Enhanced Biocatalysis in Nanofluidic Membrane Reactors With Goldilocks Nanopores for Enzymes

ABSTRACT 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.