Cofactor Regeneration of NAD⁺from NADH: Novel Water‐Forming NADH Oxidases

Abstract

Dehydrogenases with their superb enantioselectivity can be employed advantageously to prepare enantiomerically pure alcohols, hydroxy acids, and amino acids. For economic syntheses, however, the co‐substrate of dehydrogenases, the NAD(P)(H) cofactor, has to be regenerated. Whereas the problem of regenerating NADH from NAD+ can be considered solved, the inverse problem of regenerating NAD+ from NADH still awaits a definitive and practical solution. A possible solution is the oxidation of NADH to NAD+ with concomitant reduction of oxygen catalyzed by NADH oxidase (E.C. 1.6.‐.‐) which can reduce O2 either to undesirable H2O2 or to innocuous H2O. We have found and cloned two novel genes from Borrelia burgdorferi and Lactobacillus sanfranciscensis with hitherto only machine‐annotated NADH oxidase function. We have overexpressed the corresponding proteins and could prove the annotated function to be correct. As demonstrated with a more sensitive assay than employed previously, the two novel NADH oxidases reduce O2 to H2O.

Cofactor Recycling Using a Thermostable NADH Oxidase

“…One catalyzes a two-electron reduction of oxygen to give hydrogen peroxide, and the other catalyzes a four electron reduction of oxygen to give water with NADH oxidation. So far, NOXs have been isolated from anaerobic bacteria such as Streptococcus (Matsumoto et al, 1996;Higuchi et al, 1993Higuchi et al, , 1994Ross and Claiborne, 1992;Schmidt et al, 1986), Thermotoga (Yang & Ma, 2005, Clostridium (Kawasaki et al, 2004;Maeda et al, 1992), Eubacterium (Herles et al, 2002), and Lactobacillus (Hummel et al, 2003b;Riebel et al, 2002Riebel et al, , 2003, and from archaea such as…”

Immobilized redox mediators for electrochemical NAD(P)+ regeneration

“…No reagents or by-products of the regeneration/substitution step should interfere with product isolation, and they should be compatible with the rest of the reaction system. In the literature, several enzyme-coupled (Geueke et al 2003;Riebel et al 2002Riebel et al , 2003Seelbach et al 1996) and substrate-coupled (Kroutil et al 2004b;Mertens et al 2003;Schumacher et al 2006;Stampfer et al 2002) cofactor regeneration systems are described. There are some disadvantages using an enzyme-coupled regeneration system: costs for a second enzyme and a co-substrate, interferences during product purification caused by secondary products, and reaction conditions have to be optimized for both enzyme reactions.…”

Novel Developments Employing Redox Enzymes: Old Enzymes in New Clothes

“…Among the two subclasses of these enzymes-two-electron-transfer/H 2 O 2 -forming and four-electron-transfer/H 2 Oforming-the latter have a clear advantage for not producing a highly reactive coproduct. Numerous H 2 O-forming NADH-oxidases were recently identified in various microorganisms, such as Pyrococcus furiosus [95], L. brevis [96], Borrelia burgdorferi , and Lactobacillus sanfranciscensis [97]. The latter is able to utilize not only NADH but also NADPH as substrate with an activity ratio of approximately 3 : 1 [97].…”

“…Numerous H 2 O-forming NADH-oxidases were recently identified in various microorganisms, such as Pyrococcus furiosus [95], L. brevis [96], Borrelia burgdorferi , and Lactobacillus sanfranciscensis [97]. The latter is able to utilize not only NADH but also NADPH as substrate with an activity ratio of approximately 3 : 1 [97]. Water-forming NADH-oxidases from L. brevis and L. sanfranciscensis were employed for the oxidative kinetic resolution of rac-1-phenylethanol (Scheme 11) [98,99].…”