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

Abstract

Cutinases degrade chains of PET (polyethylene terephthalate), upon the surface of solid PET, into TPA (terephthalic acid). Alongside, degradation intermediates (DIs) such as OETs (oligoethylene terephthalate chains), BHET (bis-hydroxyethyl terephthalate),...

Rational mutagenesis of Thermobifida fusca cutinase to modulate the enzymatic degradation of polyethylene terephthalate

“…In contrast, a double mutant, G62A/F209I, showed a remarkable ~6.4‐fold improvement of activity against PET, over Tf Cut2, rather than a decrease, suggesting that the negative effect of the F209I mutation is somehow overcompensated for by the G62A mutation which ordinarily only increases activity ~3.4‐fold by itself. It is conceivable that when the F209I mutation, which is analogous to the F243I mutation previously introduced into LCC (Tournier et al, 2020), is combined with the G62A mutation, the replacement of phenylalanine by a less hydrophobic residue (F209I) allows for a higher fraction of the Tf Cut2 population to remain in solution (rather than in a PET‐bound state) through reduction of the nonspecific and catalytically non‐productive binding of PET, with this improving the overall degradation of DIs in solution by the higher fraction of enzyme that remains in solution, to improve the overall yield of TPA, with the PET‐bound enzyme fraction working synergistically with the fraction that remains in solution, akin to what has recently been demonstrated (Mrigwani et al, 2022). Notably, this possibility is supported by our observation that the activity of the F209I mutant against BHET is ~1.5‐fold higher than the activity of wild‐type Tf Cut2, as shown in Figure 2b, even though the activity of the F209I mutant against PET is ~4.4‐fold lower than that of wild‐type Tf Cut2.…”

“…Structural comparisons of Tf Cut2 with LCC and Is PETase were done using PyMOL (Schrödinger). Molecular docking and molecular dynamics (MD) simulation studies, respectively, were performed using Glide (Schrödinger) and Desmond (Schrödinger), employing parameters described previously (Mrigwani et al, 2022). Docking was performed using Tf Cut2 (PDB ID: 4CG1) and 2HE‐(MHET) 3 , containing three terephthalate moieties.…”

Counter‐intuitive enhancement of degradation of polyethylene terephthalate through engineering of lowered enzyme binding to solid plastic

“…Recently, we have shown how pure (and potentially-recyclable) TPA can be generated in high yields through such an approach. 15 A second approach, which we describe in this paper, involves mutation of an extremely-efficient PET-binding/PET-invading enzyme with the objective of reducing the efficiency of its transfer onto the surface of solid PET, through reduction of its PET-binding ability. Such a reduction could conceivably allow a fraction of the enzyme's population to remain in solution, and degrade DIs into TPA.…”

“…Solid PET is highly hydrophobic, owing to the periodic placement of terephthalate moieties along PET chains 14 . Enzymes that degrade PET are required to possess high surface hydrophobicity (in particular, in the vicinity of their PET‐binding catalytic sites), in order to partition away from aqueous solution and onto the surface of PET 15–19 . Consequently, efforts to improve PET‐degrading enzymes have focused on improving the binding of enzymes to PET through enhancement of enzyme surface hydrophobicity, or through the discovery of esterases, or cutinases, with high surface hydrophobicity, and the ability to hydrolyze ester bonds 16,17 …”

“…14 Enzymes that degrade PET are required to possess high surface hydrophobicity (in particular, in the vicinity of their PET-binding catalytic sites), in order to partition away from aqueous solution and onto the surface of PET. [15][16][17][18][19] Consequently, efforts to improve PETdegrading enzymes have focused on improving the binding of enzymes to PET through enhancement of enzyme surface hydrophobicity, or through the discovery of esterases, or cutinases, with high surface hydrophobicity, and the ability to hydrolyze ester bonds. 16,17 The problem, however, is that PET contains ester bonds between several different types of chemical groups, causing esterases and cutinases to generate numerous types of chemical (intermediate) species while degrading PET, for example, DIs such as oligoethylene terephthalate (OET), bis-hydroxyethyl terephthalate (BHET), and mono hydroxyethyl terephthalate (MHET).…”