A Computational Evaluation of IsPETase Variants: Thermodynamic Stability vs. PET Substrate Binding

PET plastic is widely used in applications, including packaging, textiles, and consumer goods, despite its slow natural degradation. A breakthrough discovery by Yoshida et al. identified Ideonella sakaiensis, a bacterium that breaks down PET via IsPETase. However, the wild-type IsPETase has slow degradation rates and is thermally unstable, leading researchers to engineer thermostable variants. Whether these stability-enhancing mutations cause unintended effects on ΔG binding, especially with smaller oligomer sizes comparable to microplastics, remains unexplored. Here, 13 IsPETase variants and 1-4mer PET oligomers were compiled for molecular docking (AutoDock Vina) to calculate ΔG binding . ChimeraX extracted structural metrics for the catalytic gap, hydrogen-bond count, and interface surface area. ΔG binding progressively became more negative as the oligomer length increased (r= -0.9837, p= 0.016), indicating stronger overall binding to longer oligomers. Among engineered mutants, ΔTm positively correlated with ΔG binding for the 2-mer and 3-mer lengths (2-mer: r= 0.7592, p= 0.018; 3-mer: r= 0.7310, p= 0.025), suggesting that more thermostable variants bound these intermediate lengths looser. This weakened binding coincided with a widened catalytic gap, consistent with the rigidification of the binding cleft as a possible effect of stabilizing mutations, which potentially limited the accommodation of intermediate PET ligands. Importantly, none of the correlations survived Benjamini-Hochberg correction for multiple comparisons, so this study’s findings should be interpreted as exploratory trends rather than established correlations. These findings nonetheless highlight a previously overlooked stability-binding trade-off, offering future research possibilities for engineering PETase variants for microplastic and PET degradation.