Thomsen_2023_ChemSusChem_16_e202300291

The rate response of poly(ethylene terephthalate) (PET)-hydrolases to increased substrate crystallinity (X(C) ) of PET manifests as a rate-lowering effect that varies significantly for different enzymes. Herein, we report the influence of X(C) on the product release rate of six thermostable PET-hydrolases. All enzyme reactions displayed a distinctive lag phase until measurable product formation occurred. The duration of the lag phase increased with X(C) . The recently discovered PET-hydrolase PHL7 worked efficiently on "amorphous" PET disks (X(C) =10 %), but this enzyme was extremely sensitive to increased X(C) , whereas the enzymes LCC(ICCG) , LCC, and DuraPETase had higher tolerance to increases in X(C) and had activity on PET disks having X(C) of 24.4 %. Microscopy revealed that the X(C) -tolerant hydrolases generated smooth and more uniform substrate surface erosion than PHL7 during reaction. Structural and molecular dynamics analysis of the PET-hydrolyzing enzymes disclosed that surface electrostatics and enzyme flexibility may account for the observed differences.