Enhanced Biocatalysis in Nanofluidic Membrane Reactors With Goldilocks Nanopores for Enzymes

Continuous-flow biocatalysis enables sustainable synthesis but suffers from diminished enzyme-substrate interactions under convective transport. Nanoconfinement addresses the challenge, yet the lack of tunable, flow-compatible nanoreactors has limited exploration of extreme confinement matching the enzyme dimension under flow. Here, we reveal an enzyme-scale confinement regime that maximizes enzymatic activity for flow reactions using enzyme-encapsulating molecular network membrane reactors with nanometer-scale pore tunability. Continuous-flow kinetic resolution identifies an optimal balance at an enzyme-to-average pore size ratio of ∼2, where confinement-enhanced substrate access within sub-minute space time is balanced against flow resistance. Under tight confinement, the apparent catalytic efficiency increases by up to 133-fold relative to free enzymes and achieves 30 000-fold higher specific space-time yield than conventional flow reactors. This work presents a tunable nanoreactor platform and establishes enzyme-scale confinement control as a key design principle for high-performance nanofluidic biocatalysis in flow-based synthesis and sensing.