Lin_2026_Front.Bioeng.Biotechnol_14_1936157

Enzymatic recycling of polylactic acid (PLA) is constrained by limited enzyme-polymer interactions, particularly insufficient adsorption onto highly hydrophobic solid surfaces. In this study, a cutinase-like enzyme from Papiliotrema nemorosa was heterologously expressed and characterized. Structural analysis suggested that Ser142 may function as a potential hydrophilic gatekeeper residue at the entrance of the substrate-binding pocket. Guided by this insight, computational screening and virtual saturation mutagenesis were employed to generate eight variants aimed at modulating enzyme-polymer interfacial recognition. Among these variants, the S142F variant exhibited the highest PLA-degrading capability. In addition to showing a 121.2% increase in PLA film adsorption capacity, S142F displayed significantly enhanced intrinsic catalytic turnover, resulting in an approximately two-fold enhancement in degradation efficiency, achieving 30.25% weight loss within 36 h at 60 degreesC. Furthermore, biochemical quantification revealed an approximately 1.58-fold increase in L-lactic acid release compared with the wild-type enzyme, providing additional biochemical support for the enhanced degradation performance. Molecular dynamics simulations suggested that the S142F substitution increased local hydrophobicity at the pocket entrance, which may strengthen enzyme-substrate interactions and contribute to improved substrate recognition. These findings suggest that structure-guided engineering of substrate-binding regions represents a promising strategy for regulating enzyme-polymer interfacial interactions and developing enhanced biocatalysts for PLA recycling.