Enzymatic Biodegradation of Chlorooxides

描述（由申请人提供）：氯氧化物（ClOx-）离子是人为的杀菌剂、漂白剂和氧化剂，由于其高溶解度和在农业、工业和军事中的广泛使用，已成为饮用水和淡水环境的主要污染物。各种氯化物都被怀疑对健康有危害，并已被记录在案。下面的建议将确定在蛋白质/酶水平上深入研究基于微生物的ClOx-解毒的可行性。美国环保署已将亚氯酸盐（CIO2-）列入其主要饮用水标准清单，其法定上限为0.8毫克/升。与慢性CIO2暴露相关的健康风险包括儿童和婴儿贫血和神经系统紊乱。高氯酸盐（CIO4-）最近被确定为井水污染物，被添加到EPA的污染物候选清单中。目前，环境保护局和其他机构正在研究其对健康的全面影响。为了应对氯化物的涌入，大自然进化出了解毒甚至利用它们的机制。厌氧呼吸氯化物的微生物，包括CIO4-、CIO3-和CIO2-，在过去的十年中已经被分离出来。从水中去除小离子的传统方法既昂贵又不具有特异性，因此氯化物的生物修复是一个新兴的生物工程目标。芳香脱氯单胞菌是唯一一种基因组测序的（ClOx-）呼吸微生物，是生化研究的明显选择（J.D. Coates, University of California, Berkeley）。此外，脱氯单胞菌在纯培养菌株中是独一无二的，因为它氧化不妥协的碳氢化合物，包括苯，一种主要的环境污染物和一种强效的人类致癌物。我们寻求[1]分离、部分测序和鉴定编码（ClOx-）代谢活性的基因；[2]从宿主和异源表达系统中获得足够数量的纯酶用于机制和x射线结构研究；和[3]部分表征内源性和表达酶。这些目标为未来（ClOx-）解毒途径的机制、光谱、结构和蛋白质工程研究奠定了基础。这项工作将成为旨在开发生物修复系统和/或用于环境监测的基于酶的传感器的更全球性努力的一部分。

DESCRIPTION (provided by applicant): The chlorooxide (ClOx-) ions are anthropogenic biocides, bleaches, and oxidants that, because of their high solubility and ubiquitous use by agriculture, industry, and the military, have become major contaminants of drinking water and fresh water environments. The various chlorooxides are both suspected and documented health hazards. The following proposal will determine the feasibility of in-depth studies of microbe-based ClOx- detoxification at the protein/enzyme level. The U.S. EPA has included chlorite (CIO2-) on its list of Primary Drinking Water Standards, with a legally mandated upper limit of 0.8 mg/L in drinking water. Health risks associated with chronic CIO2- exposure include anemia and nervous system disorders in children and infants. After its recent identification as a well water contaminant, perchlorate (CIO4-) was added to the EPA's Contaminant Candidate List. Its full health effects are currently under study by the EPA and by others. In response to an influx of chlorooxides, nature has evolved mechanisms to detoxify and even exploit them. Microorganisms that anaerobically respire chlorooxides, including CIO4-, CIO3-, and CIO2-, have been isolated over the last decade. Traditional means of removing small ions from water are expensive or nonspecific, so bioremediation of chlorooxides is an emerging bioengineering goal. Dechloromonas aromatica, the only (ClOx-)-respiring microbe with a sequenced genome, is the obvious choice for biochemical study (J.D. Coates, University of California, Berkeley). Moreover, Dechloromonas is unique among strains in pure culture, in that it oxidizes intransigent hydrocarbons including benzene, a major environmental contaminant and a potent human carcinogen. We seek [1] to isolate, partially sequence, and identify the genes encoding (ClOx-)-metabolizing activities in D. aromatica; [2] to obtain sufficient quantities of pure enzymes for mechanistic and X-ray structural studies from the host and heterologous expression systems; and [3] to partially characterize the endogenous and expressed enzymes. These objectives set the stage for future mechanistic, spectroscopic, structural, and protein-engineering studies of these components of the (ClOx-) detoxification pathway. This work will be part of a more global effort aimed at developing bioremediation systems, and/or enzyme-based sensors for environmental monitoring.