Halohydrin dehalogenases (HHDHs) (E.C. 4.5.1.-) are bacterial lyases belonging to the superfamily of short-chain dehydrogenases/reductases. While their natural function is the dehalogenation of vicinal haloalcohols with concomittant epoxide formation, they are also able to catalyze the reverse reaction, i.e. epoxide-ring opening, with a range of different nucleophiles enabling formation of C-C, C-N, C-O and C-S bonds. This has made HHDHs attractive for application in biocatalysis and various examples, e.g. for the synthesis of enantioenriched oxazolidinones, tertiary alcohols, epihalohydrines or spiroepoxyoxindoles, using HHDH-catalysis have been reported in literature. Using a database mining approach based on HHDH-specific sequence motifs, a large number of new halohydrin dehalogenases have been identified in public sequence databases in recent years and several new members have been characterized as well. While most HHDHs display similar substrate scopes, HheG from Ilumatobacter coccineus also accepts sterically more demanding cyclic epoxides as well as acyclic non-terminal epoxide substrates. Additionally, HheG displays α-regioselectivity in the ring-opening of different styrene oxide derivates, whereas other HHDHs exclusively attack the β-carbon of the epoxide ring. Solving the crystal structure of HheG revealed a much broader active site for this enzyme compared to other HHDHs, which explains the acceptance of bulkier substrates. This feature makes HheG very attractive for industrial application, e.g. for the synthesis of pharmaceutical building blocks. A major drawback for application, however, is the enzyme's low stability (apparant melting temperature Tm of only 38 °C) and its low organic solvent tolerance. Based on protein engineering of HheG, variants with amino acid exchanges at position T123 have been obtained displaying up to 14 K higher Tm as well as up to three-fold higher activity, resulting also in a slight increase in organic solvent resistance. To complement our protein engineering efforts with HheG, we herein report an orthogonal strategy to the stabilization of HheG using immobilization. Common enzyme immobilization methods include carrier binding, polymer entrapment, and carrier-free immobilization methods based on cross-linking such as cross-linked enzyme crystals (CLECs) or cross-linked enzyme aggregates (CLEAs). Since wildtype HheG crystallizes readily after a single IMAC purification step, we opted for CLEC generation to obtain an immobilized HheG preparation that would still be active at higher cosolvent concentration, and that could be reused. CLECs of various enzymes have previously been reported to display high stability regarding temperature and pH, also in extreme conditions, and have been applied for biocatalytic reactions in non-conventional reaction media. The most common cross-linker used for CLEC formation is glutaraldehyde, which reacts primarily with ε-amino groups of lysines, but is also able to cross-link arginine and tyrosine side chains. Previous attempts, however, to cross-link HheG crystals with glutaraldehyde via soaking resulted only in inactive CLECs. The high excess of glutaraldehyde during cross-linking likely also resulted in cross-linking of Tyr165 and Arg169 of the catalytic triad of HheG. In contrast, cross-linking of HheG crystals with glutaraldehyde via vapor diffusion yielded CLECs that still displayed activity in the ring opening of cyclohexene oxide (1) with azide (Scheme 1). This approach, however, is impractical in larger scales where protein crystallization is generally performed in stirred vessels. Hence, we herein attempted to use protein engineering of HheG to insert desired residues in crystal contacts that would allow us to use chemoselective cross-linkers for stable CLEC generation without compromising biocatalyst activity.