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Engineering Biocatalysts for the Next Generation of omega-Transaminase Processes

用于下一代欧米伽转氨酶工艺的工程生物催化剂

基本信息

批准号：
BB/M021947/1
负责人：
Elaine O'Reilly
金额：
$ 25.43万
依托单位：
University of Nottingham
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2015
资助国家：
英国
起止时间：
2015 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FM021947%2F1
关键词：
Engineering Biocatalysts Next Generation omega

项目摘要

Chiral amines are prevalent in natural products, which often display potent biological activity. Such chiral amine motifs are also frequently found in pharmaceutical drug compounds and chemical building blocks meaning that the development of environmentally benign and sustainable routes to produce these important motifs is extremely desirable. Nature synthesizes these complex and valuable molecules through the action of highly specialized enzymes. These natural catalysts enable an extremely efficient biosynthesis from simple starting materials, installing functional groups with exceptional levels of selectivity. Chemical catalysts are frequently designed to mimic the action of enzymes and are often capable of achieving impressive selectivity. However, unlike enzymes, processes involving these catalysts usually involve high temperatures, sub-optimal pH, organic solvent and complex purification methods. Enzymes called omega-transaminases (TAs) catalyze the conversion of commercially available or easily accessible starting materials to high-value amines. These biocatalysts require an additional donor molecule to provide the amine functional group. This donor is ultimately converted to a by-product and the desired amine product is formed. However, the reaction is freely reversible and unless this by-product is removed from the reaction, low yields of the desired amine will be isolated, as the enzyme will more readily catalyse the reverse reaction to regenerate starting materials. A number of elegant approaches have been reported which remove this ketone by-product and allow access to appreciable quantities of the chiral amine. These strategies include the addition of expensive enzymes or the use of extremely large quantities of the amine donor in combination with the technically challenging removal of ketone by-products. One such approach, which relies on an extensively modified TA, is currently used for the industrial synthesis of the antidiabetic drug compound, sitagliptin. However, the approach is far from efficient and the development of this heavily modified TA biocatalyst was enormously challenging, highlighting an immediate need for more sustainable strategies for performing these biotransformations and for developing suitable enzyme catalysts. This research will build upon recent work reported in our laboratory that describes arguably the most efficient approach to date for performing biotransformations involving TAs. The success of the approach is due to spontaneous precipitation of the by-product, which cannot regenerate starting materials. This polymer is also highly colored and has allowed the development of an effective high-throughput screening strategy that enables the rapid identification of active enzymes. Our focus now is to optimize the process further and make it more suitable for industrial application. Specifically, low cost amine donor molecules will be used that are spontaneously removed from the reaction in a similar way to our previously reported method. We will also apply a simple high-throughput screening strategy to assist in the genetic engineering of natural enzymes in order to increase the scope of the reactions that they can catalyze and make them suitable for industrial scale synthesis. The enzymes developed in this study will enable cost-effective, sustainable and environmentally neutral methods for the small/medium and industrial scale production of one of the most important compound classes.

手性胺广泛存在于天然产物中，具有很强的生物活性。此类手性胺基序也经常存在于药物化合物和化学结构单元中，这意味着开发环境友好和可持续的途径来生产这些重要的基序是极其期望的。大自然通过高度专业化的酶的作用合成这些复杂而有价值的分子。这些天然催化剂能够从简单的起始材料进行非常有效的生物合成，以特殊的选择性安装官能团。化学催化剂通常被设计成模拟酶的作用，并且通常能够实现令人印象深刻的选择性。然而，与酶不同，涉及这些催化剂的过程通常涉及高温、次优pH、有机溶剂和复杂的纯化方法。被称为ω-转氨酶（TA）的酶催化将市售或容易获得的起始材料转化为高价值的胺。这些生物催化剂需要额外的供体分子来提供胺官能团。该给体最终转化为副产物并形成所需的胺产物。然而，该反应是自由可逆的，除非从反应中除去该副产物，否则将分离出低收率的所需胺，因为酶将更容易催化逆反应以再生起始材料。已经报道了许多优雅的方法，其去除这种酮副产物并允许获得可观量的手性胺。这些策略包括添加昂贵的酶或使用极大量的胺供体，以及在技术上具有挑战性的去除酮副产物。其中一种方法依赖于广泛修饰的TA，目前用于工业合成抗糖尿病药物化合物西他列汀。然而，这种方法是远远不够的效率和这种高度改性的TA生物催化剂的发展是巨大的挑战，突出了更可持续的策略，进行这些生物转化和开发合适的酶催化剂的迫切需要。这项研究将建立在我们实验室最近报告的工作基础上，该工作描述了迄今为止进行涉及TA的生物转化的最有效方法。该方法的成功是由于副产物的自发沉淀，其不能再生起始材料。这种聚合物也是高度着色的，并且允许开发有效的高通量筛选策略，从而能够快速鉴定活性酶。我们现在的重点是进一步优化工艺，使其更适合工业应用。具体地，将使用低成本的胺供体分子，其以与我们先前报道的方法类似的方式从反应中自发除去。我们还将应用一种简单的高通量筛选策略来协助天然酶的基因工程，以增加它们可以催化的反应范围，并使其适合工业规模的合成。本研究中开发的酶将为最重要的化合物类别之一的中小规模和工业规模生产提供具有成本效益，可持续和环境中性的方法。