Computer-Aided Site-Specific\nPEGylation of PET Hydrolases\nfor Enhanced PET Degradation

The enormous accumulation of poly(ethylene terephthalate)\n(PET)\nwaste has posed a serious threat to the environment and human health,\nand biodegradation with PET hydrolase (PETase) can be a possible solution.\nHerein, we propose site-specifically modifying PETase with amphiphilic\npolymers to improve the enzyme performance at ambient temperature.\nFor this purpose, we devise a computer-aided strategy to prioritize\nthe conjugation site, and polyethylene glycol (PEG) preparations of\n0.55 to 10 kDa are site-specifically conjugated to PETase. The most\nactive conjugate PETase–PEG 5k (PETase-5K) shows an increase\nof melting temperature (3.88 °C) and significantly improves PET\ndegradation performance (3.5- and 3.1-fold increases at 30 and 40\n°C, respectively). Experimental investigation and molecular dynamics\nsimulations reveal that the site-specific PEGylation increases the\nhydrophobic solvent-accessible surface area and the binding capability\nto the PET surface, thickens the hydration layer, increases the intramolecular\nhydrogen bonding, reduces the interactions between water and the conjugated\nenzyme surface, and rigidifies the enzyme structure via hydrogen bonding\nand hydrophobic interactions between the polymer and the enzyme, thus\nleading to improved enzymatic performance of PETase-5K. We further\nvalidate the versatility of the site-specific PEGylation in one of\nthe most evolved variants of PETase, FAST-PETase, by 1.8-fold improvement\nin PET degradation at 30 °C. The presented computer-aided site-specific\nconjugation strategy has opened a new avenue to enhancing PETase performance\nat ambient temperature, and the contribution of PEGylation to PETase\nunraveled in this work laid a foundation for the rational engineering\nof PET hydrolases.