(154a) Discovery and Mechanism-Guided Engineering of BHET Hydrolases for Improved PET Recycling and Upcycling

Plastics have facilitated human life and become an integral part of packaging, healthcare, aerospace, and agriculture due to their durability, transparency, and economic viability¹. Due to the lack of or low activity of catabolic microorganisms/enzymes that can degrade these plastic constituents, many plastics persist significantly in terrestrial and marine environments². Microplastics have even been found in human feces, blood, and placenta, indicating that plastic pollution is a threat to human health and needs more attention². Polyethylene terephthalate (PET), a significant portion of plastic waste³, is exceedingly hard to degrade due to the chemical inertness of the ester linkages and the aromatic nuclei⁴. Several PET-degrading microorganisms (e.g., Ideonella sakaiensis³, Thermobifida cellulosilytica⁵, Saccharomonospora viridis⁶, Fusarium solanipisi⁷, Nocardiopsaceae⁸) and enzymes (e.g., lipase⁹, esterase¹⁰, cutinase⁷, PETase³, and mono-2-hydroxyethyl terephthalate hydrolase (MHETase)) have been well explored³. However, current PET recycling without BHETase and/or relevant bacteria still faces the bottleneck that the heterogeneous products yielded from PET degradation are unfavorable for PET recondensation and high-value derivative synthesis.

In our study, Two BHETases (ChryBHETase from Chryseobacterium sp. PET-29 and BsEst from Bacillus subtilis PET-86) are identified from the environment via enzyme mining, including three phases: (i) microorganism determination; (ii) enzyme identification; (iii) in vitro and in silico characterization. After the computational study, we found that a certain number of water molecules were present in SBC, indicating the larger BHET might be blocked by uncertain barrier structures (the particular region is called lid in hydrolase ¹¹). Subsequently, mechanism-guided barrier engineering is employed to yield two robust and thermostable ΔBHETases with up to 3.5-fold enhanced k_cat/K_M than wild-type. Coupling ΔBHETase into a two-enzyme system overcomes the challenge of heterogeneous product formation and results in up to 7.0-fold improved TPA production than seven state-of-the-art PET hydrolases (PETase, DepoPETase, FAST-PETase, ThermoPETase, DuraPETase, LCC, and LCC-ICCG), under the conditions used here. Finally, we employ a ΔBHETase-joined tandem chemical-enzymatic approach to valorize 21 commercial post-consumed plastics into virgin PET and an example chemical (p-phthaloyl chloride) for achieving the closed-loop PET recycling and open-loop PET upcycling.

Reference

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Figure. The mechanism-guided barrier engineering yielded the improved ΔBHETase. (a) Schematic representation of the mechanism-guided barrier engineering strategy. Step 1: In silico prediction of structure and solvation observables. Step 2: Design truncated variants. Step 3: Statistical normative scores by assigning weights of 10%, 30%, 40%, and 20% to the binding energy, number of hydrogen bonds, serine affinity attack distance, and cavity volume, respectively. Step 4: In vitro validation.