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

ABSTRACT

Biocatalytic oxidation is an interesting prospect for the selective synthesis of active pharmaceutical intermediates. Bubbling air or oxygen is considered as an efficient method to increase the gas-liquid interface and thereby enhance oxygen transfer. However, the enzyme is deactivated in this process and needs to be further studied and understood to accelerate the implementation of oxidative biocatalysis in larger production processes. This paper reports data on the stability of NAD(P)H oxidase (NOX) when exposed to different gas-liquid interfaces introduced by N2 (0% oxygen), air (21% oxygen), and O2 (100% oxygen) in a bubble column. A pH increase was observed during gas bubbling, with the highest increase occurring under air bubbling from 6.28 to 7.40 after 60 h at a gas flow rate of 0.15 L min-1. The kinetic stability of NOX was studied under N2, air, and O2 bubbling by measuring the residual activity, the deactivation constants (kd1) were 0.2972, 0.0244, and 0.0346 with the corresponding half-lives of 2.2, 28.6, and 20.2 h, respectively. A decrease in protein concentration of the NOX solution was also observed and was attributed to likely enzyme aggregation at the gas-liquid interface. Most aggregation occurred at the air-water interface and decreased greatly from 100 to 14.16% after 60 h of bubbling air. Furthermore, the effect of the gas-liquid interface and the dissolved gas on the NOX deactivation process was also studied by bubbling N2 and O2 alternately. It was found that the N2-water interface and O2-water interface both had minor effects on the protein concentration decrease compared with the air-water interface, whilst the dissolved N2 in water caused serious deactivation of NOX. This was attributed not only to the NOX unfolding and aggregation at the interface but also to the N2 occupying the oxygen channel of the enzyme and the resultant inaccessibility of dissolved O2 to the active site of NOX. These results shed light on the enzyme deactivation process and might further inspire bioreactor operation and enzyme engineering to improve biocatalyst performance.