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DOE OSTI · 2572546

Protocol for engineering poly(ethylene terephthalate) hydrolases via directed evolution using a high-throughput screening assay

Groseclose, Thomas Michael [Los Alamos National Laboratory (LANL), Los Alamos, NM (United States); BOTTLE Consortium, Golden, CO (United States)] (ORCID:0000000178314294)·Taylor, Zoe Kyoko [Los Alamos National Laboratory (LANL), Los Alamos, NM (United States)] (ORCID:0009000463670850)·Lujan, Lexy Amber Esmith [Los Alamos National Laboratory (LANL), Los Alamos, NM (United States)]·Dale, Taraka [Los Alamos National Laboratory (LANL), Los Alamos, NM (United States); BOTTLE Consortium, Golden, CO (United States)] (ORCID:0000000190361210)·Nguyen, Hau Thi Bich [Los Alamos National Laboratory (LANL), Los Alamos, NM (United States); BOTTLE Consortium, Golden, CO (United States)] (ORCID:0000000301749622)
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Abstract

Poly(ethylene terephthalate) (PET) hydrolases, which depolymerize PET to its monomers, have gained attention for their potential to facilitate bio-industrial recycling of this waste plastic. Here, we present a protocol for screening large, random mutagenesis enzyme libraries simultaneously for enhanced activity, solubility, and stability. We outline steps for library construction, screening using plate-based split GFP and model substrate assays, and determination of enzyme thermostability. We then detail procedures for validation assays on PET substrates and characterization of final variants.

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59 BASIC BIOLOGICAL SCIENCESBiotechnology and bioengineeringDirected EvolutionEnzymatic Plastic DegradationHigh-throughput ScreeningMolecular BiologyPET HydrolasePlastic RecyclingProtein BiochemistryProtein EngineeringProtein expression and purificationSplit GFPpoly(ethylene terephthalate) (PET)

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Groseclose, Thomas Michael [Los Alamos National Laboratory (LANL), Los Alamos, NM (United States); BOTTLE Consortium, Golden, CO (United States)] (ORCID:0000000178314294), Taylor, Zoe Kyoko [Los Alamos National Laboratory (LANL), Los Alamos, NM (United States)] (ORCID:0009000463670850), Lujan, Lexy Amber Esmith [Los Alamos National Laboratory (LANL), Los Alamos, NM (United States)], Dale, Taraka [Los Alamos National Laboratory (LANL), Los Alamos, NM (United States); BOTTLE Consortium, Golden, CO (United States)] (ORCID:0000000190361210), Nguyen, Hau Thi Bich [Los Alamos National Laboratory (LANL), Los Alamos, NM (United States); BOTTLE Consortium, Golden, CO (United States)] (ORCID:0000000301749622). 2025-07-15. Protocol for engineering poly(ethylene terephthalate) hydrolases via directed evolution using a high-throughput screening assay. https://doi.org/10.1016/j.xpro.2025.103969

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