A Coupled Ketoreductase-Diaphorase Assay for the Detection of Polyethylene Terephthalate-Hydrolyzing Activity

In the last two decades, several PET-degrading enzymes from already known microorganisms or metagenomic sources have been discovered to face the growing environmental concern of polyethylene terephthalate (PET) accumulation. However, there is a limited number of high-throughput screening protocols for PET-hydrolyzing activity that avoid the use of surrogate substrates. Herein, a microplate fluorescence screening assay was described. It was based on the coupled activity of ketoreductases (KREDs) and diaphorase to release resorufin in the presence of the products of PET degradation. Six KREDs were identified in a commercial panel that were able to use the PET building block, ethylene glycol, as substrate. The most efficient KRED, KRED61, was combined with the diaphorase from Clostridium kluyveri to monitor the PET degradation reaction catalyzed by the thermostable variant of the cutinase-type polyesterase from Saccharomonospora viridis AHK190. The PET degradation products were measured both fluorimetrically and by HPLC, with excellent correlation between both methodsts This work was supported by a grant from the Universidad Autónoma de Madrid (Project for Young Researchers, Ref. SI1/PJI/ 2019-00394). D. M. M. was supported by a Research Talent Attraction contract from the Community of Madrid (Ref. 2016-T2/ BIO-1960). We thank Carmen Ortega (Center of Molecular Biology “Severo Ochoa”, UAM) for preliminary tests with KREDs. We are also grateful to Jose Luis González Alfonso (Institute of Catalysis and Petrochemistry, CSIC) and David Rodrigo Frutos (Analiza Calidad Madrid S.L.) for technical support on HPLC analyse