<i>In Silico</i> Optimization of a Bifunctional Lipase–Polyethylene Terephthalate (PET) Hydrolase for Enhanced PET and Lipid Hydrolysis

Polyethylene terephthalate (PET) and lipid-rich waste pose urgent environmental challenges that need multifunctional biocatalysts. LipMRD9, a lidless lipase with natural PETase activity, exemplifies such a scaffold. Here, we used an integrated approach combining automated in silico mutagenesis (AsiteDesign), Protein Energy Landscape Exploration (PELE), and molecular dynamics (MD) simulations, structure-guided rational engineering, and bioprospecting of homologous sequences to improve the dual lipase–PETase activity of LipMRD9. Computational analyses pinpointed mutational hotspots extending the active-site binding region, guiding the creation of variants with better substrate binding and active-site geometry. Rationally engineered mutants such as RA18 and RA19, along with natural homologues, showed significant increases in PET breakdown (up to 2.8-fold) while maintaining or increasing lipase activity. Key design principles included aromatic substitutions that selectively expand subsite -I to boost PET affinity, surface charge adjustments, loop stabilization to promote substrate retention, and structural plasticity in evolutionary homologues that support dual reactivity. A comparison with natural homologues highlights the versatility of this scaffold for multifunctional uses. These findings demonstrate that designing lipase-derived PETases that effectively break down polyester while hydrolyzing lipids is achievable, providing a strong foundation for sustainable recycling of mixed PET–lipid waste streams.