Single-Molecule Detection of Enzymatic PET Hydrolysis Using Fluorogenic Probe-Doped PET Nanoparticles

Enzymatic depolymerization of plastics offers a sustainable route to polymer recycling, yet the mechanism of action of poly(ethylene terephthalate) (PET) hydrolases at solid polymer interfaces remains poorly understood. In particular, it is still unclear whether individual enzymes perform isolated cleavage events or catalyze multiple successive reactions during a single adsorption event. Here, we employ fluorogenic probe-doped PET nanoparticles together with a leaf-branch compost cutinase variant (LCC^ICCG) to visualize interfacial PET hydrolysis at the single-molecule level and reveal the dynamic principles governing interfacial catalysis. Fluorescence trajectories reveal multi-burst events with short intervals, consistent with semi-processive hydrolysis during a single enzyme adsorption event. Based on the quantitative correspondence between the experimentally observed fraction of multi-burst events and the simulated nearest-neighbor distance distribution, we estimate an apparent effective reaction distance of approximately 2 nm for individual enzymes. We further show that burst frequency exhibits a nonmonotonic temperature dependence governed by the surface-chain mobility near the glass transition, whereas the overall PET degradation rate increases continuously. Our findings provide a quantitative framework linking enzyme dynamics, the local reaction environment, and polymer interfacial physics, providing a basis for engineering more efficient PET hydrolases.