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

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