Evolving Inproved Formate Dehydrogenases

描述（由申请人提供）：烟酰胺辅助因子依赖酶越来越多地用于合成用于药品生产的手性中间体。随着各种氧化还原酶（biocalytics, Inc. 2003年目录，见附录）可用性的增加，以及利用氨基酸脱氢酶和醇脱氢酶制造非天然氨基酸和手性醇的现有工艺的工作，对更有效和更具成本效益的烟酰胺辅助因子方法的需求正在增加。鉴于烟酰胺辅助因子的高成本（目前NAD+的散装价格为1500美元/摩尔；NADP+的散装价格为5000美元/摩尔），有必要使用合适的回收系统再生它们。目前，葡萄糖脱氢酶用于NADP+的再循环。在这个过程中，随着反应的进行，必须加入葡萄糖，副产物葡萄糖酸以等摩尔量产生，必须从所需的产物中分离出来。每摩尔产物生成1mol的H+离子，需要控制反应混合物的pH值。利用甲酸脱氢酶回收NAD+。我们已经成功地实施了一种通过甲酸脱氢酶与氨基酸脱氢酶偶联生产非天然存在的氨基酸的商业工艺。与氨基酸脱氢酶相比，甲酸脱氢酶的活性和长期稳定性较低，NADH对甲酸脱氢酶的抑制作用也限制了这一过程。改进这些特性将大大提高现有工艺的经济性，并增加类似工艺中其他应用的机会。在第二阶段，我们计划继续改进用于NADP+和NAD+回收的甲酸脱氢酶的定向进化。对NADP+再循环的改进突变体的研究将集中在产生新的突变酶上，这些突变酶具有更高的比活性、更好的热稳定性、更低的KM和更少的NADPH抑制。用于NAD+回收的甲酸脱氢酶的进化将集中在进一步提高比活性，减少NADH对产物的抑制和更高的热稳定性上。我们将利用易出错PCR技术寻找新的突变体，并在发现的所有位点进行饱和诱变，从而改进酶以优化现有突变体。在第二阶段结束时，我们计划有两个新产品：第一个工业上有用的用于NADP+回收的甲酸脱氢酶和一个用于NAD+回收的工程FDH，它比任何现有的甲酸脱氢酶具有更高的比活性，更低的产物抑制和更大的热稳定性。

DESCRIPTION (provided by applicant): Nicotinamide cofactor dependent enzymes are increasingly finding use for the synthesis of chiral intermediates for the production of pharmaceuticals. With the increased availability of various oxidoreductases (BioCatalytics, Inc. 2003 catalog, see appendix), and the work on existing processes using amino acid dehydrogenases and alcohol dehydrogenases to make non-natural amino acids and chiral alcohols, the demands for ever more efficient and cost-effective methods for nicotinamide cofactor are increasing. Given the high cost of nicotinamide cofactors (current bulk prices are $1500/mole for NAD+; $5000/mole for NADP+), it is necessary to regenerate them using a suitable recycling system. Currently, glucose dehydrogenase is used for the recycling of NADP+. In this process, glucose must be fed as the reaction proceeds, and the byproduct, gluconic acid, is produced in equimolar quantities and must be separated from the desired product. The reaction also generates 1 mole of H+ ions per mole of product, necessitating pH control of the reaction mixture. For the recycling of NAD+, formate dehydrogenase is used. We have successfully implemented a commercial process for the production of a non-naturally occurring amino acid by coupling formate dehydrogenase with an amino acid dehydrogenase. This process is limited by the lower activity and long-term stability of formate dehydrogenase relative to the amino acid dehydrogenase, and product inhibition of formate dehydrogenase by NADH. Improving these characteristics would both significantly improve the economics of the existing process and increase the opportunities for additional applications in similar processes. In Phase II we plan to continue with the directed evolution of improved formate dehydrogenases for both NADP+ and NAD+ recycling. Work on an improved mutant for NADP+ recycling will focus on generating new mutant enzymes with increased specific activity, improved thermostability, lower KM, and reduced inhibition by NADPH. Evolution of formate dehydrogenase for NAD+ recycling will focus on further improvements in specific activity, reduced product inhibition by NADH, and greater thermostability. We will search for the new mutants using error-prone PCR and carry out saturation mutagenesis at all sites found to lead to improved enzymes to optimize the existing mutants. By the end of Phase II, we plan to have two new products: the first industrially-useful formate dehydrogenase for NADP+ recycling and an engineered FDH for NAD+ recycling that displays the higher specific activity, lower product inhibition, and greater thermostability than any existing formate dehydrogenase.