Electrified Nanoconfined Biocatalysis with Rapid Cofactor Recycling

Abstract

In living cells, the overall rates of catalytic reaction chains (cascades) are massively enhanced by nanoconfinement of enzymes in tiny enclosed volumes: presented in such a way, interdependent catalysts are highly concentrated, and distances (active site‐to‐active site) across which intermediates and cofactors must diffuse, may be tiny. In a parallel technology exploiting this principle, enzyme cascades are powered, amplified, and monitored in real time as they work in concert, being nanoconfined within the pores of an electrically conductive metal oxide electrode. The technology, nicknamed the electrochemical leaf, mimics chloroplast biosynthesis by exploiting nicotinamide adenine dinucleotide (NADP(H)) recycling catalysed by the flavoenzyme, ferredoxin NADP+ reductase (FNR). Adsorbed on the inner walls of the nanopores, FNR rapidly transfers electrons between the electrode and NADP(H). This activity is coupled to an oxidoreductase enzyme, also nanoconfined within the pores, which recycles the cofactor and selectively synthesises a desired product or senses an analyte. Use of catalyst and cofactor is very efficient. The technology is simple, inexpensive and adaptable, and scalable to both microscopic levels (diagnostics) and macroscopic levels (organic synthesis). Whereas native FNR is specific for NADP(H), the technology can be extended by genetic engineering to include NAD(H) as recycling cofactor.

The power of electrified nanoconfinement for energising, controlling and observing long enzyme cascades

“…The cyclic voltammograms-revealing how the steady-state catalytic velocity of the cascade varies with driving force-were obtained for a mixture of pyruvate, KHCO 3 , NH 4 Cl, NADP + , in the absence and presence of aspartate. They were measured after loading all the enzymes into a nanoporous layer of indium tin oxide (ITO) formed by depositing commercial nanoparticles electrophoretically onto a titanium foil support (area 1.89 cm 2 ITO layer, varying between 1 and 3 µm deep depending on the deposition time (2-10 min), comprises pores and cavities of less than 100 nm in diameter 33,35,36 . This procedure provides a fast, natural way of creating random nanospace (i.e., of ≪1 μm porosity) that can be energized electrochemically via the trapped FNR (E1), which serves as the transducer (Fig.…”

“…The cascade is energized by direct electron transfer between the electrode and the FAD group of E1 (represented as yellow circles in Fig. 1b), which drives rapid and reversible interconversion of NADP + and NADPH and acts here as the tranducer [33][34][35][36][37][38] .…”

Progress in Scaling up and Streamlining a Nanoconfined, Enzyme‐Catalyzed Electrochemical Nicotinamide Recycling System for Biocatalytic Synthesis

“…A rapid electrochemical system that comprises nano-confined components deals with these challenges and also allows the assembly of enzyme cascades that can perform multiple steps in a single reactor. [18][19][20][21][22] In the 'electrochemical leaf', a nanoporous metal oxide electrode is used to entrap at least two enzymes operating in a cascade: one of these is ferredoxin-NADP + reductase (FNR), the small photosynthetic flavoenzyme responsible for channeling light-activated electrons into biosynthesis; [23][24] the second (E2) can be any of hundreds of NADP(H)-dependent dehydrogenases. The concept is depicted in Figure 1A.…”

“…[21] The nanopores lead to a high local concentration of the enzymes and restrict the escape of NADP(H) or (in an extended cascade) intermediates that are needed for the next stage. [21][22] The resulting overall rate and progress of a biocatalytic run are observed directly through the current that flows and the charge that is passed, respectively. The (FNR + E2 + …)@ITO/support material thus forms the basis for an inexpensive, easilyaccessible "plug-in" device able to drive, interactively, a potentially unlimited number of organic reactions depending on the identity of E2 and further enzymes.…”

Bioelectrocatalytic Cofactor Regeneration Coupled to CO2 Fixation in a Redox‐Active Hydrogel for Stereoselective C−C Bond Formation

“…Co‐immobilizing enzymes of a multistep catalytic process locally concentrates the biocatalysts in a small volume. The small distances between the catalysts can provide an advantage by allowing the fast diffusive mass transport of intermediates and cofactors between active sites, leading to higher reaction rates [32] and thus enhanced electrocatalytic properties. [7] Such confinement effect was observed when comparing a system with Ccr co‐immobilized with FNR in the hydrogel, versus Ccr freely diffusing in solution (Figure S13C,D).…”