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[This corrects the article DOI: 10.1021/acssuschemeng.4c00143.].
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The discovery and engineering of novel biocatalysts capable of depolymerizing polyethylene terephthalate (PET) have gained significant attention since the need for green technologies to combat plastic pollution has become increasingly urgent. This study focuses on the development of novel substrates that can indicate enzymes with PET hydrolytic activity, streamlining the process of enzyme evaluation and selection. Four novel substrates, mimicking the structure of PET, were chemically synthesized and labeled with fluorogenic or chromogenic moieties, enabling the direct analysis of candidate enzymes without complex preparatory or analysis steps. The fluorogenic substrates, mUPET1, mUPET2, and mUPET3, not only identify enzymes capable of PET breakdown but also differentiate those with exceptional performance on the polymer, such as the benchmark PETase, LCCICCG. Among the substrates, the chromogenic p-NPhPET3 stands out as a reliable tool for screening both pure and crude enzymes, offering advantages over fluorogenic substrates such as ease of assay using UV-vis spectroscopy and compatibility with crude enzyme samples. However, ferulic acid esterases and mono-(2-hydroxyethyl) terephthalate esterases (MHETases), which exhibit remarkably high affinity for PET oligomers, also show high catalytic activity on these substrates. The substrates introduced in this study hold significant value in the function-based screening and characterization of enzymes that degrade PET, as well as the the potential to be used in screening mutant libraries derived from directed evolution experiments. Following this approach, a rapid and dependable assay method can be carried out using basic laboratory infrastructure, eliminating the necessity for intricate preparatory procedures before analysis.
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Manufactured nanoplastic particles (NPs) are indispensable for in vitro and in vivo testing and a health risk assessment of this emerging environmental contaminant is needed. The high surface area and inherent hydrophobicity of plastic materials makes the production of NPs devoid of any contaminants very challenging. In this study, we produced nanoprecipitated polyethylene terephthalate (PET) NPs (300 nm hydrodynamic diameter) with an overall yield of 0.76%. The presence of the ionic surfactant sodium dodecyl sulfate (SDS) was characterized by 1H NMR, where the relative ratio of NP/surfactant was monitored on the basis of the chemical shifts characteristic of PET and SDS. For a wide range of surfactant/NP ratios (17:100 to 1.2:100), the measured zeta potential changed from -42.10 to -34.93 mV, but with an NP concentration up to 100 µg/mL, no clear differences were observed in the cellular assays performed in protein-rich media on primary human cells. The remaining impurities contributed to the outcome of the biological assays applied in protein-free buffers, such as human red blood cell hemolysis. The presence of SDS increased the NP-induced hemolysis by 1.5% in protein-rich buffer and by 7.5% in protein-free buffer. As the size, shape, zeta potential, and contaminants of NPs may all be relevant parameters for the biological effects of NPs, the relative quantification of impurities exemplified in our work by the application of 1H NMR for PET NPs and the ionic surfactant SDS could be a valuable auxiliary method in the quality control of manufactured NPs.
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Polyethylene terephthalate (PET) is widely used material and as such became highly enriched in nature. It is generally considered inert and safe plastic, but due to the recent increased efforts to break-down PET using biotechnological approaches, we realized the scarcity of information about structural analysis of possible degradation products and their ecotoxicological assessment. Therefore, in this study, 11 compounds belonging to the group of PET precursors and possible degradation products have been comprehensively characterized. Seven of these compounds including 1-(2-hydroxyethyl)-4-methylterephthalate, ethylene glycol bis(methyl terephthalate), methyl bis(2-hydroxyethyl terephtahalate), 1,4-benzenedicarboxylic acid, 1,4-bis[2-[[4-(methoxycarbonyl)benzoyl]oxy]ethyl] ester and methyl tris(2-hydroxyethyl terephthalate) corresponding to mono-, 1.5-, di-, 2,5- and trimer of PET were synthetized and structurally characterized for the first time. In-silico druglikeness and physico-chemical properties of these compounds were predicted using variety of platforms. No antimicrobial properties were detected even at 1000 µg/mL. Ecotoxicological impact of the compounds against marine bacteria Allivibrio fischeri proved that the 6 out of 11 tested PET-associated compounds may be classified as harmful to aquatic microorganisms, with PET trimer being one of the most toxic. In comparison, most of the compounds were not toxic on human lung fibroblasts (MRC-5) at 200 µg/mL with inhibiting concentration (IC50) values of 30 µg/mL and 50 µg/mL determined for PET dimer and trimer. Only three of these compounds including PET monomer were toxic to nematode Caenorhabditis elegans at high concentration of 500 µg/mL. In terms of the applicative potential, PET dimer can be used as suitable substrate for the screening, identification and characterization of novel PET-depolymerizing enzymes.
