Novel flow systems and process optimisation for H2 driven biocatalysis

Thesis

Catalysis is essential in the pharmaceutical industry to selectively generate chiral centres, with 50 % FDA approved small molecule drugs in 2020 containing at least 1 chiral centre. In practice, this often means that expensive heavy metals such as palladium are used as catalysts at high pressures and temperatures to improve the rate of reactions. As well as the obvious safety and environmental costs, traditional metal catalysts can also have selectivity issues.

By contrast, biocatalysts operate at room temperature and exhibit excellent selectivity due to their unique active sites. However, many enzymes require additional cofactors to function, which can be expensive such as the nicotinamide cofactor NAD⁺ (£64.80 g^-1). To make biocatalysis cost efficient the cofactors must be recycled, but this can generate carbon based waste such as gluconolactone. Some enzymes will accept reduced flavins as an alternative cofactor, including ene-reductases which have previously been tested in batch using reduced flavins recycled by a Ni-Fe hydrogenase. This research shows vitamin B2, riboflavin (£3.50 ^g-1), is an excellent alternative to nicotinamide cofactors at 5.4 % of the NAD⁺ cost.

Flow chemistry removes the need for catalyst separation, ensures the reactant experiences a high local concentration of catalyst and only requires heating or pressure at the point of reaction, improving sustainability and safety. Inline analysis can also allow agile decisions to be made during the reaction to optimise conditions or preserve unreacted starting material. Although flow biocatalysis provides an excellent greener synthesis solution; there are still operational barriers such as immobilisation, cofactor recycling and heterogeneous mass transfer preventing widespread implementation in the pharmaceutical industry.

This project explores flavins as a cost-effective alternative cofactor to nicotinamide cofactors for flow biocatalysis, demonstrating a cofactor recycling system with a Ni-Fe hydrogenase in commercially available flow reactors. A focus of the project is to pair the cofactor recycling system with commercial ene-reductases to create an enzyme cascade. Flow biocatalysis is also used with Ni-Fe hydrogenase immobilised on carbon to investigate pharmaceutically relevant nitro-reductions in synthesis.

Overall, this work involves building novel flow systems, implementing existing batch biocatalytic reactions in flow and integrating flavin cofactor recycling, powered by H₂ for selective and sustainable biocatalysis.

Authors: Helin, S

• DOI: 10.5287/ora-mzdd48jdy
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: inline analysis; UV-vis; GC; hydrogen; ene-reductase; process; redox; hydrogenase; flow; biocatalysis; sustainable
• Subjects: Process control; Enzyme immobilisation; Chemistry, Inorganic; Multiphase flow; Inline analysis; Biocatalysis