Next, we assessed the thermostability of HheG, HheG2 and HheG3 under operational conditions, which typically require the application of organic cosolvents to accommodate hydrophobic epoxide substrates. Since even moderate concentrations of organic cosolvents are known to affect the stability and activity of HHDHs, we opted to use iPrOH, which was tolerated best by HheG in previous studies. Differential scanning fluorimetry of the three enzymes incubated in Tris buffer at pH 7 with 3% (v/v) glycerol revealed that different concentrations of iPrOH drastically lowered the apparent melting points (𝑇 𝑚 ) of all three enzymes (Figure ). Despite its favorable catalytic performance in previous studies, HheG2 displayed 𝑇 𝑚 values of only 29-25 °C, which translated to apparent Gibbs free energies of unfolding (Δ𝐺 𝑢 0 ) at 25 °C of 4.6 (with 0% iPrOH) to 1.6 kJ mol -1 (with 4% iPrOH). In contrast, HheG and HheG3 exhibited much higher 𝑇 𝑚 and Δ𝐺 𝑢 0 values (Figure ), with HheG3 almost performing on par with the thermostabilized variants T123G and T123W of HheG reported recently .