通过使用高通量筛选测定法进行定向进化来工程化聚对苯二甲酸乙二酯水解酶的方案。

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

作者信息

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

机构信息

Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM 87545, USA; BOTTLE Consortium, Golden, CO 80401, USA.

Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM 87545, USA.

出版信息

STAR Protoc. 2025 Jul 15;6(3):103969. doi: 10.1016/j.xpro.2025.103969.

DOI:10.1016/j.xpro.2025.103969

PMID:40668676

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC12281158/

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. and Groseclose et al..

摘要

聚对苯二甲酸乙二酯（PET）水解酶可将PET解聚为其单体，因其在促进这种废塑料的生物工业回收方面的潜力而受到关注。在此，我们提出了一种同时筛选大型随机诱变酶文库以提高活性、溶解性和稳定性的方案。我们概述了文库构建、使用基于平板的分裂绿色荧光蛋白和模型底物测定进行筛选以及酶热稳定性测定的步骤。然后，我们详细介绍了PET底物验证测定和最终变体表征的程序。有关本方案使用和执行的完整详细信息，请参考格罗斯克洛斯等人以及格罗斯克洛斯等人的文献。