Interestingly, the pH profile of the CLECs broadened compared to soluble enzyme, specifically with respect to performance at lower pH values, while the pH optimum stayed at pH=6 (Figure ). This pH optimum is in line with the catalytic mechanism of HHDHs for epoxide ring opening, with Tyr165 of the catalytic triad donating a proton to the oxyanion generated during epoxide ring opening. While acidic pH will facilitate reprotonation of Tyr165 for the next catalytic cycle, the enzyme is inactivated at low pH values (Figure ). The latter is accompanied by a reduced enzyme stability at acidic pH, which was partially mitigated by cross-linking. In line with these results, CLECs exhibited also significantly higher Tm values at lower pH compared to soluble enzyme (Figure ). Previous literature on cross-linked enzyme crystals of Candida rugosa lipase and Pseudomonas cepacia lipase, as well as CLECs of the protease subtilisin reported improved activity and stability of respective CLECs in organic solvents compared to souble enzymes. Organic co-solvents are usually required to increase substrate solubility in HHDH-catalyzed reactions. Previously, however, significantly reduced activity/stability of HheG in the presence of different water-miscible organic solvents has been observed. Hence, we also investigated the activity of HheG D114C CLECs in the presence of 25% (v/v) of various co-solvents. In comparison to soluble enzyme, D114C CLECs were still active in the presence of 25% (v/v) DMF, methanol, ethanol and isopropanol, whereas soluble enzyme seemed to be fully inactivated at this solvent concentration (Figure ). In contrast, the achieved conversion of soluble HheG D114C in the presence of 25% (v/v) DMSO for the azidolysis of 1 was similar to the respective conversion obtained with the CLECs, while for both enzyme preparations no activity could be observed with 25% (v/v) acetonitrile. Moreover, melting temperatures of HheG D114C CLECs and soluble enzyme in the presence of 10% (v/v) co-solvents have been determined (Table ), which clearly indicate that acetonitrile has the highest destabilizing effect on both enzyme preparations. In contrast, the influence of DMSO on D114C stability (soluble and CLEC form) seems only marginal. Both results are in line with obtained conversion data (Figure ). According to Tm measurements, D114C