Fluorescence-activated screening of polyester-depolymerizing enzymes based on pseudo-PET polythioester plastics

While fluorescence-based high-throughput screening (HTS) has been valuable for identifying enzymes and microorganisms for polyethylene terephthalate (PET) depolymerization, the traditional approach relies on cleaving ester bonds in small-molecule probes, limiting its ability to directly detect activity towards polymeric PET substrates. This study proposes a novel fluorescence-based screening strategy for detecting the release of sulfhydryl groups during the depolymerization of pseudo-PET polythioesters by polyester hydrolases. The strategy successfully identifies the polyester-depolymerizing activity of leaf-branch compost cutinase (LCC^ICCG), whereas porcine liver esterase (PLE) only hydrolyses small molecular substrates. Furthermore, the strategy can be integrated with a droplet microfluidic system for high-throughput screening of LCC^ICCG. This work further demonstrates the applicability of the microplate reader platform for screening polyester-depolymerizing bacteria. This novel fluorescence-based approach offers an efficient strategy to identify enzymes and microbial resources for depolymerizing polyester-like plastics.