Work

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

Year: 2025

Type: article

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. For complete details on the use and execution of this protocol, please refer to Groseclose et al. 1 and Groseclose et al. 2

Source: STAR Protocols

DOI: 10.1016/j.xpro.2025.103969

Authors: Thomas M. Groseclose, Zoe K. Taylor, Lexy A. Lujan, Taraka Dale, Hau B. Nguyen

Institution: Los Alamos National Laboratory

Language: English

Topic: Enzyme Catalysis and Immobilization

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

SDG: Responsible consumption and production

Open Access status: gold

Estimated APC paid: $1,550