Tools to investigate oxygen-related challenges with flavin-dependent enzymes

Enzymes have multiple applications in medicine but during the past decades interest in the application of enzymes as (bio)catalysts to produce a wide range of valuable molecules in various industries has increased. Many chemical compounds (from pharmaceuticals to bulk commodities) can be produced by a series of enzymatically-catalysed chemical steps, and in many cases one of these steps is an oxidation. The use of molecular oxygen as an oxidising agent in biocatalytic processes is a double-edged approach. From one side, the oxygen is supplied to the reactor in the form of air bubbling, which is cheap, highly available and non-toxic. From the other side, bubbling air into the reaction media creates a gas-liquid interface which adsorbs enzymes and compromises their stability. Moreover, the oxygen is quite insoluble in water, which often results in oxygen-limited reactions. These aspects are the main limiting factors for the stability and kinetics of enzymes that perform oxidative biocatalysis and prevent the reaction from happening at a rate that is high/competitive enough for industrial feasibility. Therefore, we need systems to mimic and understand better these factors to try and mitigate their effects upon scale-up. In this review, we present two complementary systems to study these factors: one apparatus that ensures a constant gas-liquid interface and another one that maintains a constant oxygen partial pressure. Both can provide highly valuable information regarding the maximum rate of reaction and about the deactivation profiles of enzymes in the presence of bubbles.