trinitrotoluene and Aminodinitrotoluene Degradation

用于三硝基甲苯和氨基二硝基甲苯降解的芳香族双加氧酶的定向进化

• 批准号: 0606195
• 负责人: Thomas Wood
• 金额: $ 9.46万
• 依托单位: Texas A&M Engineering Experiment Station
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-10-15 至 2006-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0606195&HistoricalAwards=false
• 关键词: Directed; Evolution; Aromatic; Dioxygenases; Trinitrotoluene

0114126 Smets The objective of this research project is to use an innovative protein engineering approach to create dioxygenases that initiate aerobic attack on trinitrotoluene (TNT) and aminodinitrotoluenes (ADNT), products of natural attenuation of TNT. The approach will be to expand the substrate range of nitroarene dioxygenases to allow oxidative transformation of ADNT and/or TNT. The gene encoding the large subunit of the terminal oxygenase will be the focus of this work. DNA shuffling will be used to create variants of two 2,4-dinitrotoluene (DNT) dioxygenases individually. Burkholdria sp. strain DNT will be used as the initial host to create a complete pathway for TNT/ADNT degradation. Creation of a complete, successful ADNT and/or TNT mineralization pathway will be initiated. Hence, the enzymatic steps for the down-stream processing of expected transformation products will also be addressed. Using a novel, directed-evolution approach and metabolic engineering, this project aims to create the first oxidative enzyme and pathway for the biomineralization of TNT/ADNT. The constructed strain will be invaluable in developing sustainable strategies for remediation of explosives contaminated environments. ***

0114126 Smets这项研究项目的目标是使用一种创新的蛋白质工程方法来产生双加氧酶，该双加氧酶对三硝基甲苯(TNT)和氨基二硝基甲苯(ADNT)发起有氧攻击，三硝基甲苯和氨基二硝基甲苯是TNT自然衰减的产物。该方法将扩大硝基芳烃双加氧酶的底物范围，以允许ADNT和/或TNT的氧化转化。编码末端加氧酶大亚基的基因将是这项工作的重点。DNA改组将被用来分别创造两个2，4-二硝基甲苯(DNT)双加氧酶的变体。Burkholdria sp.菌株DNT将作为初始宿主，为TNT/ADNT的降解建立完整的途径。将启动一条完整、成功的ADNT和/或TNT成矿途径。因此，还将讨论预期转化产物的下游加工的酶处理步骤。利用一种新的定向进化方法和代谢工程，该项目旨在为TNT/ADNT的生物矿化创造第一个氧化酶和途径。构建的菌株将在制定修复受爆炸物污染的环境的可持续战略方面发挥非常重要的作用。***