Correlation of Structure and Properties of Enzyme Crystals in Consideration of Downstream Processing and Formulation

The immobilization of enzymes is standardly applied to enhance enzyme stability and to enable their reuse over multiple reaction cycles. In this context, cross-linked enzyme crystals (CLECs) exhibit a high volumetric catalytic activity along with significant chemical and thermal stability, making them highly attractive for use as immobilized biocatalysts. However, during their application, these biocatalysts are exposed to mechanical stresses such as stirring or filtration. Therefore, the mechanical properties of CLECs are critical for their usability. These properties stem from two primary sources: the three-dimensional protein structure, including the amino acid sequence, and the chemical cross-linking of enzyme molecules within the crystal, which is essential to prevent the crystals from dissolving. This article summarizes the results of our investigations into the mechanical properties of CLECs, that have been produced from two model enzymes, penicillin G acylase (PGA) and halohydrin dehalogenase HheG. It explores the influence of various enzyme engineering strategies that lead to changes in enzyme structure and crystal contact formation, as well as the impact of cross-linking parameters on CLEC behavior. Finally, a computational model based on the crystal structure of HheG was developed to describe the mechanical relationships investigated, such as anisotropic crystal behavior and the influence of linkers or mutations on micromechanical properties, which may be useful for a tailor-made production of CLECs.