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Applied and Environmental Microbiology, July 2009, p. 4711-4719, Vol. 75, No. 14
0099-2240/09/$08.00+0     doi:10.1128/AEM.02674-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Rapid Methods for High-Throughput Detection of Sulfoxides{triangledown}

Janna Shainsky,1 Netta-Lee Derry,1 Yael Leichtmann-Bardoogo,1 Thomas K. Wood,2 and Ayelet Fishman1*

Department of Biotechnology and Food Engineering, Technion-Israel Institute of Technology, Haifa 32000, Israel,1 Departments of Chemical Engineering, Biology, and Civil Engineering, 220 Jack E. Brown Building, Texas A&M University, College Station, Texas 77843-31222

Received 22 November 2008/ Accepted 14 May 2009

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.

* Corresponding author. Mailing address: Department of Biotechnology and Food Engineering, Technion-Israel Institute of Technology, Haifa 32000, Israel. Phone: 972-4-829-5898. Fax: 972-4-829-3399. E-mail: afishman{at}tx.technion.ac.il

{triangledown} Published ahead of print on 22 May 2009.

Applied and Environmental Microbiology, July 2009, p. 4711-4719, Vol. 75, No. 14
0099-2240/09/$08.00+0     doi:10.1128/AEM.02674-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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