The effects of various reaction parameters regarding activity, stability and apparent enantioselectivity of recombinant epoxide hydrolase (EH) from Aspergillus niger in organic media were investigated using the racemic para-chlorostyrene oxide as a model substrate. Depending on the nature of the organic solvent, EH responded differently to variations in initial a(w) and no correlation between the activity or enantiomeric ratio (E) and the log P of solvent could be found. The most appropriate solvent medium and initial a(w) value was heptane and 0.9, respectively. The agitation speeds, where external substrate diffusional limitations were minimized, were defined. The study of internal diffusional limitations by mixing EH extract with BSA as an "inert" protein revealed the negative direct effect of BSA on the apparent enantioselectivity of EH when its relative concentration was too high. An acidic shift in the "pre-drying pH memory" of EH was observed in the heptane medium as compared to the pH activity profile in aqueous media. The temperature effect on enantioselectivity showed an increase in the apparent E-value with decreasing reaction temperature. From those data, the differential activation parameters Delta Delta H-double dagger and Delta Delta S-double dagger were determined to have the same negative sign and therefore their effects counteracted each other, however the former dominates at ambient temperature. The use of organic medium increased the half-life of EH by 2.0-13.8-fold as compared to aqueous medium at 27-59 degrees C, but it decreased the catalytic efficiency which was mainly due to an increase in the K-m(app) value. (c) 2005 Elsevier Inc. All rights reserved.