Phase-separated dual-enzyme protein condensates promote biodegradation of polyethylene terephthalate and phthalate...

The enzymatic degradation of phthalates (PAEs) and polyethylene terephthalate (PET) represents a green approach to addressing white pollution and upcycling plastic waste. Despite developing numerous new enzymes over the past decade, engineering reaction processes that efficiently biodegrade PET and PAEs remain an unresolved yet crucial problem. This report proposes self-assembled dual-enzyme condensates based on liquid-liquid phase separation (LLPS) induced by intrinsically disordered regions (IDRs) to enhance PAEs and PET biodegradation. Specifically, two degrading enzymes, GoEst15-GoEstM1 for PAEs degradation and FAST-PETase (LCC^ICCG)-IsMHETase for PET degradation, are colocalized within LLPS droplets. This colocalization enhanced degradation rates of PAEs, nano-PET, and micro-PET by 3.3-, 12.3-, and 2.8-fold, respectively, when compared with the equivalent free enzyme system. The accelerated degradation rate correlated positively with the extent of phase separation. Increasing the ionic strength in the dual-enzyme system weakened phase separation, which reduced the reaction rate. In contrast, the free enzyme system was not influenced by ionic strength. The experimental results indicate that the possible reasons for the enhanced degradation rates are (1) altered enzyme kinetics and improved molecular transport because of the proximity of enzymes within the condensed droplets and (2) the enrichment of PAEs and nano/micro-PET within the dual-enzyme condensates, thereby increasing the local substrate and enzyme concentrations. The confinement of multiple enzymes within microscale LLPS droplets provides a simple and general approach to enhancing the degradation of various macromolecules.