The Depth-Dependent Mechanical Behavior of Anisotropic Native and Cross-Linked HheG Enzyme Crystals

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.