Computational Engineering of a Thermostable Enzyme for the Degradation of PET Through Manipulation of Disulfide Bonds

Plastic pollution is a significant problem, accounting for approximately 12% of global solid waste [1], and plastic production and waste have more than doubled since 2000. Most plastics do not biodegrade and persist in the environment, slowly breaking down into toxic microplastics that harm ecosystems and create lasting adverse impacts. One of the most widely used plastics is polyethylene terephthalate (PET), accounting for 18% of global plastic production [2]. Currently, the most sustainable recycling method is biocatalysis, in which enzyme-catalyzed degradation of the polymer backbone generates low-molecular-weight fragments, enabling mild depolymerization. However, less than 1% of global plastic waste processing facilities use this process due to the limited thermostability of PETase enzymes, which limits the approach's general applicability and hinders its broad adoption [3]. One strategy by which nature enhances the thermal stability of enzymes is by the incorporation of disulfide bonds, a defining feature of thermophilic bacteria that thrive at elevated temperatures [4]. This study investigated whether increasing disulfide bond density could improve the thermostability of FAST-PETase by testing the hypothesis that additional disulfide bonds increase PETase thermostability. The FAST-PETase structure was mutated in PyMOL to introduce additional disulfide bonds, and the resulting mutants' thermal stability was evaluated. Engineered variants demonstrated enhanced thermal robustness compared to existing PETase benchmarks, indicating that rational disulfide bond engineering can stabilize PETase enzymes. These computational results demonstrate a novel enzyme-design approach that addresses a key bottleneck in biocatalytic plastic recycling and sets the stage for advancing enzymatic PET degradation toward global commercial viability.