Biocatalysis has recently emerged as a powerful and eco-friendly technology in waste plastic recycling, especially for the widely used polyethylene terephthalate (PET). So far, however, a high-throughput screening assay specifically toward PET-hydrolyzing activity has rarely been applied. This hinders the identification of new polyester hydrolases and their variants with adequate activities fulfilling the requirements for industrial applications. This chapter describes the detailed procedure for assaying terephthalate as a major product of enzymatic PET hydrolysis in a 96-well microtiter plate format. Using PET nanoparticles derived readily from waste food packaging as a substrate, an active thermophilic PET hydrolase was clearly distinguished from an inactive variant by a Fenton chemistry-mediated fluorimetric detection. The assay uses enzymes in crude cell lysates, obtained by a simple freeze–thaw protocol. The experimental work validates the applicability of this method for screening mutant libraries of novel PET hydrolases and will thus facilitate the identification of promising variants useful for effective plastic waste recycling.
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