DIRECTED EVOLUTION OF A THERMOSTABLE PHOSPHITE DEHYDROGENASE

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。NAD（P）H依赖性氧化还原酶是合成手性化合物的重要工具。然而，辅因子的昂贵成本需要原位辅因子再生以用于纯化应用。我们试图开发一种基于施氏假单胞菌亚磷酸脱氢酶的酶系统，以再生还原型烟酰胺辅因子NADH和NADPH。定向进化已被用于产生具有更高热稳定性的突变体。圆二色性和差示扫描量热法等技术将用于阐明个体稳定突变的机制。

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. NAD(P)H-dependent oxidoreductases are valuable tools for synthesis of chiral compounds. The expensive cost of the cofactors, however, requires in situ cofactor regeneration for preparitive applications. We have attempted to develop an enzymatic system based on phosphite dehydrogenase from Pseudomonas stutzeri to regenerate the reduced nicotinamide cofactors NADH and NADPH. Directed evolution has been used to generate mutants with higher thermal stability. Techniques such as Circular Dichroism and Differential Scanning Calorimetry will be used to elucidate the mechanism of individual stabilizing mutations.