Engineering PHL7 for Improved Poly(Ethylene Terephthalate) Depolymerization via Rational Design and Directed Evolution

Journal Article · Mon May 26 04:00:00 EDT 2025 · Chem Catalysis

DOI:https://doi.org/10.1016/j.checat.2025.101399· OSTI ID:2574707

Groseclose, Thomas ^[1]; Kober, Erin ^[1]; Clark, Matilda ^[2]; Moore, Benjamin ^[2]; Jha, Ramesh ^[1]; Taylor, Zoe ^[3]; Lujan, Lexy ^[3]; Beckham, Gregg ^[4]; Pickford, Andrew ^[2]; Dale, Taraka ^[1]; Nguyen, Hau ^[1]

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1. BOTTLE Consortium; Los Alamos National Laboratory
2. BOTTLE Consortium; University of Portsmouth
3. Los Alamos National Laboratory
4. National Renewable Energy Lab., Golden, CO (United States)

Enzymatic depolymerization of poly(ethylene terephthalate) (PET) has emerged as a promising approach for polyester recycling, and, to date, many natural and engineered PET hydrolase enzymes have been reported. For industrial use, PET hydrolases must achieve high depolymerization extent and exhibit excellent thermostability. Here, we engineered a natural PET hydrolase, Polyester Hydrolase Leipzig #7 (PHL7), through rational design and directed evolution using a high-throughput screening platform. Four new enzymes were engineered with enhanced properties compared with the parent enzyme, wild-type PHL7 (PHL7-WT), and other benchmark PET hydrolases, under the tested conditions. In bioreactors, the exemplary engineered enzyme, PHL7-Jemez, exhibited improved ability to depolymerize amorphous PET film compared with PHL7-WT at 2.9% and 20% substrate loadings, with 37% and 270% higher hydrolysis, respectively, after 48 h. This study develops several state-of-the-art PET hydrolases and demonstrates a directed evolution platform to engineer high-performance enzymes, which can accelerate enzyme discovery toward improved biocatalytic recycling.

• Research Organization: National Renewable Energy Laboratory (NREL), Golden, CO (United States)
• Sponsoring Organization: USDOE Office of Energy Efficiency and Renewable Energy (EERE), Energy Efficiency Office. Advanced Manufacturing and Materials Technologies Office (AMMTO)
• DOE Contract Number: AC36-08GO28308
• OSTI ID: 2574707
• Report Number(s): NREL/JA-2A00-91746
• Journal Information: Chem Catalysis, Journal Name: Chem Catalysis Journal Issue: 8 Vol. 5
• Country of Publication: United States
• Language: English

Related Subjects

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY
PET hydrolase
directed evolution
enzymatic plastic degradation
high-throughput screening
plastic recycling
poly(ethylene terephthalate) (PET)
protein engineering
split GFP