• Protocol for engineering poly(...

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

Title:
Protocol for engineering poly(ethylene terephthalate) hydrolases via directed evolution using a high-throughput screening assay.
Authors:
Groseclose TM; Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM 87545, USA; BOTTLE Consortium, Golden, CO 80401, USA., Taylor ZK; Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM 87545, USA., Lujan LA; Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM 87545, USA., Dale T; Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM 87545, USA; BOTTLE Consortium, Golden, CO 80401, USA., Nguyen HB; Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM 87545, USA; BOTTLE Consortium, Golden, CO 80401, USA. Electronic address: hau@lanl.gov.
Source:
STAR protocols [STAR Protoc] 2025 Sep 19; Vol. 6 (3), pp. 103969. Date of Electronic Publication: 2025 Jul 15.
Publication Type:
Journal Article
Language:
English
Journal Info:
Publisher: Cell Press Country of Publication: United States NLM ID: 101769501 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 2666-1667 (Electronic) Linking ISSN: 26661667 NLM ISO Abbreviation: STAR Protoc Subsets: MEDLINE
Imprint Name(s):
Original Publication: [Cambridge, MA] : Cell Press, [2020]-
MeSH Terms:
Polyethylene Terephthalates*/metabolism , Polyethylene Terephthalates*/chemistry , High-Throughput Screening Assays*/methods , Directed Molecular Evolution*/methods , Hydrolases*/genetics , Hydrolases*/metabolism , Hydrolases*/chemistry , Protein Engineering*/methods
Contributed Indexing:
Keywords: Biotechnology and bioengineering; High Throughput Screening; Molecular Biology; Protein Biochemistry; Protein expression and purification
Substance Nomenclature:
0 (Polyethylene Terephthalates)
EC 3.- (Hydrolases)
Entry Date(s):
Date Created: 20250716 Date Completed: 20250923 Latest Revision: 20250923
Update Code:
20250923
PubMed Central ID:
PMC12281158
DOI:
10.1016/j.xpro.2025.103969
PMID:
40668676
Database:
MEDLINE

Weitere Informationen

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. <sup>1</sup> and Groseclose et al. <sup>2</sup> .
(Copyright © 2025 The Author(s). Published by Elsevier Inc. All rights reserved.)

Declaration of interests H.B.N., T.D., and T.M.G. declare competing financial interests. The high-throughput screening platform, methods, and enzyme variants are the subject of domestic and foreign patent applications by Los Alamos National Laboratories on behalf of the Department of Energy and Triad National Security, LLC.