Exposure Experiments to Test the Kinetic Stability of 5-hydroxymethylfurfural Oxidase (HMFO) in Different Reactor Environments

The impact of agitation on protein aggregation is often misattributed to shear stress rather than related phenomena such as cavitation and entrainment. For some time now, it has been known that shear is unlikely to harm most proteins directly. Rather, interfacial phenomena, particularly those involving dynamic gas-liquid interfaces, are critical contributors to protein damage, which leads to aggregation and compromises stability. This work investigated the kinetic stability of 5-hydroxymethylfurfural oxidase (HMFO; EC: 1.1.3.47) in a 2 L stirred tank reactor. Exposure experiments revealed that the leading cause of enzyme deactivation was exposure to the gas-liquid interface, either produced when sparging gas into the system or by air entrainment from the overhead space due to mechanical stirring. This was further proven by experiments using the BioThrust membrane module, which enabled bubble-free aeration, thus confirming that exposure to the gas-liquid interface is the leading cause of deactivation rather than dissolved oxygen.