Evolving Inproved Formate Dehydrogenases

不断改进的甲酸脱氢酶

• 批准号: 6832896
• 负责人: JAMES DAVID ROZZELL
• 金额: $ 37.5万
• 依托单位: BIOCATALYTICS, INC.
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-01-01 至 2006-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6832896
• 关键词: NAD(H) phosphate; NAD(P)H dehydrogenase; biotechnology; centrifugation; enzyme activity; formates; gas chromatography; gel electrophoresis; high performance liquid chromatography; hybrid enzyme; liquid chromatography mass spectrometry; oxidoreductase; peptide chemical synthesis; polymerase chain reaction; protein engineering; site directed mutagenesis; thermostability

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.

描述（由申请人提供）：烟酰胺辅因子依赖性酶越来越多地发现用于合成药物生产的手性中间体。随着各种氧化还原酶的可用性（Biocatalytics，Inc。2003目录，请参阅附录），以及使用氨基酸脱氢酶和酒精脱氢酶在现有过程中的工作，以使非天然氨基酸和手性酒精对尼古丁氨基酸酯的效率和成本效益方法的需求增加。鉴于烟酰胺辅因子的高成本（当前的散装价格为$ 1500/摩尔的NAD+; NADP+5000美元/摩尔），有必要使用合适的回收系统再生它们。当前，葡萄糖脱氢酶用于回收NADP+。在此过程中，必须随着反应的进行，葡萄糖必须以等效量产生副产品，葡萄糖酸，并且必须与所需产物分离。该反应还会产生每摩尔产物的H+离子1摩尔，因此需要对反应混合物的pH控制。对于NAD+的回收，使用甲酸脱氢酶。我们通过将造型脱氢酶与氨基酸脱氢酶耦合，成功实施了生产非天然氨基酸的商业过程。该过程受到甲酸脱氢酶相对于氨基酸脱氢酶的较低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。改善这些特征将显着改善现有过程的经济学，并增加在类似过程中其他应用的机会。在第二阶段，我们计划继续进行NADP+和NAD+回收的改善甲酸脱氢酶的定向演变。在改进的NADP+回收的改进突变体上的工作将集中在产生具有比活性增加，改善热稳定性，较低KM以及NADPH抑制减少的新型突变酶。 NAD+回收利用的甲酸甲酸盐脱氢酶的演变将集中在特定活性的进一步改进，NADH抑制产物抑制以及更大的热稳定性上。我们将使用容易发生的PCR搜索新的突变体，并在所有发现会导致改进酶以优化现有突变体的酶进行饱和诱变。到第二阶段结束时，我们计划拥有两种新产品：NADP+回收利用的第一个工业用甲酸甲酸盐脱氢酶和用于NAD+回收的工程FDH，它显示出比任何现有的甲酸脱氢酶的更高特异性活性，较低的产品抑制和更大的热稳定性。