A Versatile High Throughput Screen for Dioxygenase Activity Using Solid-Phase Digital Imaging

We have developed a solid-phase, high throughput (10,000 clones/day) screen for dioxygenase activity. The cis-di- hydrodiol product of dioxygenase bioconversion is converted to a phenol by acidification or to a catechol by reaction with cis-dihydrodiol dehydrogenase. Gibbs reagent reacts quickly with these oxygenated aromatics to yield colored products that are quantifiable using a microplate reader or by digital imaging and image analysis. The method is reproducible and quantitative at product concentrations of only 30,uM, with essentially no background from media components. This method is an effective general screen for aromatic oxidation and should be a useful tool for the discovery and directed evolution of oxygenases.