Use of image analysis to understand enzyme stability in an aerated stirred reactor

Efficient regeneration of NAD(P)⁺ cofactors is essential for large-scale application of alcohol dehydrogenases due to the high cost and chemical instability of these cofactors. NAD(P)⁺ can be regenerated effectively using NAD(P)H oxidases (NOXs) that require molecular oxygen as a cosubstrate. In large-scale biocatalytic processes, agitation and aeration are needed for sufficient oxygen transfer into the liquid phase, both of which have been shown to significantly increase the rate of enzyme deactivation. As such, the aim of this study was to identify the existence of a correlation between enzyme stability and gas-liquid interfacial area inside the bioreactor. This was done by measuring gas-liquid interfacial areas inside an aerated stirred reactor, using an in situ optical probe, and simultaneously measuring the kinetic stability of NOXs. Following enzyme incubation at various power inputs and gas-phase compositions, the residual activity was assessed and video samples were analyzed through an image processing algorithm. Enzyme deactivation was found to be proportional to an increase in interfacial area up to a certain limit, where power input appears to have a higher impact. Furthermore, the presence of oxygen increased enzyme deactivation rates at low interfacial areas. The areas were validated with defined glass beads and found to be in the range of those in large-scale bioreactors. Finally, a correlation between the enzyme half-life and specific interfacial area was obtained. Therefore, we conclude that the method developed in this contribution can help to predict the behavior of biocatalyst stability under industrially relevant conditions, concerning specific gas-liquid interfacial areas.