The impact of agitation on protein aggregation is often misattributed to shear stress rather than related phenomena such as cavitation and gas entrainment from the surface. 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 deliberately when sparging gas into the system or by accidental air entrainment from the overhead space due to mechanical stirring. This was further proven by experiments using the Bio Thrust membrane module, which enabled bubble‐free aeration thus, confirming that exposure to the gas‐liquid interface is the leading cause of deactivation.

Enzymes have several excellent catalytic features, and the last few years have seen a revolution in biocatalysis, which has grown from using one enzyme to using multiple enzymes in cascade reactions, where the product of one enzyme reaction is the substrate for the subsequent one. However, enzyme stability remains an issue despite the many benefits of using enzymes in a catalytic system. When enzymes are exposed to harsh process conditions, deactivation occurs, which changes the activity of the enzyme, leading to an increase in reaction time to achieve a given conversion. Immobilization is a well-known strategy to improve many enzyme properties, if the immobilization is properly designed and controlled. Enzyme co-immobilization is a further step in the complexity of preparing a biocatalyst, whereby two or more enzymes are immobilized on the same particle or support. One crucial problem when designing and using co-immobilized enzymes is the possibility of using enzymes with very different stabilities. This paper discusses different scenarios using two co-immobilized enzymes of the same or differing stability. The effect on operational performance is shown via simple simulations using Michaelis–Menten equations to describe kinetics integrated with a deactivation term. Finally, some strategies for overcoming some of these problems are discussed.