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.