Ultrahigh-throughput screening assay for PET-degrading enzymes

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.