INVESTIGATION OF THE ACTIVE SITE INTEGRITY AND REACTIVITY OF PROTEINS USED IN IN

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 人们对在工业应用中使用金属酶越来越感兴趣，因为它们往往可以提供一种进行催化过程的有效手段，同时减少对人类健康和环境的影响。然而，为了使这些酶有效，活性部位必须能够经受住恶劣的条件，这些条件可能包括高温、极端的pH和/或存在螯合剂。必须保持蛋白质活性部位的完整性，以保持所需的催化活性。因此，工业用金属酶的优化需要一种在所要求的应用条件下表征活性中心几何结构和电子结构的手段。XAS EDGE和EXAFS光谱为研究生物催化剂活性中心的变化提供了一种理想的方法，因此我们建议将这些方法应用于一些工业上感兴趣的酶系统。我们的初步研究将集中在依赖锌和钙的中性蛋白酶(NPR)和嗜热分解酶(Thr)，以及它们在甜味剂和其他工业应用中的优化。我们的XAS研究稍后将扩展到包括依赖锌、锰或钴的有机磷水解酶(OPH)，它们已被开发用于农药的生物修复和VX神经毒气等生物战剂的分解；以及依赖镁、钴或锰的D-葡萄糖异构酶(GI)(也用于甜味剂应用)。这些研究应对工业生物技术作出重要贡献，并将推动对工业过程采取可持续的化学方法。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. There is growing interest in the use of metalloenzymes in industrial applications, as they can often provide an efficient means of carrying out catalytic processes, while reducing the impact on human health and the environment. However, for these enzymes to be effective, the active sites must be able to withstand harsh conditions, which may include high temperature, extreme pH, and/or the presence of chelators. The integrity of the protein active sites must be maintained to preserve the desired catalytic activity. Hence the optimization of metalloenzymes for industrial use requires a means of characterizing the active site geometric and electronic structure under the required application conditions. XAS edge and EXAFS spectroscopy provides an ideal means for examining changes in the active site of biocatalysts, and hence we propose to apply these methods to a number of enzyme systems of industrial interest. Our initial studies will focus on the zinc- and calcium-dependent neutral protease (NPR) and thermolysin (THR), and their optimization for sweeteners and other industrial applications. Our XAS studies will later be extended to include the zinc-, manganese- or cobalt-dependent organophosphorus hydrolases (OPH) which have been developed for bio-remediation of pesticides and the breakdown of biological warfare agents such as VX nerve gas; and the magnesium-, cobalt- or manganese-dependent D-glucose isomerases (GI) (also used in sweetener applications). These studies should make an important contribution to industrial biotechnology and will further a sustainable chemistry approach to industrial processes.