Microfluidic enzyme reactors using redox-reversible artificial metalloenzymes

背景：酶在化学合成中的应用因其可持续性和环境兼容性而备受关注。然而，对于许多应用，合适的自然产生的酶是不可用的。量身定制的人造金属酶将酶的选择性和生物相容性与合成催化剂的反应性结合在一起，从而有可能扩大生物催化剂的应用范围。人工金属酶尚未进入普遍应用，主要是因为蛋白质和催化剂的生产具有挑战性和昂贵，而且当人工酶不再需要或不再具有活性时，其有价值的成分不容易回收。我们开发了一种新的铁基金属酶，可以通过化学还原铁离子来分解。因此，蛋白质和合成催化剂都可以回收和循环使用。该项目将蛋白质支架固定在固体载体上，使其能够整合到微流控流动系统中。这样，就有可能移除和更换失去活性的催化剂。随后用不同的催化剂进行替换将特别令人感兴趣，因为这不仅可以回收蛋白质，还可以从一种催化反应切换到另一种催化反应。目的和目的：本项目的目的是将合适的蛋白质支架固定在固体载体上，并使用氧化还原控制和流动技术来指导人工酶的组装和催化。1.展示一种稳健的方法，将选定的蛋白质支架固定在固体载体上，并通过氧化还原控制指导人造金属酶的组装和拆卸，从而实现催化剂交换和成分回收。将固定化的人造金属酶转化为微流控流动系统，用于高通量筛选和自动化产品合成。研究方法：表达我们的蛋白质支架的聚组氨酸标签将通过附着到功能化的固体载体上来固定化人造金属酶。将进行顺序的氧化还原控制催化剂“捕捉和释放”循环，以允许回收和循环使用过的铁载体-催化剂结合物，并使不同催化剂的后续反应能够在相同的支架和固体载体上进行。由于固定化的蛋白质支架也可以从其载体上分离出来，因此支架的重复使用应该是可能的。利用微制造技术，将产生流动反应器阵列，以允许并行测试几个反应，以增加吞吐量，加速人工酶的开发和反应条件的优化。长期目标是扩大反应范围并解决生物催化领域的关键挑战，特别是酶的固定化、成分回收和纳入连续流程处理。调整EPSRC战略：预计到2020年，全球特种酶市场将达到约9.5亿美元。使用量身定制的人工酶作为生物催化剂进行化学转化特别有吸引力，因为它们具有可持续性和环境兼容性。在这项日益变革性的技术中，提高国家研究形象并进一步增加英国市场份额的一个可能方法是扩大生物催化工具箱；这就是本项目旨在实现的目标。从长远来看，目标是扩大人造酶的应用范围，从而增强生物催化的范围和可持续性。拟议的工作支持UKRI和EPSRC确定的几个战略重要性领域，特别是面向未来的制造、催化、化学生物和生物化学。合作者：Anne Duhme-Klair教授，约克大学化学系。

Context: The application of enzymes in chemical syntheses is attractive because of their sustainability and environmental compatibility. However, for many applications, suitable naturally occurring enzymes are not available. Tailor-made artificial metalloenzymes combine the selectivity and biocompatibility of enzymes with the reactivity of synthetic catalysts and thus have the potential to expand the range of applications in which biocatalysts can be used. Artificial metalloenzymes have not yet progressed into general use, mainly because the proteins and the catalysts are challenging and expensive to produce and, when the artificial enzyme is no longer required or active, its valuable components cannot easily be recycled.We have developed a new iron-based metalloenzyme that can be disassembled by chemical reduction of the iron ion. Hence both the protein and the synthetic catalyst can be recovered and recycled. This project will immobilise the protein scaffolds on solid supports to enable their integration into microfluidic flow systems. In this way, the removal and replacement of catalysts that have lost activity becomes possible. Subsequent replacements with different catalysts would be of particular interest since this would not only allow the protein to be recycled but also enable switching from one catalysed reaction to another. Aims and Objectives: The aim of this project is to immobilise appropriate protein scaffolds on solid supports and to use redox-control and flow techniques to direct artificial enzyme assembly and catalysis. 1. Demonstrate a robust approach to immobilise selected protein scaffolds on solid supports and direct the assembly and disassembly of artificial metalloenzymes via redox control, thereby enabling catalyst exchange and component recycling.2. Translate immobilised artificial metalloenzymes into microfluidic flow systems for high-throughput screening and automated product synthesis.Research Methodology: Polyhistidine tags, with which our protein scaffolds are expressed, will be used to immobilise artificial metalloenzymes via attachment to functionalised solid supports. Sequential redox-controlled catalyst 'catch-and-release' cycles will be performed to allow used siderophore-catalyst conjugates to be reclaimed and recycled and to enable subsequent reactions with different catalysts to be performed on the same scaffold and solid support. Since the immobilised protein scaffolds can also be cleaved from their support, the reuse of the scaffolds should be possible.Using microfabrication techniques, arrays of flow reactors will be produced to allow several reactions to be tested in parallel, in order to increase throughput and to accelerate artificial enzyme development and the optimisation of reaction conditions. The long-term aim is to widen the reaction scope and address key challenges in biocatalysis, in particular enzyme immobilisation, component recycling and incorporation into continuous flow processing.Alignment to EPSRC strategy: The speciality enzymes market is predicted to reach ~$950 million globally by 2020. The use of tailor-made artificial enzymes as biocatalysts for chemical transformations is particularly attractive because of their sustainability and environmental compatibility. A possible way of enhancing the national research profile and furthering the increase in the market share of the UK in this increasingly transformative technology is the expansion of the biocatalytic toolbox; this is what this project aims to achieve. In the long term, the aim is to extend the range of applications of artificial enzymes, thereby enhancing the scope and sustainability of biocatalysis. The proposed work supports several areas of strategic importance identified by UKRI and the EPSRC, in particular manufacturing for the future, catalysis, chemical biology and biological chemistry. Collaborators: Prof. Anne Duhme-Klair, Department of Chemistry, University of York.