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Ultrahigh-throughput screening assay for PET-degrading enzymes.

Álvaro Lorente-Arévalo¹, María Gimeno-Pérez², Carmen Ortega¹

¹Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid-Consejo Superior de Investigaciones Científicas (UAM-CSIC), Nicolás Cabrera 1, Madrid, Spain; Department of Molecular Biology, Universidad Autónoma de Madrid, Campus de Cantoblanco, Madrid, Spain.

Methods in Enzymology

|April 27, 2025

Summary

A new screening method directly detects polyethylene terephthalate (PET) degradation using coupled enzymes and fluorescence. This advance enables ultrahigh-throughput screening of PET-degrading enzymes from microbial and metagenomic libraries.

Keywords:

Coupled reaction Fluorescence assay Ketoreductases Microdroplets PET degradation Ultrahigh-throughput

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Area of Science:

Biotechnology
Environmental Science
Enzymology

Background:

Polyethylene terephthalate (PET) accumulation is a significant environmental concern.
Existing screening methods for PET-degrading enzymes often rely on indirect surrogate substrates.
There is a need for direct, high-throughput methods to identify novel PET-hydrolyzing enzymes.

Purpose of the Study:

To develop a novel, direct, and ultrahigh-throughput screening method for PET-degrading enzymes.
To couple ketoreductases (KREDs) and diaphorase for sensitive detection of PET hydrolysis products.
To demonstrate the utility of this method for screening metagenomic and enzyme libraries.

Main Methods:

A coupled enzyme assay was designed using a metagenomic ketoreductase (KRED) and diaphorase from Clostridium kluyveri.
The assay detects PET degradation products by producing the fluorescent compound resorufin.
The reaction was optimized and implemented in water-in-oil microdroplets, encapsulating single E. coli cells.

Main Results:

The coupled KRED-diaphorase system successfully detected PET degradation products catalyzed by Bacillus subtilis BS2 esterase.
The method demonstrated high sensitivity and direct measurement of PET hydrolysis.
The microdroplet-based system is suitable for ultrahigh-throughput screening of large libraries.

Conclusions:

A novel, direct, and efficient screening assay for PET-degrading enzymes has been established.
This method overcomes limitations of surrogate substrate-based assays.
The developed system holds significant potential for discovering and engineering enzymes for PET bioremediation.