Biochemical and structural characterization of an aromatic ring-hydroxylating dioxygenase for terephthalic acid catabolism.

SignificanceMore than 400 million tons of plastic waste is produced each year, the overwhelming majority of which ends up in landfills. Bioconversion strategies aimed at plastics have emerged as important components of enabling a circular economy for synthetic plastics, especially those that exhibit chemically similar linkages to those found in nature, such as polyesters. The enzyme system described in this work is essential for mineralization of the xenobiotic components of poly(ethylene terephthalate) (PET) in the biosphere. Our description of its structure and substrate preferences lays the groundwork for in vivo or ex vivo engineering of this system for PET upcycling.

A flexible kinetic assay efficiently sorts prospective biocatalysts for PET plastic subunit hydrolysis.

Esterase enzymes catalyze diverse hydrolysis reactions with important biological, commercial, and biotechnological applications. For the improvement of these biocatalysts, there is a need for widely accessible, inexpensive, and adaptable activity screening assays that identify enzymes with particular substrate specificities. Natural systems for biopolymer bioconversion, and likely those designed to mimic them, depend on cocktails of enzymes, each of which specifically targets the intact material as well as water-soluble subunits of varying size. In this work, we have adapted a UV/visible assay using pH-sensitive sulfonphthalein dyes for the real-time quantification of ester hydrolysis of bis-(2-hydroxyethyl) terephthalate (BHET), a subunit of polyethylene terephthalate (PET) plastic. We applied this method to a diverse set of known PET hydrolases and commercial esterases in a microplate format. The approach identified four PET hydrolases and one commercial esterase with high levels of specificity for BHET hydrolysis. Five additional PET hydrolases and three commercial esterases, including a thermophilic enzyme, effectively hydrolyzed both BHET and its monoester product MHET (mono-(2-hydroxyethyl) terephthalate). Specific activities were discernible within one hour and reactions reached an unequivocal endpoint well within 24 hours. The results from the UV/visible method correlated well with conventional HPLC analysis of the reaction products. We examined the suitability of the method toward variable pH, temperature, enzyme preparation method, mono- and multi-ester substrate type, and level of sensitivity versus stringency, finding the assay to be easily adaptable to diverse screening conditions and kinetic measurements. This method offers an accurate, easily accessible, and cost-effective route towards high-throughput library screening to support the discovery, directed evolution, and protein engineering of these critical biocatalysts.