Thermostability. Thermodynamic stability of all variants was assessed by nano-differential scanning fluorimetry (Fig. , Table ). The apparent melting temperatures for the variants were in the range of 45°C-60°C. AncDhaA13, AncDhaA14, AncDhaA16 and AncDhaA20 surpassed Template_2 in terms of apparent melting temperature. The highest ΔTm of 9°C was measured for AncDhaA20. Protein aggregation was observed for the variants AncDhaA1 and AncDhaA20, showing the onset at 45.5°C and 48.5°C, respectively (Fig. ). Temperature profiles. We then proceeded to measure temperature profiles (Fig. ). Most variants showed the Tmax (temperature at which maximum activity was detected) of 40°C, which is in agreement with previously determined temperature profiles for DhaA . Notably, AncDhaA13 showed Tmax of 50°C, which aligns with its increased thermostability. The temperature profiles for AncDhaA11 and AncDhaA16 were not obtained, as the activities were below the detection limit. Due to compromised activity and folding, both variants were excluded from the subsequent substrate specificity profiling. Substrate specificity. The temperature of 35°C was selected for the subsequent specificity characterization for being below the onset of denaturation (Table ) for most of the variants and close to their Tmax values. To explore the obtained substrate specificities in the context of the haloalkane dehalogenase family, the principal component analysis (PCA) has conducted by augmenting the previously used dataset comprising substrate specificities for 32 wild-type dehalogenases with the newly obtained data. The PCA of raw data showed that the highest overall activity (Fig. ) was observed for AncDhaA20, surpassing both the Template_2 and DbjA. The overall activity was retained for AncDhaA10, while for the other variants, it was substantially decreased (Fig. ). The analysis of log-transformed activity data (Fig. ) further showed that AncDhaA1, AncDhaA10, AncDhaA13, and AncDhaA14 differed only very slightly from templates in their substrate preferences. The profiles of AncDhaA18 and AncDhaA20 resembled more closely the specificity profile of DbjA, which is not unexpected in the case of AncDhaA20 due to its high sequence similarity. AncDhaA16 differed significantly from all other variants due to the low number of converted substrates (10 out of 27), which is a unique property for haloalkane dehalogenases known for their broad substrate specificity.