For the stable CLECs, the activity in the conversion of cyclohexene oxide (1) with azide was subsequently examined and compared to soluble enzyme preparations (Figure ). As mentioned previously, CLECs of HheG wild type cross-linked with glutaraldehyde via soaking displayed only negligible residual activity in the range of the chemical background. In contrast, HheG D114C CLECs cross-linked with BMOE via soaking displayed high conversion in the azidolysis of 1, albeit at 4 h reaction time conversion is slightly lower compared to the soluble enzyme (Figure ). The latter, however, might be due to diffusion limitations within the crystal, reducing substrate accessibility and product release. On the other hand, the limited enzyme motion within the CLEC could also explain the observed activity decrease. Literature reports indicated that cross-linking can also have a positive effect on the enantioselectivity of an enzyme. Thus, the enantioselectivity of our CLECs in the conversion of 1 was investigated as well. Interestingly, HheG D114C CLECs formed azidoalcohol 2 with slightly higher product enantiomeric excess than the respective soluble enzyme (56.0% vs 49.9%, respectively). This might be explained by reduced molecular motion of enzyme molecules within the crystal due to cross-linking. Moreover, an 8 K increase in apparent melting temperature (Tm) compared to soluble enzyme was observed for D114C CLECs cross-linked with BMOE (Figure ). This gain in melting temperature was also obtained when using only 0.5 mM BMOE and a cross-linking time of 24 h, as well as with 2 mM BMOE and 3 h cross-linking time (Figure ). This demonstrates the effectiveness of BMOE as crosslinker for HheG D114C crystals. Using higher cross-linker concentrations or longer cross-linking times did not enhance CLEC stability further (Figure ). For comparison, cross-linking of wild-type HheG crystals with glutaraldehyde yielded a Tm increase by 9 K (data not shown).