Nanoconfined Protein Scaffold as a Generalizable Platform Empowers Synergistic Enzyme–Nanozyme Catalysis for Cascade Biotransformation

Abstract Enzymatic and chemoenzymatic cascades hold immense potential for industrial applications but are hindered by inherent instability, low atom economy, inefficient intermediate transfer, and complex enzyme immobilization processes. Here, we repurpose encapsulin, which naturally loads cargo, as a programmable nanoreactor to assemble an enzyme–nanozyme cascade inside the nanoconfined cavity (EcENN). d-Amino acid oxidase (DAAO) was fused with a targeting peptide for selective encapsulation during self-assembly, while palladium (Pd) nanozymes were biomineralized inside the same compartment for in situ toxic byproduct elimination. Molecular dynamics simulations revealed Pd nanozyme enrichment directly on the enzyme surface, minimizing intermediate diffusion losses. Consequently, EcENN showed a 7-fold increase in pyruvic acid titers in laboratory settings and presented up to a 93.1% conversion rate (versus 49.7% for free DAAO) in pilot-scale assays. Overall, this work provides a generalizable precision nanoconfinement strategy for constructing robust biocatalytic cascades, with potential in the synthesis of α-keto acid and pharmaceutical intermediates.