A cell-based adrenaline assay for automated high-throughput activity screening of epoxide hydrolases.
Herein we describe the development of a cell-based pretest for high-throughput screening of the activity of epoxide hydrolases (EHs) by using the chemistry of the known adrenaline test. It is a practical tool for the directed evolution of EHs as catalysts in synthetic organic chemistry and/or for assaying culture strains. The pretest can be applied to a wide variety of epoxide substrates, thereby circumventing the need for cell rupture and enzyme purification. In the present study we focus on parameters to improve assay specificity and sensitivity in order to obtain a robust and reproducible cell-based test. The introduction of threshold values allows a clear and reliable hit identification which is a prerequisite for industrial or academic prescreens probing thousands of samples in strain collections or in directed evolution libraries.
Kahakeaw
Daniel
D
Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470 Mülheim/Ruhr, Germany.
Reetz
Manfred T
MT
D004837:Epinephrine / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D004851:Epoxide Hydrolases / Q000378:metabolism*; D015394:Molecular Structure; D012680:Sensitivity and Specificity
D004851:Epoxide Hydrolases; D004837:Epinephrine