Enzyme‐Loaded Nanoreactors Enable the Continuous Regeneration of Nicotinamide Adenine Dinucleotide in Artificial Metabolisms

Nicotinamide adenine dinucleotide (NAD) is an essential coenzyme for numerous biocatalytic pathways. While in nature, NAD + is continuously regenerated from NADH by enzymes, all synthetic NAD + regeneration strategies require a continuous supply of expensive reagents and generate byproducts, making these strategies unattractive. In contrast, we present an artificial enzyme combination that produces NAD + from oxygen and water continuously; no additional organic substrates are required once a minimal amount pyruvate is supplied. Three enzymes, i.e., LDH, LOX, and CAT, are covalently encapsulated into a substrate‐permeable silica nanoreactor by a mild fluoride‐catalyzed sol–gel process. The enzymes retain their activity inside of the nanoreactors and are protected against proteolysis and heat. We successfully used NAD + from the nanoreactors for the continuous production of NAD + i) to sense glucose in artificial glucose metabolism, and ii) to reduce the non‐oxygen binding methemoglobin to oxygen‐binding hemoglobin. This latter conversion might be used for the treatment of Methemoglobinemia . We believe that this versatile tool will allow the design of artificial NAD + ‐dependent metabolisms or NAD + ‐mediated redox‐reactions.