DIRECTED EVOLUTION OF A THERMOSTABLE PHOSPHITE DEHYDROGENASE

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。NAD(P)H依赖的氧化还原酶是合成手性化合物的有价值的工具。然而，昂贵的辅因子成本需要原位辅因子再生以用于制备应用。我们试图开发一种以斯氏假单胞菌亚磷酸脱氢酶为基础的酶系统，以再生还原的烟酰胺辅助因子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。圆二色谱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.