Nitrate Respiration of the Hyperthermophile Pyrobaculum aerophilum

该项目的目的是研究极端嗜热条件下的反硝化机理。研究的重点将是反硝化的第一步酶——硝酸还原酶。以最适生长温度为100℃的超嗜热古菌为模型系统进行研究。本课题的目的包括：(1)改善嗜气假单胞菌的细胞培养条件。这将使用加压瓶和/或进料批发酵罐来完成，以防止硝酸盐呼吸过程中亚硝酸盐的毒性积累。预计一旦对生物体的营养需求有了更好的了解，细胞产量就可以增加。我们将尝试确定额外的碳源、电子供体和可能提高细胞产量的微量元素。(2)硝酸还原酶的纯化和生理特性将对高温酶的生物化学有深入的了解。除了现有的人工电子给体酶分析外，还将开发以醌类为电子给体的酶分析来表征膜结合酶。该酶将用洗涤剂从膜中提取，并使用适当的技术进行纯化。该高温酶的生化特性将被检验：高温硝酸还原酶是否像已知的嗜中温细菌硝酸还原酶一样是含钼酶？或者，它是否像来自嗜热古菌的其他金属酶一样含有钨？电子向酶转移的本质是什么？(3)为进一步获得硝酸盐还原酶基因的结构、调控和进化信息，将对其进行克隆和测序。将建立一个基因库，并利用反向遗传学或PCR探针克隆nar基因。DNA序列应该揭示相当多的信息，与中温细菌的对应关系。这个研究项目将有助于我们对极端嗜热生物的能量代谢的理解，并扩大我们对能量代谢如何进化的认识。这个项目关注的是了解在高温下生长的被称为古菌的不寻常细菌。嗜气焦杆菌在100℃下生长最好，也可以在没有氧气的情况下生长，使用硝酸盐代替。本文对硝酸还原酶这一利用硝酸的关键酶进行了研究。酶将被纯化，其性质将被确定。这将为高温下的生命提供新的和有趣的信息，并可能导致利用高温酶的生物技术应用。＊＊＊

9631006 Schroder The objective of this project is to study the mechanism of denitrification under extremely thermophilic conditions. The focus of the research will be the enzyme of the first step of denitrification, nitrate reductase. The hyperthermophile archaeon which has an optimal growth temperature of 100 C will be Pyrobaculum aerophilum studied as the model system. The objectives of this project include: (1) the improvement of cell culture conditions for P. aerophilum. This will be done using pressurized vials and/or a fed-batch fermentor to prevent the toxic accumulation of nitrite during nitrate respiration. It is anticipated that the cell yields can be increased once the nutritional needs of the organism are better known. We will attempt to identify additional carbon sources, electron donors, and trace elements which may improve cell yields. (2) the purification and physiological characterization of the nitrate reductase will give insight into the biochemistry of a high temperature enzyme. Besides the already existing enzyme assay using artificial electron donor, additional enzyme assays involving quinones as electron donor will be developed to characterize the membrane-bound enzyme. The enzyme will be extracted from the membrane with detergents and purified using appropriate techniques. The biochemical properties of this high temperature enzyme will be examined: Is the high temperature nitrate reductase a molybdenum containing enzyme like the known mesophilic bacterial nitrate reductases? Alternatively, does it contain tungsten as do other metallo-enzymes from thermophilic archaea? What is the nature of electron transfer to the enzyme? (3) to gain further structural, regulatory and evolutionary information, the nitrate reductase genes will be cloned and sequenced. A gene library will be prepared and the nar genes will be cloned using reverse genetics or PCR probing. The DNA sequence should reveal considerable information as to the relatedness to mesophilic counterparts. This res earch project will contribute to our understanding of the energy metabolism in extremely thermophilic organisms and widen our knowledge as to how energy metabolism has evolved. %%% This project is concerned with understanding unusual bacteria known as archae that grow at very high temperatures. Pyrobaculum aerophilum grows best at 100 C and can also grow in the absence of oxygen using nitrate instead. In this work the enzyme nitrate reductase, a key enzyme for using nitrate, will be studied. The enzyme will be purified, and its properties will be determined. This will provide new and interesting information about life at high temperatures and may lead to biotechnology applications that make use of high temperature enzymes. ***