Enhancing Multi‐Enzyme Cascade Activity in Metal‐Organic Frameworks via Controlled Enzyme Encapsulation

To position multi-enzymes in a core-shell structure, the conventional layer-by-layer approach is often used. However, this method is time-consuming and complex, requiring multiple steps and the isolation of intermediates at each stage. To address this challenge, a sequential strategy is introduced for the controlled encapsulation of multi-enzymes within metal-organic frameworks (MOFs), achieving a core-shell structure without the need for intermediate isolation. Synchrotron Terahertz-Far-Infrared (THz-Far-IR) spectroscopy is employed to monitor this encapsulation process. The results revealed that the first enzyme is co-precipitated within the MOFs, followed by biomineralization upon the addition of a second enzyme, achieving distinct enzyme positioning. This approach is applicable to both two-enzyme and three-enzyme cascade systems. The results demonstrate that multi-enzyme cascade activity is significantly enhanced compared to conventional one-pot and layer-by-layer approaches, owing to optimal spatial arrangement, increased surface area, and improved enzyme conformation. Furthermore, the encapsulated enzymes exhibit strong resistance to high temperatures, proteolysis, and organic solvents, along with excellent reusability, making this method highly promising for industrial biocatalytic applications.