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ABSTRACT Polyethylene Terephthalate (PET) is an extensively used plastic whose durability and resistance to degradation contribute to growing environmental pollution and concerns. Enzymatic PET degradation, particularly via PETase from Ideonella sakaiensis , has emerged as a sustainable approach due to its ability to depolymerize PET under mild conditions. While research has largely focused on enhancing the enzyme’s thermal stability through distal mutations, less attention has been given to active-site engineering aimed at directly improving catalytic efficiency. Here, we used an automated in silico protein engineering platform called Gene Discovery and Enzyme Engineering (GDEE), designed to systematically explore mutations at the active site. By leveraging FastPETase (FP) as scaffold, we perform a high throughput generation of thousands of variants, evaluated them via docking studies with a PET substrate analogue, and ranked candidates based on binding affinity and catalytic geometry. We identified S238Y as a key mutation that enhanced PET film degrading performance at 40 °C when inserted in two of the most active PETase variants reported to date: 2.2-fold increase in the FP scaffold and 3.4-increase in the ThermoStable-PETase (TSP) background. Compared to wild type PETase, FP S238Y showed a 14.8-fold increase in bulk activity, translating into 9.4-fold more TPA and 20-fold more MHET by UPLC, while TSP S238Y reached a 25.8-fold increase (14.4-fold more TPA and 42.6-fold more MHET). This mutation also enhanced catalytic efficiency and resistance to enzyme concentration inhibition, especially in the TSP scaffold. Molecular dynamics confirm position 238 as a relevant modulator of ligand stabilisation. These findings underscore the potential of targeted active-site engineering, combined with structure-guided prediction, to accelerate the development of efficient mesophilic biocatalysts for plastic waste remediation.