Effect of Nitrogen, Air, and Oxygen on the Kinetic Stability of NAD(P)H Oxidase Exposed to a Gas-Liquid Interface

Biocatalytic oxidation is an interesting prospect forthe selectivesynthesis of active pharmaceutical intermediates. Bubbling air oroxygen is considered as an efficient method to increase the gas-liquidinterface and thereby enhance oxygen transfer. However, the enzymeis deactivated in this process and needs to be further studied andunderstood to accelerate the implementation of oxidative biocatalysisin larger production processes. This paper reports data on the stabilityof NAD-(P)H oxidase (NOX) when exposed to different gas-liquidinterfaces introduced by N-2 (0% oxygen), air (21% oxygen),and O-2 (100% oxygen) in a bubble column. A pH increasewas observed during gas bubbling, with the highest increase occurringunder air bubbling from 6.28 to 7.40 after 60 h at a gas flow rateof 0.15 L min(-1). The kinetic stability of NOX wasstudied under N-2, air, and O-2 bubbling by measuringthe residual activity, the deactivation constants (k (d1)) were 0.2972, 0.0244, and 0.0346 with the correspondinghalf-lives of 2.2, 28.6, and 20.2 h, respectively. A decrease in proteinconcentration of the NOX solution was also observed and was attributedto likely enzyme aggregation at the gas-liquid interface. Mostaggregation occurred at the air-water interface and decreasedgreatly from 100 to 14.16% after 60 h of bubbling air. Furthermore,the effect of the gas-liquid interface and the dissolved gason the NOX deactivation process was also studied by bubbling N-2 and O-2 alternately. It was found that the N-2-water interface and O-2-water interfaceboth had minor effects on the protein concentration decrease comparedwith the air-water interface, whilst the dissolved N-2 in water caused serious deactivation of NOX. This was attributednot only to the NOX unfolding and aggregation at the interface butalso to the N-2 occupying the oxygen channel of the enzymeand the resultant inaccessibility of dissolved O-2 to theactive site of NOX. These results shed light on the enzyme deactivationprocess and might further inspire bioreactor operation and enzymeengineering to improve biocatalyst performance.