Structure-guided engineering of a gatekeeper residue enhances polylactic acid depolymerization by a cutinase from Papiliotrema nemorosa

Abstract

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 °C. 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.