Abstract
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.
Highlights
Nicotinamide adenine dinucleotide (NAD+; the oxidized form of NAD) is an attractive bio-based oxidizing agent in synthesis, its regeneration from NADH requires stoichiometric amounts of reagents or the use of organometallic catalysts
We present a NAD+-regeneration strategy that relies on three enzymes co-encapsulated into semipermeable silica nanoreactors (SiNRs) prepared via a mild fluoride-catalyzed sol–gel chemistry in the microemulsion
We realized the continuous production of NAD+ by the combination of lactate dehydrogenase (LDH), lactate oxidase (LOX), and catalase (CAT) encapsulated in a substratepermeable silica nanoreactor (Figure 1 a)
Summary
Nicotinamide adenine dinucleotide (NAD+; the oxidized form of NAD) is an attractive bio-based oxidizing agent in synthesis, its regeneration from NADH (the reduced form of NAD) requires stoichiometric amounts of reagents or the use of organometallic catalysts. Angewandte Chemie International Edition published by Wiley-VCH GmbH We demonstrate the continuous production of NAD+ by an artificial enzyme-combination inside a substrate-permeable and robust silica nanoreactor by water and oxygen as the necessary stoichiometric reagents with a minimal amount of pyruvate, without any additional substrates and without generating byproducts. Encapsulation or immobilization of the enzymes is essential to increase stability and ease purification It is accompanied by denaturation and reduced enzyme activity, for example, acidic/basic pH, non-selective chemistry, or organic solvents.[9].
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