To determine specific activities of HheG and its variants, conversions of 20 mM cyclohexene oxide (1) with 40 mM sodium azide were investigated in 1.5 ml reaction volumes in 50 mM Tris•SO4, pH 7.0 at 22°C (900 rpm in Eppendorf ThermoMixer C). After starting reactions by addition of 50 µg enzyme, samples were taken after 1, 3, 5, 10, 15, 30, and 60 min and extracted with an equal volume of tertbutyl methyl ether (TBME) containing 0.1% (v/v) n-dodecane. Organic phases were dried over MgSO4 and samples were analyzed by achiral GC (Table ). When measuring the conversion of CLECs and comparison to soluble enzyme, reactions were performed similarly but this time in 1 ml reaction volumes with 100 µg biocatalyst. To evaluate enantioselectivities, product enantiomeric excesses were obtained from 4 h samples by chiral GC described elsewhere. To assess activities in different organic co-solvents, reactions contained 25% (v/v) of co-solvents such as ethanol, methanol, 2-propanol, acetonitrile, dimethylsulfoxide, dimethylformamide. Sampling of duplicate reactions was performed after 4 and 24 h and analyzed by achiral GC.