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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 Å in the wild type to 3.8 Å 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., (Copyright © 2022 Elsevier B.V. All rights reserved.)

[Display omitted] • Cell membrane protection strategy enhances PET hydrolase (4Mz) production. • Phospholipase activity could damage cell membrane, reducing the yield of 4Mz. • The production of 4Mz is the highest level among PET hydrolases. • The raw fermented broth of 4Mz can directly apply on PET degradation. Biodegradation, particularly via enzymatic degradation, has emerged as an efficient and eco-friendly solution for Poly (ethylene terephthalate) (PET) pollution. The production of PET hydrolases plays a role in the large-scale enzymatic degradation. However, an effective variant, 4Mz, derived from Thermobifida fusca cutinase (Tfu_0883), was previously associated with a significant reduction in yield when compared to the wild-type enzyme. In this study, a novel cell membrane protection strategy was developed to enhance the yield of 4Mz. This approach increased the yield of 4Mz by 18.2-fold from shaken flasks to 3-L bioreactors, reaching a yield of 3.1 g·L−1, the highest yield of a PET hydrolase described thus far. In addition, the raw culture broth from 4Mz was applied directly for the enzymatic degradation of PET bottles, achieving a 91.2 % degradation rate. These advancements render the large-scale enzymatic degradation of PET more feasible, thus contributing to the more sustainable management of plastic waste. [ABSTRACT FROM AUTHOR]