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-dihydrodiol 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 microM, 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.
Joern
J M
JM
California Institute of Technology, Pasadena, CA 91125, USA.
Sakamoto
T
T
Arisawa
A
A
Arnold
F H
FH
D001331:Automation; D002722:Chlorobenzenes / Q000737:chemistry; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D007091:Image Processing, Computer-Assisted; D008956:Models, Chemical; D010088:Oxidoreductases / Q000737:chemistry; D010100:Oxygen / Q000378:metabolism; D010105:Oxygenases / Q000032:analysis* / Q000737:chemistry*; D016133:Polymerase Chain Reaction; D011485:Protein Binding
D002722:Chlorobenzenes; D010088:Oxidoreductases; D010105:Oxygenases; C048338:trans-1,2-dihydrobenzene-1,2-diol dehydrogenase; C031294:chlorobenzene; D010100:Oxygen