Electro enzymatic NADPH regeneration using the thioredoxin-based reaction cascade

NADPH-dependent oxidoreductases are central to biocatalysis and industrial biotransformations, including alcohol dehydrogenases, imine reductases, monooxygenases and ene-reductases. However, their application is often limited by the requirement of an efficient NADPH regeneration system. While NADH can be efficiently be regenerated by formate dehydrogenase on industrial scale, NADPH regeneration systems suffer from lower activities, sacrificial substrates and increased waste stream formation due to side products. The TR/Trx1 cascade from Thermus thermophilus enables in situ recycling of NADPH and is highly stable under alkaline conditions, elevated temperatures, and organic solvents, however produces oxidised dithiothreitol as an unavoidable side-product. To address this limitation an electrochemical approach was chosen to directly reduce NADP + to NADPH in an electro enzymatic setup. In this work, we present the characterisation and application of a TR/Trx1-based electroenzymatic NADPH regeneration system immobilised on carbon felt electrodes. The enzymes were embedded in an agarose- or alginate-based immobilisation matrix, enabling operation in a bio electrochemical cuvette setup. An e-cuvette setup is developed for spectroelectrochemical measurements in a three-electrode system. NADPH regeneration was detected at only -450 mV vs. Ag/AgCl. A proof-of-concept reaction demonstrated functional coupling of electroenzymatic NADPH synthesis and downstream biocatalysis with an NADPH-dependent alcohol dehydrogenase.