Fundamentals and applications of a nanoconfined electrochemical reactor for rapid biosynthesis

This thesis presents novel electrochemically driven enzyme-catalysed strategies which focus on fundamental studies and application by exploiting an established electrochemical nicotinamide cofactor recycling technology, the Electrochemical Leaf, taking it into new directions.
Inspired by nanoconfinement in Nature, in the Electrochemical Leaf, enzyme cascades are entrapped in high concentrations within the nanopores of an indium tin oxide electrode where they are driven and controlled with their rate being monitored in real time as electrical current. The key concept exploits the photosynthetic enzyme, ferredoxin-NADP+ reductase (FNR) which rapidly interconverts electrons (supplied from a potentiostat) and hydride – stored on the nicotinamide cofactor, NADP(H). This activity is coupled to a second NADP(H)-dependent enzyme which recycles the cofactor back to FNR during its catalytic cycle to produce a desired product.
In this work, the ‘Leaf’ has been exploited to develop new ‘green’ strategies for redox biocatalysis e.g. synthesis of an amine or an alcohol) by using either hydrogen to drive reductions, or O2 in the air to drive oxidations, in each case linking the ‘Leaf’ to a platinised carbon electrode in a fuel cell set-up. The catalytic rate was continuously monitored through the current that flows between the two electrodes. Capitalising on the nanoconfinement and enantioselective nature of enzymes, high total turnover numbers were achieved and enantiomerically pure compounds were obtained.
Hypotheses were proposed by conducting systematic investigations into the thermodynamics, kinetics, stability and enantioselectivity of the enzyme nanoreactors that are now emerging to be of considerable fundamental and practical significance. Three effects were found to be important: (i) the order of introducing FNR and E2 into the nanopores can lead to a difference in enzyme-limitation and ultimately rate determination; (ii) potential switch experiments were designed and used to relieve to product inhibition of the downstream E2 enzyme. The elegant convergence of timecourses when using (S)- and (R)-alcohols separately suggests that a de-racemiser or racemiser might be operated using one electrode; (iii) the nanoconfined environment of the electrode led to a surprising result in which, unlike behaviour in dilute solution, the highly concentrated and nanoconfined enzyme system strongly favours the thermodynamic outcome, racemization. These reversible reactions occur rapidly and independently of FNR, which directly results in an acceleration of homogeneous racemisation, counteracting the enantioselective nature of E2.
Building on these results, a unique two-stage electrochemically driven deracemisation technology scheme using two enantio-complementary enantioselective alcohol dehydrogenase enzymes was proposed and successfully implemented. Enantioselective oxidation and reduction reactions were conducted sequentially on a racemic mixture of enantiopure secondary alcohols, leading to >90\% enantiomeric excess in both (R) and (S).

Keywords: Biocatalysis
Deracemisation
Fuel Cell
Nanoconfined Enzymes
Electrochemical Leaf
Bioelectrochemistry
Advisor: Armstrong, F