| Title | Discovery and Surface Charge Engineering of Fungal Cutinases for Enhanced Activity on Poly(ethylene terephthalate) |
|---|---|
| ID_Doc | 13099 |
| Authors | Brinch-Pedersen, W; Keller, MB; Dorau, R; Paul, B; Jensen, K; Borch, K; Westh, P |
| Title | Discovery and Surface Charge Engineering of Fungal Cutinases for Enhanced Activity on Poly(ethylene terephthalate) |
| Year | 2024 |
| Published | Acs Sustainable Chemistry & Engineering, 12.0, 19 |
| Abstract | Bioprocessing of poly(ethylene terephthalate) (PET) waste has emerged as a promising approach to a circular economy. The key to this development has been the discovery of enzymes that effectively depolymerize PET to its constituent monomers, and hitherto, the most promising candidates are quite closely related, bacterial cutinases. Fungal enzymes have been less investigated, although a few examples of hydrolytic activity against PET have been reported. To further assess this, we have produced 24 fungal cutinases that are homologues to known PET hydrolases and identified three previously uncharacterized enzymes. The activity of the newly discovered enzymes was comparable to or higher than that of the fungal cutinase from Humicola insolens (HiC). One enzyme from Thermocarpiscus australiensis (TaC) appeared particularly proficient on PET, with up to four times higher activity than that of HiC, but required high salt concentrations (about 0.5 M NaCl) to reach maximal activity. Biochemical and biophysical measurements suggested that this salt dependence could be ascribed to electrostatic repulsion between TaC and the PET, and to mitigate this, we designed and produced 40 TaC variants with a reduced (negative) net charge. Several variants performed up to 2.5-fold better in salt-free buffer compared to wild-type TaC, and a few of them entirely escaped the dependence of salt but retained the maximal catalytic performance of the wild type. Overall, we expanded the diversity of fungal PET hydrolases, and learnings from this study may be of more general relevance for engineering balanced enzyme-substrate interaction strength in PET hydrolases. |
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