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 bis‐maleimidoethane 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.

The generation of cross-linked enzyme crystals is a very attractive method for immobilization of enzymes displaying high crystalizability. However, the commonly used cross-linker glutaraldehyde is not always compatible with enzyme activity. Therefore, we previously reported the engineering of halohydrin dehalogenase HheG from Ilumatobacter coccineus to enable thiol-specific cross-linking during CLEC generation by insertion of cysteine residues in the crystal contact. To broaden the applicability of this approach, herein crystal contact engineering of HheG has been performed to incorporate additional lysine residues as defined cross-linking sites for CLEC generation. Using the primary amine-specific cross-linker dithiobis(succinimidyl propionate) (DSP), CLECs of HheG variant V46K were obtained that displayed a high gain in thermal stability compared to wild-type HheG, while using only a low cross-linker concentration. Moreover, respective V46K CLECs exhibited a 10 K higher reaction temperature optimum as well as significantly improved activity and stability at acidic pH and in the presence of organic co-solvents. Overall, our study demonstrates that lysine-specific cross-linkers can also be used as an alternative to glutaraldehyde for stable CLEC generation of halohydrin dehalogenases, and that cross-linking efficiency is significantly improved upon crystal contact 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 predefined cross-linking sites. Variant HheG D114C displayed improved crystallizability and yielded stable and catalytically active CLECs using bis-maleimidoethane 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.

Enzymes are able to catalyze various specific reactions under mild conditions and can, therefore, be applied in industrial processes. To ensure process profitability, the enzymes must be reusable while ensuring their enzymatic activity. To improve the processability and immobilization of the biocatalyst, the enzymes can be, e.g., crystallized, and the resulting crystals can be cross-linked. These mechanically stable and catalytically active particles are called CLECs (cross-linked enzyme crystals). In this study, the influence of cross-linking on the mechanical and catalytic properties of the halohydrin dehalogenase (HheG) crystals was investigated using the nanoindentation technique. Considering the viscoelastic behavior of protein crystals, a mechanical investigation was performed at different indentation rates. In addition to the hardness, for the first time, depth-dependent fractions of elastic and plastic deformation energies were determined for enzyme crystals. The results showed that the hardness of HheG enzyme crystals are indentation-rate-insensitive and decrease with increases in penetration depth. Our investigation of the fraction of plastic deformation energy indicated anisotropic crystal behavior and higher irreversible deformation for prismatic crystal faces. Due to cross-linking, the fraction of elastic energy of anisotropic crystal faces increased from 8% for basal faces to 68% for prismatic crystal faces. This study demonstrates that mechanically enhanced CLECs have good catalytic activity and are, therefore, suitable for industrial use.

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