Rate Response of Poly(Ethylene Terephthalate)-Hydrolases to Substrate Crystallinity: Basis for Understanding the Lag...

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.