The Role of Protein Folding in Prenucleation Clusters on the Activity of Enzyme@metal-Organic Frameworks

Enzymes present a promising green chemistry alternative for accelerating chemical reactions in industry. However, many industrial applications require harsh synthetic conditions (i.e. high temperature and/or organic solvents), causing enzymes to denature. Metal-organic frameworks (MOFs) offer a promising method to overcome the structural limitations of potentially revolutionary enzymes through enzymatic immobilization. However, for high activity of immobilized enzymes to be achieved, a comprehensive understanding of the factors influencing enzyme performance is critical. While extensive efforts have demonstrated that final MOF properties such as crystallinity, porosity, and network defects significantly influence enzyme activity, here we propose a separate, but equally impactful, factor in determining enzyme@MOF activity: enzyme folding in prenucleation clusters. To investigate both MOF structural property and enzyme folding factors, two model enzymes, glucose oxidase and catalase, were encapsulated into ZIF-8. The biocomposite formation and prenucleation clusters were examined through electron microscopy, advanced spectroscopy, and scattering techniques, respectively. Results from these studies indicate enzyme folding with MOF precursors is a crucial aspect to consider when pairing enzymes with selected MOF precursors. To assist in this consideration, we provide alternative strategies for retaining enzyme activity in denaturing conditions. Finally, a roadmap is provided for tuning activity of immobilized enzymes, paving the way for industrial-scale production of enzyme@MOFs. Two steps must be satisfied to achieve high activity enzyme@MOFs: proper enzyme folding and low MOF crystallinity.