Enantiopure sulfoxides can be prepared via the asymmetric oxidation of sulfides using sulfide monooxygenases. Five different organic-aqueous biphasic systems were investigated for their abilities to increase the bio-oxidation efficiency of a water-insoluble sulfide (phenyl methyl sulfide, PMS). Isooctane was found to be the most suitable organic phase solvent. In this isooctane-aqueous system, the oxidation reaction was not sensitive to pH, giving high yields over the entire range of the tested pH values (5.0-11.0). In contrast, cell concentration significantly affected the reaction rate. (S)-Phenyl methyl sulfoxide ((S)-PMSO) was formed at a higher concentration and a greater enantiomeric excess (18.3 mM and >99.0% ee(S)) in this isooctane-aqueous biphasic system than in an aqueous single-phase system (2.17 mM and 59.9% ee(S)). The asymmetric oxidation of 150 mM PMS in the isooctane phase was successfully carried out in a mechanically stirred bioreactor (2.4 L) and resulted in a 49.0% yield of (S)-PMSO with enantiomeric excess greater than 99.0%. (C) 2010 Elsevier Ltd. All rights reserved.