Engineering surface electrostatics affords control over morphological preference, synergy, and activity in polymer degrading enzymes

Abstract

The biocatalytic recycling of plastics, such as polyethylene terephthalate (PET), promises a sustainable alternative to our present open-loop cycles. Engineering of PET-hydrolases for this purpose has focused on improving activity near the glass-transition temperature of the polymer by increasing their thermostability, neglecting other features of the protein-polymer system that affect enzymatic activity. Here, we isolate the effect of electrostatics on the activity of a thermophilic PETase by rationally redesigning its surface charge, while preserving its thermodynamic properties. The enzyme variant, SfInv, shows orders of magnitude improvements in binding affinity and in activity towards untreated plastic films, with inverted morphological preference. When combined, the wildtype enzyme and SfInv act synergistically, revealing an entirely new mechanism for cooperative activities driven by complimentary electrostatic interactions at the PET surface. These findings highlight unexplored avenues in improving PETase function through the control of morphological preference or introduction of protein cooperativity by exploiting protein electrostatics.