Kinetic Stability of Oxygen-dependent Enzymes in Scale-down Reactors

Despite enzymes' many advantages, stability remains an issue. While conditions such as temperature, pH, and solvent are well-studied, other conditions, such as aeration and agitation, are often overlooked despite being present in almost all industrial processes. One of the many benefits of using enzymes to catalyze reactions is the ability to alter the enzyme through protein engineering to overcome reaction limitations. However, before protein engineering, it is essential to understand the deactivation mechanism and underlying causes to work toward targeted stability strategies.

In this thesis, a scale-down approach was used to investigate the deactivation trend of oxidases. This becomes a particularly interesting case study when investigating the effect of a gas-liquid interface, as these enzymes require oxygen as a co-substrate. Due to oxygen having a low solubility in water, a continuous supply is required, which is often done by gas-bubbling to the reactor, creating a gas-liquid interface and damaging the enzymes. Even without bubbling, a gas-liquid interface can be present in the system, including situations with an overhead space between the liquid and head plate or mixing, leading to air entrainment. Thus, the gas-liquid interface is present in almost every bioprocess on an industrial and laboratory scale. This thesis consists of case studies investigating the stability study of HMF oxidase and NAD(P)H oxidase, working towards understanding the deactivation caused by industrial representative conditions in several specifically scale-down reactor designs. The driving force of enzyme deactivation, is a protein loss both to the interface, but also in the solution.

This thesis observes a two-stage deactivation trend for both investigated enzymes. Findings throughout the experimental work provide evidence that the two-stage deactivation trend is caused by protein loss to the interface, which disrupts the hydrogen bond in the enzyme's ligand interactions. In the first stage, a rapid protein loss is observed, which stops at the transition time. The protein concentration remains constant for the remaining time of the experiment. Thus, the protein loss is related to the activity loss in the first stage. In the second stage, the protein concentration in solution is constant, but the deactivation rate occurs much faster. The reason for this was found to be the disruption of the hydrogen bonds binding the cofactor (FAD) to the enzyme, which is required for enzyme activity. Removing the interface by operating in a column apparatus with no interface only showed a one-stage deactivation decay beyond the transition time, with no loss of FAD, thus providing the final evidence that the interface causes the two-stage deactivation trend.