Enantiopure sulfoxides are prevalent in drugs and are useful chiral auxiliaries in organic synthesis. The biocatalytic enantioselective oxidation of prochiral sulfides is a direct and economical approach for the synthesis of optically pure sulfoxides. The selection of suitable biocatalysts requires rapid and reliable high-throughput screening methods. Here we present four different methods for detecting sulfoxides produced via whole-cell biocatalysis, three of which were exploited for high-throughput screening. Fluorescence detection based on the acid activation of omeprazole was utilized for high-throughput screening of mutant libraries of toluene monooxygenases, but no active variants have been discovered yet. The second method is based on the reduction of sulfoxides to sulfides, with the coupled release and measurement of iodine. The availability of solvent-resistant microtiter plates enabled us to modify the method to a high-throughput format. The third method, selective inhibition of horse liver alcohol dehydrogenase, was used to rapidly screen highly active and/or enantioselective variants at position V106 of toluene ortho-monooxygenase in a saturation mutagenesis library, using methyl-p-tolyl sulfide as the substrate. A success rate of 89% (i.e., 11% false positives) was obtained, and two new mutants were selected. The fourth method is based on the colorimetric detection of adrenochrome, a back-titration procedure which measures the concentration of the periodate-sensitive sulfide. Due to low sensitivity during whole-cell screening, this method was found to be useful only for determining the presence or absence of sulfoxide in the reaction. The methods described in the present work are simple and inexpensive and do not require special equipment.The growing demand for green catalytic processes has increased the utilization of enzymes as industrial biocatalysts for the synthesis of fine chemicals (6,19,20). As a consequence, there is a continuous search for novel or improved biocatalysts. In order to find an appropriate candidate for a process, various sources of enzymes must be screened for activity (23). Therefore, a sensitive, reproducible, accurate, and simple highthroughput screening method is a key prerequisite for the development of biocatalytic processes on an industrial scale (32, 39).Screening systems are divided into three different classes. The first class contains assays applicable to testing growing or resting microbial colonies for enzymatic activity directly on agar plates (23), for example, detection of epoxide hydrolase activity on butane oxide by use of safranin O. Oxidation of the 1,2-diol product by Escherichia coli modified the membrane potential and led to accumulation of the red dye in the colonies producing active enzyme (34). In another study, the spontaneous oxidation of substituted catechols to brown-red quinones was used to screen random libraries of whole cells expressing toluene monooxygenases (TMOs) for regioselective oxidation of substituted phenols (12, 30). The positive cl...
