Biocatalytic reactions using purified enzymes (HheG wild type and selected variants) were performed under the same reaction conditions as described for library screening but using 5 to 400 µg mL -1 of purified enzyme. Samples were taken after specific time points (2 h for epoxide 1 + azide; 24 h for epoxide 1 + cyanide, cyanate and nitrite; 10 min for epoxide 3 + azide; 30 min for epoxide 5 + azide), extracted with an equal volume of TBME containing 0.1% dodecane as internal standard and analyzed by achiral and chiral GC (Table ). Time points were selected to minimize chemical background reactions. All reactions were performed in duplicate. E values were calculated according to Chen et al. Reactions to compare soluble HheG wild-type and soluble variant M45F-D114C with CLECs of HheG M45F-D114C were performed in 1 mL 50 mM Tris•SO4 buffer, pH 7.0 containing 20 mM cyclohexene oxide (1), 40 mM azide and 200 µg biocatalyst. Reactions were incubated for 1 h at 22 °C and 900 rpm. Reactions were performed in duplicate, extracted with an equal volume of TBME containing 0.1% dodecane as internal standard and analyzed by achiral and chiral GC (Table ).