Biocatalytically Active and Stable Cross‐Linked Enzyme Crystals of Halohydrin Dehalogenase HheG by Protein Engineering

Abstract A major drawback for practical application of halohydrin dehalogenase HheG in biocatalysis is its rather low thermal stability and organic solvent tolerance. We therefore pursued a stabilization via immobilization as cross‐linked enzyme crystals. Since glutaraldehyde inactivates HheG, we introduced cysteine residue the crystal interface, which enabled thiol‐specific cross‐linking at well‐defined sites. Variant D114C displayed improved crystallizability yielded stable catalytically active CLECs using bis‐maleimidoethane cross‐linker. Effective predefined site could be confirmed CLEC structure. Compared to soluble enzyme, significantly activity higher temperatures, lower pH values presence water‐miscible solvents, their reuse over 21 days azidolysis cyclohexene oxide.