Next, all generated mutants were screened for their dehalogenation and epoxide ring-opening activity in 96-well format. To this end, well-established model substrates for both the dehalogenation (dichloropropanol 1f for HheC and chlorocyclohexanol 2f for HheG) and the ring-opening reaction (epichlorohydrin 1 for HheC and cyclohexene oxide 2 for HheG) were used. For fast activity screening, we employed pH-based assays which have previously been reported in literature and make use of either phenol red or bromothymol blue as pH-indicators to detect qualitatively the amount of released (dehalogenation) or consumed free protons (epoxide ring opening) during catalysis (Figure and Table ). For the exchange of the threonine (T7 in HheC and T13 in HheG) and glycine (G14 in HheC and G20 in HheG) in both enzymes only mutants carrying a chemically similar amino acid (serine instead of threonine, alanine instead of glycine) still exhibited detectable activity. In contrast, F12 in HheC displayed a much higher variability (Figure ) with HheC mutants F12G, F12A, F12C, F12S, F12Q, F12H, and F12Y being active in both dehalogenation and epoxide ring opening reactions. Interestingly, this was not the case for position Y18 of HheG. Here, only the exchange of tyrosine by phenylalanine yielded a mutant with significant activity. Those activity data are in full agreement with our results regarding the soluble expression of the generated mutants, with the only exception that not all soluble HheC F12X mutants were indeed also active. For all further tests, active HheC and HheG mutants of motif 1 were produced in larger scale and applied as FPLC-purified proteins (for yields see Table ).