Aer_2024_J.Hazard.Mater_476_134887

Although many efforts have been devoted to the modification of polyethylene terephthalate (PET) hydrolases for improving the efficiency of PET degradation, the catalytic performance of these enzymes at near-ambient temperatures remains a challenge. Herein, a multi-enzyme cascade system (PT-EC) was developed and validated by assembling three well-developed PETases, PETase(EHA), Fast-PETase, and Z1-PETase, respectively, together with carboxylesterase TfCa, and hydrophobic binding module CBM3a using scaffold proteins. The resulting PT-EC(EHA), PT-EC(FPE), PT-EC(ZPE) all demonstrated outstanding PET degradation efficacy. Notably, PT-EC(EHA) exhibited a 16.5-fold increase in product release compared to PETase(EHA), and PT-EC(ZPE) yielded the highest amount of product. Subsequently, PT-ECs were displayed on the surface of Escherichia coli, respectively, and their degradation efficiency toward three PET types was investigated. The displayed PT-EC(EHA) exhibited a 20-fold increase in degradation efficiency with PET film compared to the surface-displayed PETase(EHA). Remarkably, an almost linear increase in product release was observed for the displayed PT-EC(ZPE) over a one-week degradation period, reaching 11.56 +/- 0.64 mM after 7 days. TfCa(I69W/L281Y) evolved using a docking-based virtual screening strategy showed a further 2.5-fold increase in the product release of PET degradation. Collectively, these advantages of PT-EC demonstrated the potential of a multi-enzyme cascade system for PET bio-cycling.