In biotechnological processes, the use of immobilized enzymes in the form of cross-linked protein crystals (CLECs) offers many advantages, such as high volume-specific catalytic activity and higher resistance to chemical and enzymatic attack. However, a prerequisite for processability and reusability of the immobilizates, in addition to maintaining the enzymatic activity of the protein crystals, is their mechanical stability. Both mechanical stability and enzyme activity are closely related to the crystal structure and thus, to the amino acid sequence of the proteins on the one hand, and to the selected cross-linking reagent and degree of cross-linking on the other. Therefore, the aim of this work was to elucidate relationships between enzyme structure and the resulting application-relevant properties of cross-linked protein crystals. Since the structure formation of CLECS is influenced by the individual process steps of the entire manufacturing process, starting with genetic modification, production and purification of the enzymes, protein crystal generation and characterization, and finally formulation, this must also be included in the considerations as a whole. As a basis for the investigations, amino acids were exchanged on the surface of the folded wild type enzyme structure by means of various protein engineering methods in order to incorporate new potential cross-linking sites within the subsequently produced protein crystals and thus, improve the mechanical crystal properties. The industrially relevant model protein used for this purpose, halohydrin dehalogenase, was genetically modified, produced and characterized as part of a collaboration at the Institute of Biochemistry at the Technical University of Braunschweig. The focus of this work was the development of methods for protein crystallization, cross-linking and subsequent mechanical characterization. Based on the influence of genetic mutation as well as selected formulation parameters and the resulting structural protein properties, a statistical model was derived to elucidate the structure-property relationship of CLECs. The basis of the statistical model is the three-dimensional crystal structure with all relevant amino acid residue pairs that can form possible cross-linking bridges with the selected cross-linking reagent within the specific length of the linker. The fraction of all possible cross-linking bridges within the protein crystal structure in the respective loading direction were summed up to describe the mechanical behavior of the CLECs. This allowed to reproduce the correlations, such as the anisotropic crystal behavior or the influence of the linker or mutation on the mechanical behavior. To validate the validity of the statistical model, mechanical properties of native crystals and the CLECs, such as hardness, Young’s modulus, and elasto-plastic deformation work on different size scales were investigated using atomic force microscopy and nanoindentation. A particular challenge was the establishment of methods for reliable, statistically validated mechanical characterization of bioparticles in low liquid volume. The results of the present work provide an understanding of the underlying interactions, relationships and limitations between the structure and properties of cross-linked protein crystals and can therefore be used as a fundamental building block for the production of tailored CLECs.weiterlesen