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

The rate response of poly(ethylene terephthalate) (PET)-hydrolases to increased substrate crystallinity (XC ) of PET manifests as a rate-lowering effect that varies significantly for different enzymes. Herein, we report the influence of XC 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 XC . The recently discovered PET-hydrolase PHL7 worked efficiently on "amorphous" PET disks (XC ≈10 %), but this enzyme was extremely sensitive to increased XC , whereas the enzymes LCCICCG , LCC, and DuraPETase had higher tolerance to increases in XC and had activity on PET disks having XC of 24.4 %. Microscopy revealed that the XC -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.