Comparative Performance of PETase as a Function of Reaction Conditions, Substrate Properties, and Product Accumulation

Abstract

There is keen interest to develop new technologies to recycle the plastic poly(ethylene terephthalate) (PET). To this end, the use of PET-hydrolyzing enzymes has shown promise for PET deconstruction to its monomers, terephthalate (TPA) and ethylene glycol (EG). Here, the Ideonella sakaiensis PETase wildtype enzyme was compared to a previously reported improved variant (W159H/S238F). The thermostability of each enzyme was compared and a 1.45 Å resolution structure of the mutant was described, highlighting changes in the substrate binding cleft compared to the wild-type enzyme. Subsequently, the performance of the wild-type and variant enzyme was compared as a function of temperature, substrate morphology, and reaction mixture composition. These studies showed that reaction temperature had the strongest influence on perform-ance between the two enzymes. It was also shown that both enzymes achieved higher levels of PET conversion for substrates with moderate crystallinity relative to amorphous substrates. Finally, the impact of product accumulation on reaction progress was assessed for the hydrolysis of both PET and bis(2hydroxyethyl) terephthalate (BHET). Each enzyme displayed different inhibition profiles to mono(2-hydroxyethyl) terephthalate (MHET) and TPA, while both were sensitive to inhibition by EG. Overall, this study highlights the importance of reaction conditions, substrate selection, and product accumulation for catalytic performance of PET-hydrolyzing enzymes, which have implications for enzyme screening in the development of enzyme-based polyester recycling.

Molecular and Biochemical Differences of the Tandem and Cold-Adapted PET Hydrolases Ple628 and Ple629, Isolated From a Marine Microbial Consortium

“…Similar studies have found that PET degradation by TfCut2, a cutinase isolated from Thermobifida fusca KW3, leads to the production of mainly MHET (the counterpart of BT in PET degradation) (60-70%), followed by BHET (14%) and T (8-32%) (Wei et al, 2016). Other studies also showed that some PETases, such as IsPETase, Mors1, PE-H and LCC, produce mainly MHET as the primary hydrolysis product rather than T after PET degradation (Yoshida et al, 2016;Bollinger et al, 2020;Blázquez-Sánchez et al, 2021;Erickson et al, 2022). However, other studies have observed T as the main hydrolysis product rather than MHET when IsPETase was incubated with amorphous PET films (Joo et al, 2018;Blázquez-Sánchez et al, 2021).…”

Sabatier Principle for Rationalizing Enzymatic Hydrolysis of a Synthetic Polyester

“… 48 This substrate has previously been characterized with a reported M w of 33 kDa and % crystallinity of 38. 49 The powder was suspended in 50 mM sodium phosphate (NaPi) buffer pH 8.0. The surfactant CTAB (57-09-0) and the substrate p NP-Bu(2635-84-9) were both purchased from Sigma.…”

Conversion of polyethylene terephthalate into pure terephthalic acid through synergy between a solid-degrading cutinase and a reaction intermediate-hydrolysing carboxylesterase

“…We would also like to mention that it is accepted in the field of PET degradation that enzymes that are easily produced in large amounts, soluble at high concentrations and stable at high temperatures (reducing requirements for replenishment), significantly reduce the cost factor associated with degradation. 41 It is also known that the use of temperatures in the range of 40–80 °C does not add anything to energy costs associated with other extant processes for PET degradation through a combination of mechanical and chemical means. 41 The new element added in this paper, to ongoing research and development involving LCC, is the enzyme TTCE.…”

“…41 It is also known that the use of temperatures in the range of 40–80 °C does not add anything to energy costs associated with other extant processes for PET degradation through a combination of mechanical and chemical means. 41 The new element added in this paper, to ongoing research and development involving LCC, is the enzyme TTCE. We have found that TTCE is produced at significantly higher yields over LCC (Fig.…”

“…This need can appear to be fulfilled by a recombinant system imitating the natural system present in I. sakaiensis (a mesophile bacterium), in which a PETase (capable of degrading PET, OETs, and BHET) cooperates with a MHETase (capable of degrading MHET). 40 Unfortunately, (i) both enzymes undergo facile denaturation, 5,7,41 at temperatures below the known T g s of semi-crystalline PET, 19,42 making this system useful mainly for the degradation of amorphous PET; (ii) TPA is generated in 40–60 times lower yield than that of the best cutinases by this system. 7,43…”