Designing a mesoporous cascade reactor for enhanced enzymatic performance.

Designing biomimetic catalytic systems with enhanced activity, stability, and reusability remains a grand challenge in the field of biocatalysis. Here, we report a hierarchical and modular strategy for constructing robust biocatalytic cascade reactors by spatially organizing dual enzymes, -amino acid oxidase (DAAO) and cytochrome c (Cyt c), within defect-engineered covalent organic frameworks (COFs), followed by surface encapsulation with a polydopamine (PDA) shell to mimic cellular compartmentalization. The defective COFs provide highly tunable pore architectures and versatile surface functionalities, enabling site-specific enzyme immobilization via both physical infiltration and covalent conjugation. Concurrently, the PDA coating provides an additional protective barrier, mitigating structural denaturation and preserving enzymatic conformational stability even under harsh operational conditions. Compared to their free counterparts, the COF-immobilized enzymes exhibited significantly improved catalytic performances, with apparent DAAO activity and catalytic efficiency (k/K) representing 165 % and 430 % of those of the free enzymes, respectively. Kinetic studies revealed that positively charged COF surfaces promoted substrate enrichment and electrostatic stabilization, while the PDA coating further improved enzyme resilience to high temperatures, organic solvents, proteases, and alkaline pH. Notably, the engineered bioreactors retained >85 % of initial activity after 22 reaction cycles, outperforming conventional enzyme immobilization approaches. This work introduces a generalizable platform for constructing artificial enzymatic systems with cellular-level organization, offering new opportunities in continuous biomanufacturing, biosensing, and synthetic biology under demanding operational environments.