Characterization of an Extradiol Dioxygenase Using Integrating Cavity Spectrophotometry

The development of synthetically-useful biocatalysts requires characterizing the behavior of an enzyme under conditions amenable to preparative-scale reactions. Whole cells harboring the catalyst of interest are often used in such reactions, as protein purification is laborious and expensive. However, monitoring reaction rates when using whole cells is challenging, as cellular debris precludes the use of a continuous assay. Herein, we describe an approach to continuous monitoring of whole cell reactions that is enabled by the use of an integrating cavity spectrophotometer. This approach was used to directly profile the kinetic behavior of a previously uncharacterized extradiol dioxygenase DfdB from Rhodococcus sp. YK2. Data obtained from these experiments were compared with traditional in vitro assays. In both whole cell and purified enzyme kinetic assays, similar enzymatic behaviors were observed, including substrate inhibition. Measured K_M values and observed trends in catalytic efficiency were comparable for both types of enzyme preparation. Information provided by this analysis was leveraged to optimize preparative-scale whole cell reactions with DfdB, enabling the synthesis of two DfdB products in high yield. Importantly, this work showcases the potential for ICS-based methods to rapidly evaluate kinetic trends on analytical scale and thereby enables efficient reaction optimization for preparative-scale synthesis.