NAD + Cofactor Regeneration by TMB‐Mediated Horseradish‐Peroxidase‐Catalyzed Reactions

The regeneration of nicotinamide adenine dinucleotide (NAD + ) has attracted substantial interest in the enzymatic chemical transformations. In this work, we report the use of a traditional horseradish peroxidase (HRP)/H 2 O 2 system for the catalytic transformation of reduced nicotinamide adenine dinucleotide (NADH) into NAD + , in the presence of a common HRP substrate, 3,3′,5,5′‐tetramethylbenzidine (TMB), as an electron mediator. The effect of H 2 O 2 , HRP or TMB concentration on the oxidization rate of NADH was systematically investigated. The results show that a small amount of HRP and TMB resulted in high efficient oxidization of NADH into NAD + , through the switching between the oxidized and reduced states of TMB. The enzymatic system also exhibited pH‐dependent catalytic properties, so that a XOR logic gate was constructed with H 2 O 2 and H + as the gate inputs, and the generated TMB charge transfer complex as the output signals. The addition of glucose dehydrogenase (GDH)/glucose allows the cycling between the NADH and NAD + and the coupled activation of HRP and GDH. This work provides a strategy for efficient regeneration of the NAD + cofactor and enzyme‐driven transformations.