Reaction pathways for the enzymatic degradation of poly(ethylene terephthalate): What characterizes an efficient PET-hydrolase?

Abstract

Bioprocessing of polyester waste has emerged as a promising tool in the quest for a cyclic plastic economy. One key step is the enzymatic breakdown of the polymer, and this entails a complicated pathway with substrates, intermediates, and products of variable size and solubility. We have elucidated this pathway for poly(ethylene terephthalate) (PET) and four enzymes. Specifically, we combined different kinetic measurements and a novel stochastic model, and found that the ability to hydrolyze internal bonds in the polymer (endo-lytic activity) was a key parameter for overall enzyme performance. Endo-lytic activity promoted the release of soluble PET fragments with two or three aromatic rings, which, in turn, were broken down with remarkable efficiency (kcat/KM-values of about 105 M-1s-1) in the aqueous bulk. This meant that about 70% of the final, monoaromatic products was formed via soluble di- or tri-aromatic intermediates.

Relationships of crystallinity and reaction rates for enzymatic degradation of poly (ethylene terephthalate), PET

“…This interpretation parallels conclusions based on kinetic modeling of the degradation of nanoPET. [22] Such a mode of action that predominantly proceeds via endo-type activity is also in line with the occurrence of longer soluble fragments (OETs), as observed in Fig. 3.…”

“…[34,35] The structural data discussed above have offered valuable insights into the causes of recalcitrance, and the current work aims to investigate this topic further from a functional perspective. Our starting point was numerous observations of an initial lag phase for PET-hydrolases, [9,22,[36][37][38] which has been reported to be especially pronounced for crystalline substrates. This was also evident in the current study, as progress curves for soluble products formation (Corg, Fig.…”

“…Products with 2, 3, or 4 aromatic rings were also detected by HPLC; however, the different species within each of these groups (e.g., the di-aromatic products TET, TETE, and ETETE) exhibited similar retention times and eluted as one peak using the current experimental procedure. The peaks for di-, tri-, and tetra-aromatic compounds were identified and quantified using in-house synthesized standards TETE, [22] TETET, [22] and TETETET (described herein). Reactions were conducted in duplicates and included control experiments without enzyme.…”

“…[1,4,21] To assess its catalytic activity, we employed two complementary assays described previously. [22] This involved continuous pH-stat measurements to detect the release of protons resulting from cleavage of ester bonds and HPLC for the analysis of the profile of soluble, organic products. Combined analysis of these parameters for substrates with different XC, and special focus on the initial stage of the reaction, provided a better understanding of how LCCICCG attacks PET and the role of crystallinity.…”

From Bulk to Binding: Decoding the Entry of PET into Hydrolase Binding Pockets

“…This is also consistent with a recent study by Schubert et al, in which chain mobility and the number of available sites for endo-type chain scission, which depend on the degree of crystallinity, are postulated as the activity-limiting factor causing the initial lag phase for the release of the soluble product. 7 Another biotechnological approach to mitigate this hindering effect could be to design enzymes to introduce residues that enhance enzyme-PET interactions, which would promote and counterbalance the loss of PET-PET interactions upon entry into the binding cleft.…”