Directional-path modification strategy enhances PET hydrolase catalysis of plastic degradation

Poly (ethylene terephthalate) (PET) is a widely used type of general plastic that produces a significant amount of waste due to its non-degradable properties. We propose a novel directional-path modification (DPM) strategy, involving positive charge amino acid introduction and binding groove remodeling, and apply it to Thermobifida fusca cutinase to enhance PET degradation. The highest value of PET degradation (90%) was achieved in variant 4Mz (H184S/Q92G/F209I/I213K), exhibiting values almost 30-fold that of the wild-type. We employed molecular docking, molecular dynamics simulations, and QM/MM MD for the degradation process of PET, accompanied by acylation and deacylation. We found that the distance of nucleophilic attack was reduced from about 4.6 angstrom in the wild type to 3.8 angstrom in 4Mz, and the free energy barrier of 4Mz dropped from 14.3 kcal/mol to 7.1 kcal/mol at the acylation which was the rate-limiting step. Subsequently, the high efficiency and universality of the DPM strategy were successfully demonstrated in LCC, Est119, and BhrPETase enhancing the degradation activity of PET. Finally, the highest degradation rate of the pretreated commercial plastic bottles had reached to 73%. The present study provides insight into the molecular binding mechanism of PET into the PET hydrolases structure and proposes a novel DPM strategy that will be useful for the engineering of more efficient enzymes for PET degradation.

Automated summary

PET degradation efficiency can be significantly enhanced by the novel DPM strategy proposed in this study. Molecular simulations revealed the mechanism behind the improved degradation and demonstrated the effectiveness and universality of the DPM strategy in various enzymes.