Design of a Programmable and Recyclable Protein Scaffolding Material with Geometrically Precise Enzyme Patterning for Improved Cascade Catalysis

Abstract Efficient spatial organization of enzymes into multi‐enzyme complexes via scaffolding is vital for boosting catalytic performance. However, the development of programmable and reusable scaffolding systems remains challenging, as current methods typically yield stochastic spatial distribution rather than deterministic positioning of enzyme placement, limiting cascade efficiency. Here, an entirely protein‐based and genetically programmable scaffold is developed by integrating ultrastable, self‐assembling γ‐prefoldin (γPFD) filaments with rigid protein bridges constructed from orthogonal SpyTag/SpyCatcher and SnoopTag/SnoopCatcher pairs. This assembly forms macroscale materials that are easily isolatable and reusable. By alternately aligning SpyTagged and SnoopTagged γPFD filaments and modulating the length of bridge proteins, the scaffold enables geometrically precise co‐immobilization and tunable spatial organization of cargo proteins. Förster resonance energy transfer (FRET) analysis validates the accuracy of protein positioning. As a critical demonstration, this scaffold enhances PETase and MHETase thermal half‐lives by 25‐fold and 15‐fold, respectively. In PET degradation, co‐immobilized enzymes achieve a 3.2‐fold higher terephthalic acid yield compared to free enzymes, due to optimized proximity and potential substrate channeling. The biocatalyst retains 75% activity after 10 reuse cycles owing to facile separation. This work provides a strategy for designing programmable protein scaffolds that concurrently improves enzyme stability, activity, and reusability, advancing sustainable biomanufacturing applications.