A Biocatalytic Route to Nitrile Reduction

腈还原的生物催化途径

• 批准号: 7162893
• 负责人: Dirk Iwata-Reuyl
• 金额: $ 9.97万
• 依托单位: QUZYME, LLC
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-01 至 2007-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7162893
• 关键词: Bacillus subtilis; bacterial proteins; bioengineering /biomedical engineering; cyanides; directed evolution; enzyme mechanism; high throughput technology; method development; mutant; peptide library; protein engineering; protein structure function; reduction; transfection /expression vector

DESCRIPTION (provided by applicant): Project Summary/Abstract: The recently identified ykvM gene family encodes an enzyme belonging to a unique family of nitrile oxido-reductases that catalyze the reduction of 7-cyano-7-deazaguanine to 7-aminomethyl-7-deazaguanine, a transformation never before observed in biology. Nitrile containing compounds are ubiquitous in the pharmaceutical, agricultural, and chemical industries, where they often serve as intermediates in the synthesis of commercially important acids, amides and amines. Biocatalysis has become an important option for catalyzing the conversion of nitriles to acids and amides due to the existence of the enzymes nitrilase and nitrile hydratase, respectively. While the chemical conversion of nitriles to amines suffers some of the same drawbacks as hydrolysis (high energy costs, reactivity of other functional groups, generation of hazardous waste), the absence of a known nitrile oxido-reductase has precluded the application of biocatalysis to this important chemical process. The overall goal of this multi-phase SBIR project is to reengineer the YkvM protein from Bacillis subtilis into an efficient biological catalyst for the industrially important conversion of phenylacetonitrile to phenylethyl amine. Our goal for phase I is to test the feasibility of this concept by creating a mutant enzyme with enhanced activity for the reduction of phenylacetonitrile to phenylethylamine. This will be achieved by the development of a high-throughput assay for screening YkvM mutant libraries, the construction of an appropriate expression system for creating mutant libraries, the application of the parallel and complementary approaches of rational design and directed evolution to create the desired catalyst, and the screening of the libraries to identify the catalyst. The successful realization of the project goals will not only result in the creation of a valuable industrial process, but will also serve as proof-of-concept that this family of enzymes can serve as a platform for the engineering of designed catalysts that carry out the reduction of a variety of commercially important organonitriles. Project Narrative: Relevance: The reduction of nitrile containing compounds to primary amines is a fundamental chemical transformation in the pharmaceutical, commodity, and fine chemical industries. Like many other processes, it is associated with high energy costs, the generation of hazardous waste, and lack of specificity. We propose to use the recently discovered nitrile oxido-reductase enzymes from bacteria as a platform for the design of novel biocatalysts capable of the environmentally benign reduction of targeted nitriles.

描述(由申请人提供)：项目摘要/摘要：最近发现的ykvM基因家族编码一种属于独特的氰基氧化还原酶家族的酶，该酶能催化7-氰基-7-去氮鸟嘌呤还原为7-氨甲基-7-去氮鸟嘌呤，这一转化在生物学上从未观察到。含腈化合物在制药、农业和化学工业中普遍存在，在这些行业中，它们经常作为合成具有商业重要性的酸、胺和胺的中间体。由于腈水合酶和腈水合酶的存在，生物催化已成为催化腈转化为酸和酰胺的重要选择。尽管氰化物的化学转化为胺的过程存在一些与水解相同的缺点(能源成本高、其他官能团的反应性、产生危险废物)，但由于没有已知的丁腈氧化还原酶，生物催化在这一重要的化学过程中的应用受到了阻碍。这个多阶段SBIR项目的总体目标是将枯草芽孢杆菌的YkvM蛋白重新设计为一种高效的生物催化剂，用于将具有重要工业意义的苯乙腈转化为苯乙胺。我们第一阶段的目标是通过创造一种具有增强活性的突变酶来测试这一概念的可行性，以将苯乙腈还原为苯乙胺。这将通过发展筛选YkvM突变体库的高通量试验，构建用于创建突变体库的适当表达系统，应用合理设计和定向进化的并行和互补方法来创建所需的催化剂，以及筛选文库来鉴定催化剂来实现。项目目标的成功实现不仅将创造一个有价值的工业过程，而且还将作为概念证明，这一系列酶可以作为设计催化剂的平台，执行各种具有商业重要性的有机腈的还原。项目简介：相关性：将含腈化合物还原为伯胺是制药、日用品和精细化工行业的基本化学转变。与许多其他过程一样，它与高昂的能源成本、危险废物的产生和缺乏专用性有关。我们建议使用最近从细菌中发现的丁腈氧化还原酶作为平台来设计能够对环境友好地还原目标腈的新型生物催化剂。