Redox-reversible artificial metalloenzymes

As natural biocatalysts, enzymes have evolved over billions or years to be highly efficient and selective. Their application in both bioconversions and 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, on the other hand, can combine the selectivity and biocompatibility of proteins with the reactivity and the reaction scope of synthetic catalysts and thus have the potential to expand the range of applications in which biocatalysts can be used, for example by making new-to-nature transformations accessible. 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.

Inspired by the way bacteria acquire essential iron, we have developed a new iron-based anchor unit that connects synthetic catalysts to proteins, creating artificial enzymes, but on chemical reduction of the iron centre, the anchor unit disconnects and triggers the disassembly of the artificial enzymes. Hence both the protein and the synthetic catalyst can be recovered and recycled.

In this project, we will explore the wider scope of these recyclable artificial enzymes and immobilise the protein scaffolds on solid supports to enable their integration into 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 enabling an easy switch from one catalysed reaction to another.

The application of this 'catch-and-release' approach to immobilised artificial metalloenzyme design will provide a flexible toolbox for their preparation that allows catalysts, protein scaffolds and solid supports to be mixed, matched and recycled, for us and others to use, adapt and explore further, both in batch processes and in continuous flow.

Long-term economic and societal impacts: Recently, the speciality enzyme market was projected to reach just under $950 million globally by 2020. The use of biocatalysts for chemical synthesis is particularly attractive because of their sustainability and environmental compatibility. A possible way of furthering the increase in the share of the UK in biocatalysis is the expansion of the biocatalytic toolbox; this is what this proposal aims to achieve. By integrating synthetic chemocatalysts in protein scaffolds, artificial metalloenzymes combine the beneficial features of both, performing synthetic reactions with new levels of selectivity and, importantly, add new-to-nature transformations to the range of reactions that are accessible to this increasingly transformative technology.

Thus far, however, artificial metalloenzymes have not progressed into mainstream use, mainly because both the protein scaffolds and the organometallic catalysts are challenging and expensive to produce and, when the artificial enzyme is no longer required or active, these valuable components are very difficult to recycle. The new redox-reversible anchoring system that we have developed is now opening up new opportunities for component recycling, since it enables the controlled assembly and disassembly of artificial metalloenzymes and therefore the recovery and reuse of the individual components. This redox-reversible approach will be particularly beneficial for applications that use flow processes, where the protein scaffold is immobilised on a solid support and sequential catalyst 'catch-and-release' steps can easily be implemented. Our long-term goal is to bring artificial metalloenzymes closer to commercial viability by developing a flexible toolbox for their preparation that allows catalysts, protein scaffolds and solid supports to be mixed, matched and recycled, thereby contributing to enhancing the scope and sustainability of biocatalysis.

Since the move towards greener technology is of great current interest and importance, the proposed work provides opportunities to engage with the public to raise awareness of environmental and sustainability issues. In addition, biocatalysis and artificial enzymes will be integrated into our undergraduate and graduate teaching and we are offering several undergraduate research and work experience projects in these areas, which proved to be very successful. We intend to continue and extend these and related activities further. The work will result in the training of highly skilled PDRAs, who will be well prepared for employment in either the academic or the industrial sector. In addition, new graduates and final year undergraduates working on related projects will benefit from the training provided.