A broader exploitation of enzymes in organic synthesis can be achieved by increasing their tolerance toward organic solvents. In this study, the stability and activity of Baeyer-Villiger monooxygenases from Thermobifida fusca (PAMO) and Acinetobacter sp. (CHMO) in the presence of water miscible organic solvents were compared. PAMO was more stable than CHMO. The concentration of solvent (v/v) at which it halved its activity (C-50) was 4- to 16-fold higher than that observed for CHMO. For PAMO, the C-50 varied from 16% to 55% of solvent and followed the destabilizing order methanol < ethanol < 1,4-dioxane < acetonitrile < trifluoroethanol. In the case of CHMO, the maximal C-50 was 7% with methanol and even lower with the other solvents. Therefore, methanol was the most tolerated solvent. In the case of PAMO, methanol induced a significant increase of enzyme activity (up to fivefold), which was optimal at 20% (v/v) solvent. Only minor spectral variations were observed with PAMO in 20% methanol, suggesting that the increase of activity observed in this condition is not due to marked conformational changes. Fluorescence and circular dichroism analyses showed that the lower stability of CHMO toward organic solvent correlates with a more pronounced destructive effect on its secondary and tertiary structure. A possible rationale for the higher stability of PAMO could be inferred from inspection of the PAMO and CHMO (two enzymes of similar size) structure, which revealed a higher (up to twofold) number of ionic bridges in PAMO with respect to CHMO. Biotechnol. Bioeng. 2011; 108: 491-499. (C) 2010 Wiley Periodicals, Inc.