Customizable colloidosomes confining enzymes for continuous-flow biocatalysis

Enzyme confinement at the molecular scale reveals catalytic performance that is previously unseen at the ensemble level, but all such delicate enzyme hosts now available encounter their intrinsic issues. Here, we present a mild, one-pot emulsification-directing approach to customize colloidosomes (CSs) with pores matching enzyme size as enzyme hosts for optimizing continuous-flow biocatalysis. By means of the dissolution-induced inside-out self-assembly, uniform and enzyme-matched pores can be readily achieved. Typically, 5.8-nm pores obtained using 22-nm SiO₂ nanoparticles (SiO₂ NPs) as building blocks are optimal for confining Candida antarctica lipase B (CALB, ∼5 nm), conferring 98.3% activity recovery and minimal leaching of only 2.1%. The catalytic efficiency of CALB-confined colloidosomes (CALB/CSs) is dozens of times higher than that of free CALB (fCALB) in batch mode. Moreover, a 21.3-fold catalytic efficiency was achieved by integrating robust CALB/CSs with a continuous-flow platform. Meanwhile, the desired long-term stability was confirmed, with over 85% of the initial efficiency maintained after 200-h of consecutive operation. This study offers a potentially generalizable strategy that establishes a CS platform as an enzyme-confined host that is competent for continuous flow manufacturing, hoping to unlock the full potential of enzymatic reactions.