Engineering of unspecific peroxygenases for the β-hydroxylation of amines on a preparative scale.

用于胺β-羟基化的非特异性过氧化酶的工程在制备规模上。

• 批准号: 505185500
• 负责人: Professor Dr. Jan von Langermann
• 金额: --
• 依托单位: Institut für Chemie (ICH)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/505185500?language=en
• 关键词: Engineering; unspecific; peroxygenases; hydroxylation; amines

We are convinced that unspecific peroxygenases (UPOs) represent outstanding enzymes for C–H functionalizations with an extraordinary synthetic potential. By combining protein and process engineering, UPOs’ potential will be demonstrated by synthesizing pharmaceutically important building blocks on a gram-scale. UPOs are fungal enzymes that transfer one peroxide-borne oxygen to sp3-carbons. UPO-catalyzed reactions display an impressive substrate scope with more than 400 known examples. They exhibit excellent enantioselectivities and striking total turnover numbers of up to 300,000 for benzylic hydroxylations. Approx. four thousand putative UPO genes have been annotated, but less than 20 different UPO enzymes were studied in detail due to their challenging heterologous expression. These production limitations also significantly hampered targeted protein engineering efforts, and hence the current substrate scope consists primarily of wild-type activities. It would be of utmost importance to utilize UPOs’ catalytic machinery towards new industrially relevant substrates. In particular, substrates carrying aliphatic amines are ubiquitous in active pharmaceutical ingredients (API), but there are only scarce examples of UPOs hydroxylating these compounds. The molecular class of vicinal amino alcohols is of special interest as these groups could be synthesized by UPOs from amines and represent exciting scaffolds for the pharmaceutical industry. The proposed research project directly addresses the current limitations of UPOs towards amine substrates and aims to utilize an integrated approach of biochemical and process engineering towards the development and application of engineered UPOs. In the field of protein engineering, the methodology includes developing a rapid analysis system for the detection of amino alcohols and a re-shaping of the active site incl. amine anchored regions for the design of potent amine-converting UPOs. On the process engineering side, on-line methodologies for the control of hydrogen peroxide and integration of ion exchange and crystallization techniques will be tailor-made to fit the requirements of UPO-catalyzed reactions. Implementing the developed biocatalysts and methods will be investigated explicitly concerning a high substrate width and in gram-scale.The research project is organized into eight aims and four milestones, which will address the issue in a structured manner by both project partners. It starts from the developments and engineering of assays, proteins, real-time analysis, and selective product removals and culminates in preparative syntheses in gram-scale.

我们相信，非特异性过氧合酶（UPO）代表了杰出的酶C-H功能与非凡的合成潜力。通过结合蛋白质和工艺工程，UPO的潜力将通过在克级上合成药学上重要的构件来证明。UPO是真菌酶，其将一个过氧化物携带的氧转移到sp3-碳。UPO催化的反应显示出令人印象深刻的底物范围，已知的例子超过400个。它们表现出优异的对映体选择性和惊人的总周转数高达300，000苄羟基化。约已经注释了4000个推定的UPO基因，但是由于它们具有挑战性的异源表达，详细研究了不到20种不同的UPO酶。这些生产限制也显著阻碍了靶向蛋白质工程的努力，因此目前的底物范围主要由野生型活性组成。将UPO的催化机制用于新的工业相关底物将是至关重要的。具体地，携带脂肪族胺的底物在活性药物成分（API）中普遍存在，但仅有很少的UPO羟基化这些化合物的实例。邻位氨基醇的分子类别是特别感兴趣的，因为这些基团可以由胺通过UPO合成，并且代表了制药工业的令人兴奋的支架。拟议的研究项目直接解决了目前的局限性UPOs对胺底物，旨在利用生物化学和工艺工程的综合方法对工程UPOs的开发和应用。在蛋白质工程领域，该方法学包括开发用于检测氨基醇的快速分析系统和活性位点的重塑，包括蛋白质合成。胺锚定区域用于设计有效的胺转化UPO。在工艺工程方面，将专门制定控制过氧化氢的在线方法以及离子交换和结晶技术的一体化，以满足UPO催化反应的要求。实施开发的生物催化剂和方法将明确涉及高基板宽度和克级。该研究项目分为八个目标和四个里程碑，将由项目合作伙伴以结构化的方式解决这个问题。它从分析，蛋白质，实时分析和选择性产物去除的开发和工程开始，并在克级制备合成中达到高潮。