Programmable Carrier‐Free All‐Enzyme Beads for Modular Continuous‐Flow Biocatalysis

Carrier-free enzyme materials offer maximal catalytic density but are typically limited to monolithic or amorphous architectures with restricted process compatibility. Here, we introduce programmable carrier-free all-enzyme beads as structurally defined, porous biocatalytic particles for modular continuous-flow operation. The beads are generated via droplet-based self-assembly of complementary enzyme building blocks, followed by cryogenic consolidation, yielding mechanically robust and monodisperse protein particles that retain full catalytic competence after drying and rehydration. Using mechanistically distinct model systems, including cofactor-independent decarboxylation and metal-dependent C─C bond formation, we demonstrate stable long-term continuous-flow operation exceeding 80 h and compatibility with biphasic solvent systems at elevated substrate concentrations. Beyond single-enzyme catalysis, the bead architecture accommodates binary cofactor-regenerating assemblies, integrated ternary cascades with dual cofactor recycling, and modular combinations of distinct bead populations enabling sequential nucleotide phosphorylation. Notably, flow-induced restructuring leads to adaptive consolidation of packed beds, enhancing effective catalytic volume without compromising mass transport. By uniting structural programmability, high catalytic density, architectural modularity, and adaptive behavior under flow, carrier-free all-enzyme beads bridge molecular enzyme engineering and reactor design, expanding the materials toolbox for scalable and reconfigurable continuous biocatalysis.