Protocol for engineering poly(ethylene terephthalate) hydrolases via directed evolution using a high-throughput screening assay
Summary: 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...
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| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Elsevier
2025-09-01
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| Series: | STAR Protocols |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2666166725003752 |
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| Summary: | Summary: 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 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. |
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| ISSN: | 2666-1667 |