Relationship between cross-linking reaction time and anisotropic mechanical behavior of enzyme crystals

The application of protein particles as useful biocatalysts in the chemical or pharmaceutical industry is restricted due to their fragility and sensitivity to changes of environmental conditions. The cross-linking method for enzyme crystal immobilization provides an enhancement of crystal stability. However, cross-linking conditions and the linker type strongly affect the mechanical properties of the cross-linked protein particles. Our work examines the cross-linking reaction time’s influence on mechanical properties of cross-linked HheG crystals. An atomic force microscopy-based nanoindentation is used for mechanical measurements. Our results show that cross-linking of amino acid residues takes place inhomogeneously within the crystal lattice. Hardness and Young’s modulus of prismatic crystal faces remain constant above 24 h of cross-linking. At this time, however, basal crystal faces show around 30% higher mechanical stability compared to prismatic faces. The detection of anisotropies between prismatic ...