Electro-enzymatic viologen-mediated substrate reduction using pentaerythritol tetranitrate reductase and a parallel, segmented fluid flow system

Many redox enzymes require expensive reduced cofactors like NAD(P)H which need to be recycled during catalysis, presenting a major cost and technical barrier to industrial exploitation. An electrochemical biphasic microfluidic setup is presented here, in which these cofactors are replaced by a mediator (methyl viologen) that acts by feeding electrons into the active site of the enzyme pentaerythritol tetranitrate reductase (PETNR). In this microfluidic recirculation setup, both enzyme and mediator remain in the reactor for reuse, allowing easy product recovery. System optimisation studies were performed using 2-cyclohexen-1-one as a model substrate prior to the investigation of a variety of different substrates whose reduction rates were determined to be 15-70% of those obtained when NADPH was used as sole electron donor. Additional data obtained with a thermophilic 'ene' reductase (TOYE) support the potential universality of this device for possible industrial applications.