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 (k cat /K M values of about 10 5 M À 1 s À 1 ) in the aqueous bulk. This meant that approximatly 70 % of the final, monoaromatic products were formed via soluble di-or triaromatic intermediates.

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

“…These observations clearly confirm that the duration of the lag phase varied profoundly for the different enzymes. The data concur with a recent study from our lab [42] where it was found that HiC and TfC were inferior to LCC by yielding a longer lag phase on nano PET particles. [42] Steady-state rate of the product formation Once the lag phase had occurred, the product formation for all reactions maintained a constant rate (Figure 3A-F).…”

“…The samples were quantified against authentic standards of terephthalic acid (TPA), mono(hydroxyethyl terephthalate) (MHET), BHET, and a di‐aromatic compound consisting of two ethylene glycol (EG) and two TPA moieties (TETE). TPA and BHET were purchased from Sigma‐Aldrich, while MHET and TETE were synthesized in house as described previously [42] …”

“…This phenomenon has previously been referred to as a "lag phase". [22,29,42] The enzymatic degradation of PET evidently takes place at a solid-liquid interface, and thus depends on both the enzyme, for example, activity, stability, active site structure, overall molecular flexibility, [18,43] and the physical state of the substrate. Recent work from various research groups has emphasized the significance of the substrate surface area [43] and "plasticization" of the polymer surface layer, respectively, to foster high enzymatic PET-hydrolyzing activity.…”

A high‐throughput expression and screening platform for applications‐driven PETase engineering

“…The fluorescence of oxidized monomers was therefore found to not be adequate in explaining PETase activity, likely because monomers are not the only output of PETase activity (Pirillo et al, 2021). Indeed, PETase also produces oligo (ethylene terephthalate) (OET) (Schubert et al, 2022) defined here as any soluble PET hydrolysis product containing more than one terephthalate unit. Because OET has, by definition, multiple aromatic groups, its oxidation products will in turn have higher extinction coefficients than its monomeric counterparts, increasing fluorescent signal.…”

Microbial Enzyme Biotechnology to Reach Plastic Waste Circularity: Current Status, Problems and Perspectives

“…In addition, the enzymatic capacity to adsorb and turn-over during PET chain hydrolysis were recently observed to follow a tradeoff [ 143 ]. For these reasons, the biochemical kinetics and the mechanism of enzymatic PET degradation should be studied in detail to select the best enzymes and reaction conditions under which both the PHE activity is optimal and the PET polymer is most degradable, prior to scaling up the process [ 144 ]. Moreover, it is critical to set up a standard assay condition to compare different PHEs (different conditions are used for the enzymes reported in Table 4 ).…”