Enabling Unspecific Peroxygenases for Applications in Industrial Biocatalysis

使非特异性过氧化酶能够应用于工业生物催化

• 批准号: 1941478
• 金额: --
• 依托单位: University of York
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2017
• 资助国家: 英国
• 起止时间: 2017 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-1941478
• 关键词: Enabling; Unspecific; Peroxygenases; Applications; Industrial

Oxygenation reactions catalysed by microorganisms are valued in industry for the selective functionalisation of complex pharmaceuticals such as steroids. In these cases, the hydroxylations are catalysed by cytochromes P450 (P450), usually associated with the cell-membranes of the fungi that are employed for the reactions. The potential of P450 for industrial hydroxylations has thus resulted in a large volume of research on the enabling of these enzymes for many other selective reactions on a range of industrially relevant substrates. However, many of the envisaged applications require the heterologous expression of P450 in a convenient laboratory host, such as E. coli, but the complexities associated with this expression strategy have frustrated efforts to provide usable catalysts. The reasons include poor solubility (especially of eukaryotic P450) and stability, very low catalyst turnover, but especially the requirements of P450 for nicotinamide cofactors and redox transfer proteins for activity. These difficulties have now stimulated the search both for alternative hosts for P450 expression, but also for more stable and active enzymes that catalyse equivalent processes. The field of biocatalytic oxygenations was recently energised by the discovery of 'unspecific peroxygenases' (UPOs), which are secreted by filamentous fungi as part of their catalytic arsenal for the degradation of recalcitrant plant matter. UPOs have attracted a great deal of interest as, although they are 'unspecific' - meaning that a wide range of substrates is transformed - they are also highly selective in terms of both regio and enantioselectivity. However, the most compelling advantages of UPOs over P450s are their stability - as secreted proteins they have evolved to be stable under challenging conditions of pH, temperature, and also organic solvents; activity - turnovers of 200 s-1 can be achieved, and their dependence only on hydrogen peroxide for full activity, removing the need for either expensive nicotinamide cofactors or redox transfer proteins. As UPO work is in its infancy, most groups have dependent on growing the wild-type strain of fungus Agrocybe aegerita for its production, with the attendant complexities of fungal fermentation and purification of the enzyme from the complex mix of secreted proteins. In recent work, we have expressed AaUPO in a heterologous system - the yeast Pichia pastoris. This gives the advantage of superior control of fermentative production of the enzyme, and also huge improvements in yield and simplicity of purification. We are currently characterising the recombinant UPO and assessing its suitability for scaleable asymmetric oxygenations of benzylic carbons, alkenes, and sulfides. In this studentship project, we will look to exploit the superior activity of recombinant UPOs in the transformation of a range of synthetically relevant organic substrates.

微生物催化的氧化反应在工业上对复杂药物（如类固醇）的选择性功能化很有价值。在这些情况下，羟基化是由细胞色素P450 （P450）催化的，通常与用于反应的真菌的细胞膜有关。因此，P450在工业羟基化方面的潜力导致了对这些酶在一系列工业相关底物上进行许多其他选择性反应的大量研究。然而，许多设想的应用需要在方便的实验室宿主（如大肠杆菌）中异源表达P450，但是这种表达策略的复杂性阻碍了提供可用催化剂的努力。其原因包括溶解性差（特别是真核生物P450）和稳定性差，催化剂周转率非常低，尤其是P450对烟酰胺辅助因子和氧化还原转移蛋白的活性要求。这些困难现在刺激了寻找P450表达的替代宿主，以及催化等效过程的更稳定和更活跃的酶。生物催化氧化领域最近因“非特异性过氧酶”（UPOs）的发现而活跃起来，该酶由丝状真菌分泌，作为其降解顽固植物物质的催化武器库的一部分。upo引起了人们极大的兴趣，因为尽管它们是“非特异性的”——这意味着广泛的底物被转化——它们在区域和对映体选择性方面也具有很高的选择性。然而，与p450相比，upo最引人注目的优势是它们的稳定性——作为分泌蛋白，它们在pH、温度和有机溶剂等具有挑战性的条件下已经进化成稳定的；活性-可以实现200 s-1的周转率，并且它们仅依赖于过氧化氢来获得充分的活性，从而不需要昂贵的烟酰胺辅助因子或氧化还原转移蛋白。由于UPO工作尚处于起步阶段，大多数小组依赖于种植野生型真菌Agrocybe aegerita菌株进行生产，随之而来的是真菌发酵和从复杂的分泌蛋白质混合物中纯化酶的复杂性。在最近的工作中，我们在酵母毕赤酵母的异种系统中表达了AaUPO。这使酶的发酵生产具有优越的控制优势，并且在产量和纯化的简单性方面也有很大的提高。我们目前正在对重组UPO进行表征，并评估其在苯碳、烯烃和硫化物的可伸缩不对称氧化反应中的适用性。在这个学生项目中，我们将着眼于利用重组UPOs在一系列合成相关有机底物转化中的优越活性。