Recently, we reported the efficient immobilization of HheG (especially variant D114C) in the form of cross-linked enzyme crystals (CLECs), which yielded an HheG preparation displaying high process stability as well as easy operability in different chemical reactor systems (batch and continuous flow). Thus, we herein aimed to combine mutation M45F with D114C with the goal to prepare enantioselective HheG CLECs. The resulting double mutant HheG M45F-D114C could be obtained in high yield (231 mg L -1 in comparison to 140 mg L -1 for HheG M45F) and displayed a similar specific activity in the azidolysis of 1 (18.3 U mg -1 ) as HheG M45F (16.5 U mg -1 ). Crystallization of this double mutant using the optimized crystallization conditions of HheG D114C led to hexagonal-shaped crystals after 24 h (Figure ). Cross-linking with BMOE yielded stable HheG M45F-D114C CLECs that achieved 92% conversion in the ring opening of 20 mM 1 with azide already after 1 h and a high product enantiomeric excess (eeP) of 95% (in comparison to 82% conversion and 49%eeP using wildtype HheG; Figure ). Therefore, respective CLECs were subsequently applied in a semipreparative reaction using 50 mM cyclohexene oxide (1) and 2 eq azide in 10 mL scale. After 2 h, full conversion was reached and (1S,2S)-2-azidocyclohexan-1-ol (2a) was obtained in 74% isolated yield (52 mg) with an eeP of 96%. This demonstrates that HheG variant M45F can be stabilized successfully via CLEC formation yielding a stable and highly enantioselective HheG preparation for future application in repetitive batch and continous flow.