In Situ Cofactor Regeneration Using NAD(P)H Oxidase: Enzyme Stability in a Bubble Column

NAD(P)H and NAD(P) are important cofactors and currently receiving great interest in biocatalytic synthesis (e.g. the resolution of chiral alcohols and the synthesis of keto chemicals with high selectivity), where their regeneration is necessary in order to ensure the economic sustainability of syntheses based on NAD(P)H or NAD(P)(+) dependent enzymes. This paper reports new details on the kinetic stability of a water-forming NAD(P)H oxidase (NOX) for in-situ cofactor regeneration in a bubble column. Two-stage deactivation kinetics of NOX was observed at gas flowrates of 0.25 vvm and 0.50 vvm with half-life of 29 h and 32h, respectively. Single-stage deactivation kinetics occurred at a higher gas flowrate of 0.75 vvm, while the half-life of NOX was longer (40 h) because of the shorter residence time of the gas-liquid interface. Finally, results from SDS-PAGE suggest that there is no dissociation of NOX into its subunits but rather that NOX unfolding (and aggregation) at the interface led to the observed deactivation.