A Biocatalytic Route to Nitrile Reduction

描述（由申请人提供）：项目摘要/摘要：最近鉴定的ykvM基因家族编码一种酶，该酶属于腈氧化还原酶的独特家族，催化7-氰基-7-脱氮鸟嘌呤还原为7-氨甲基-7-脱氮鸟嘌呤，这是生物学中从未观察到的转化。含腈化合物在制药、农业和化学工业中普遍存在，其中它们通常在商业上重要的酸、酰胺和胺的合成中用作中间体。由于腈水解酶和腈水合酶的存在，生物催化已经成为催化腈转化为酸和酰胺的重要选择。虽然腈到胺的化学转化具有与水解相同的一些缺点（高能量成本、其它官能团的反应性、危险废物的产生），但是已知腈氧化还原酶的缺乏已经排除了生物催化在该重要化学过程中的应用。这个多阶段SBIR项目的总体目标是将枯草芽孢杆菌的YkvM蛋白重新设计成一种有效的生物催化剂，用于工业上重要的苯乙腈转化为苯乙胺。我们的第一阶段的目标是测试这一概念的可行性，通过创建一个突变的酶与增强活性的还原苯乙腈苯乙胺。这将通过开发用于筛选YkvM突变体文库的高通量测定、构建用于产生突变体文库的适当表达系统、应用合理设计和定向进化的平行和互补方法来产生所需的催化剂以及筛选文库以鉴定催化剂来实现。该项目目标的成功实现不仅将导致创造一个有价值的工业过程，而且还将作为概念验证，该酶家族可以作为设计催化剂的工程平台，进行各种商业上重要的有机腈的还原。项目叙述：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中的基本化学转化。与许多其他过程一样，它与高能源成本，产生危险废物和缺乏特异性有关。我们建议使用最近发现的腈氧化还原酶从细菌作为一个平台，用于设计新的生物催化剂，能够对环境友好的目标腈类还原。

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.