Microfluidic enzyme reactors using redox-reversible artificial metalloenzymes

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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.