To further characterize the most beneficial HheG variants observed during screening, respective enzymes were produced on 100 mL scale and purified via immobilized metal ion affinity chromatography (IMAC). Subsequently, their specific activities and selectivities in respective epoxide ring opening reactions, for which they had been identified during screening, as well as their apparent melting temperatures were determined (Table and). Additionally, ring opening of 1 by HheG variants M45F, M45Y and M45W was also investigated using cyanate and nitrite, as HheG was recently shown to accept a broader range of nucleophiles. Table . Characterization data of purified HheG variants in epoxide ring opening reactions using different nucleophiles. Reactions were performed at 22 °C in 50 mM Tris•SO4 buffer, pH 7.0 for the indicated amount of time and analyzed by achiral and chiral GC. All reactions were performed in duplicate.