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

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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.