Evolving Inproved Formate Dehydrogenases

不断改进的甲酸脱氢酶

• 批准号: 6942735
• 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/6942735
• 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用于合成药物生产的手性中间体。随着各种氧化还原酶（BioCatalytics，Inc. 2003目录，参见附录），以及关于使用氨基酸脱氢酶和醇脱氢酶制备非天然氨基酸和手性醇的现有方法的工作，对烟酰胺辅因子的更有效和成本有效的方法的需求正在增加。鉴于烟酰胺辅因子的高成本（NAD+的当前批量价格为1500美元/摩尔; NADP+为5000美元/摩尔），有必要使用合适的再循环系统使其再生。目前，葡萄糖脱氢酶用于NADP+的再循环。在这个过程中，葡萄糖必须随着反应的进行而加入，并且产生等摩尔量的副产物葡萄糖酸，并且必须与所需产物分离。反应还产生1摩尔H+离子/摩尔产物，需要控制反应混合物的pH。对于NAD+的再循环，使用甲酸脱氢酶。我们已经成功地实施了一种商业方法，通过偶联甲酸脱氢酶与氨基酸脱氢酶来生产非天然存在的氨基酸。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以及NADH对甲酸脱氢酶的产物抑制的限制。改善这些特性将显著提高现有工艺的经济性，并增加在类似工艺中的额外应用的机会。在第二阶段，我们计划继续定向进化改进的甲酸脱氢酶，用于NADP+和NAD+的再循环。关于NADP+再循环的改进突变体的工作将集中于产生具有增加的比活性、改善的热稳定性、较低的KM和减少的NADPH抑制的新突变体酶。用于NAD+再循环的甲酸脱氢酶的发展将集中在比活性的进一步改进、减少NADH对产物的抑制以及更高的热稳定性上。我们将使用易错PCR寻找新的突变体，并在发现的所有位点进行饱和诱变，以优化现有的突变体，从而改进酶。到第二阶段结束时，我们计划有两个新产品：第一个用于NADP+再循环的工业上有用的甲酸脱氢酶和一个用于NAD+再循环的工程化FDH，其显示出比任何现有甲酸脱氢酶更高的比活性，更低的产物抑制和更高的热稳定性。