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An NMR look at an engineered PET depolymerase

Abstract : Plastic environmental pollution is a major issue that our generation must face to protect our planet. Plastic recycling has the potential not only to reduce the pollution but also to limit the need for fossil-fuel-based production of new plastics. Enzymes capable of breaking down plastic could thereby support such a circular economy. Polyethylene terephthalate (PET) degrading enzymes have recently attracted considerable interest and have been subjected to intensive enzyme engineering to improve their characteristics. A quadruple mutant of Leaf-branch Compost Cutinase (LCC) was identified as a most efficient and promising enzyme. Here, we use NMR to follow the initial LCC enzyme through its different mutations that lead to its improved performance. We experimentally define the two calcium-binding sites and show their importance on the all-or-nothing thermal unfolding process, which occurs at a temperature of 72°C close to the PET glass transition temperature. Using various NMR probes such as backbone amide, methyl group, and histidine side-chain resonances, we probe the interaction of the enzymes with mono-(2-hydroxyethyl)terephthalic acid. The latter experiments are interpreted in terms of accessibility of the active site to the polymer chain.
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https://hal-cnrs.archives-ouvertes.fr/hal-03739009
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Submitted on : Tuesday, July 26, 2022 - 6:10:19 PM
Last modification on : Wednesday, July 27, 2022 - 8:44:34 AM

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Cyril Charlier, Sabine Gavalda, Vinciane Borsenberger, Sophie Duquesne, Alain Marty, et al.. An NMR look at an engineered PET depolymerase. Biophysical Journal, Biophysical Society, In press, ⟨10.1016/j.bpj.2022.07.002⟩. ⟨hal-03739009⟩

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