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

Abstract

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.

Ultrahigh-Throughput Directed Evolution of Polymer-Degrading Enzymes Using Yeast Display

“…For both enzymes, a linear trend was established in the first 3 h, followed by an increase in rate at 6 h for ICCG and 3 h for ICCG (H218Y). This increase in the rate of PET film degradation, which was also observed in previous studies, , may be attributable to a change in the surface morphology of the film partway through the degradation, resulting in a substantial increase in the accessible PET surface area . At 6 h, ICCG (H218Y) generated 8.53 mM of released monoaromatic products, while the parent ICCG enzyme produced only 3.34 mM (2.6-fold lower).…”

“…This increase in the rate of PET film degradation, which was also observed in previous studies, 33,34 may be attributable to a change in the surface morphology of the film partway through the degradation, resulting in a substantial increase in the accessible PET surface area. 35 At 6 h, ICCG (H218Y) generated 8.53 mM of released monoaromatic products, while the parent ICCG enzyme produced only 3.34 mM (2.6-fold lower). After this time point, the rate of product release from the parent ICCG enzyme increased, although the differential in monomer released between the two variants continued to grow throughout the 12-h experiment.…”

Unveiling PET Hydrolase Surface Dynamics through Fluorescence Microscopy

“…PHL7 has a negatively charged patch on one side of the enzyme, implying potential repulsion from the partial negative charge on a PET surface. [27] This could facilitate a more orderly protein arrangement on the surface, with the same face of the enzyme binding to the PET. Consequently, it allows for an increased capacity of protein binding to the plastic surface.…”

Elucidating the Effect of Polyethylene Terephthalate Chain Structure on Its Enzymatic Degradation Behavior

“…While most attributes of PET degradation (mechanism, enzyme modeling, the effect of crystallinity, degradation of other aromatic polyesters with PETase, the effect of degradation on a plastic surface, and physical and mechanical properties) have been studied and some bioengineering methods to improve PETases have been developed, ,,− ,− the effect of branching and kinking in PET on enzymatic degradation remains unexplored. This may hold a crucial insight and may help us understand if PET can be chemically modified to improve enzymatic degradation or if such chemical modification would affect the interactions between enzyme and substrate and inhibit the degradation.…”

“…The interesting observation is that lower crystallinity does not lead to higher degradation as previously observed. 17,20,21,39,42 This suggests that the chemical structure of the substrate influences its enzymatic degradation, and its effect is stronger than the effect of crystallinity. This observation is not surprising, as the modified polymers are not pure PET.…”