Methods for the detection and analysis of dioxygenase catalyzed dihydroxylation in mutant derived libraries

Rieske non-heme iron dioxygenases (ROs) are promising candidates to perform dihydroxylation reactions, since they are capable to incorporate both atoms of molecular oxygen into vicinal non-activated CH bonds, endowing valuable products for pharmaceutical and chemical applications. ROs harbor attractive features such as, striking activity in combination with remarkable regio- and stereo-selectivity, wide reaction spectrum, and broad substrate scope. In order to identify, characterize, and enhance targeted features of dioxygenases and related oxygen dependent enzymes via enzyme engineering and evolution approaches, proper screening and analytical methods are essential to detect and to analyze the expected dihydroxylation activity. This chapter presents different methodologies suitable for the study of dihydroxylation reactions. Detailed descriptions of our established analytical protocols for both gas and liquid chromatography, as well as a colorimetric assay to detect dioxygenase activity are provided. In addition, a novel and reliable system for real-time detection of oxygen consumption, in vivo, is reported.