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的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 在工业应用中使用金属酶的兴趣越来越大，因为它们通常可以提供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，活性位点必须能够承受苛刻的条件，这些条件可能包括高温、极端pH和/或螯合剂的存在。 必须保持蛋白质活性位点的完整性以保持所需的催化活性。因此，优化金属酶的工业用途需要一种手段，在所需的应用条件下，活性部位的几何和电子结构的特征。XAS边缘和EXAFS光谱提供了一个理想的手段，用于检查生物催化剂的活性位点的变化，因此，我们建议将这些方法应用到一些酶系统的工业利益。 我们的初步研究将集中在锌和钙依赖性中性蛋白酶（NPR）和嗜热菌蛋白酶（THR），以及它们在甜味剂和其他工业应用中的优化。 我们的XAS研究以后将扩展到包括锌，锰或钴依赖性有机磷水解酶（OPH），已开发用于农药的生物补救和生物战剂，如VX神经毒气的分解;和镁，钴或锰依赖性D-葡萄糖异构酶（GI）（也用于甜味剂应用）。这些研究应能对工业生物技术作出重要贡献，并将促进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.