Evolving Inproved Formate Dehydrogenases

说明(申请人提供)：烟酰胺辅因子依赖酶越来越多地用于合成手性中间体，用于药物生产。随着各种氧化还原酶(BioCatalytics，Inc.2003目录，见附录)的增加，以及利用氨基酸脱氢酶和乙醇脱氢酶来制造非天然氨基酸和手性醇的现有工艺的工作，对烟酰胺辅因子的更有效和更具成本效益的方法的需求正在增加。鉴于烟酰胺辅因子的成本很高(目前NAD的批发价为1500美元/摩尔，NADP为5000美元/摩尔)，有必要使用适当的回收系统对其进行再生。目前，葡萄糖脱氢酶用于回收NADP。在这个过程中，葡萄糖必须随着反应的进行而进料，副产物葡萄糖酸是等摩尔量的，必须从所需产品中分离出来。该反应还会产生每摩尔产物1摩尔的H离子，这就需要对反应混合物进行pH控制。为了回收NAD，使用了甲酸脱氢酶。我们通过将甲酸脱氢酶与氨基酸脱氢酶偶联，成功地实现了一种非自然产生的氨基酸的商业生产过程。这一过程受到甲酸脱氢酶相对于氨基酸脱氢酶较低的活性和长期稳定性的限制，以及NADH对甲酸脱氢酶的产物抑制。改进这些特点将大大提高现有工艺的经济性，并增加在类似工艺中进行更多应用的机会。在第二阶段，我们计划继续改进甲酸脱氢酶的定向进化，用于NADP和NAD循环。研究一种用于NADP回收的改进突变体的工作将集中于产生新的突变酶，具有更高的比活、更好的热稳定性、更低的Km，并减少NADPH的抑制。用于NAD循环的甲酸脱氢酶的发展将集中在比活性的进一步提高、NADH减少产物抑制以及更好的热稳定性。我们将使用容易出错的PCR寻找新的突变体，并在所有发现导致酶改进的位置进行饱和诱变，以优化现有的突变体。到第二阶段结束时，我们计划推出两种新产品：第一种用于NADP循环的工业用甲酸脱氢酶和一种用于NAD循环的工程FDH，它比现有的任何甲酸脱氢酶都表现出更高的比活性、更低的产物抑制和更高的热稳定性。

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.