Efficient polyethylene terephthalate degradation at moderate temperature: a protein engineering study of LC‐cutinase highlights the key role of residue 243

Abstract

Enzymatic degradation of poly(ethylene terephthalate) (PET) is becoming a reality because of the identification of novel PET‐hydrolysing enzymes (PHEs) and the engineering of evolved enzyme variants. Here, improved variants of leaf‐branch compost cutinase (LCC), a thermostable enzyme isolated by a metagenomic approach, were generated by a semi‐rational protein engineering approach. Starting from a deleted LCC form lacking the secretion signal (ΔLCC), single and double variants possessing a higher activity on PET were isolated. The single‐point F243T ΔLCC variant partially (~ 67%) depolymerized amorphous PET film producing ~ 21.9 mm of products after 27 h of reaction at 72 °C. The S101N/F243T ΔLCC double variant reached a further increase in activity on PET. Notably, for both single and double variants the highest conversion yield was obtained at 55 °C. Kinetics studies and molecular dynamics simulations support that a slight decreased affinity for PET is responsible for the superior degradation performance of the S101N/F243T variant and that this stems from a slightly higher flexibility of the active site region close to position 243. Furthermore, our findings question the need for a high reaction temperature for PET degradation, at least for LCC: at ≥ 70 °C, the conversion of amorphous PET into a more crystalline polymer, resistant to enzymatic hydrolysis, is favoured. The evolved S101N/F243T ΔLCC variant is able to depolymerize fully 1.3 g of untreated postconsumer PET waste in ≤ 3 days at 55 °C (using 1.25 mg of enzyme only), this making PET enzymatic degradation by engineering LCC a more ecofriendly and sustainable process.

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

“…Second, we used copper-catalyzed azide-alkyne cycloaddition (CuAAC) to functionalize the −N 3 groups with an aromatic ester-containing biotin probe. We hypothesized that the aromatic ester-containing probe would serve as a better mimic of PET than previously employed nitro-phenolate-based probes, the hydrolysis of which correlates poorly with activity for PET plastic degradation . Highly active surface-displayed enzymes cleave the proximal aromatic ester chains, releasing biotin from the yeast surface.…”

“…Efforts to further improve the thermostability of LCC-ICCG have resulted in variants such as ICCG_RIP and ICCG_I6M. , These respective efforts employed rational design focused on improving internal hydrophobic interactions or machine learning to predict residues influencing thermostability. In another example, in silico site-saturation mutagenesis was used to identify a double mutant of wild-type LCC with improved activity . The PET-depolymerization activity of Is PETase has also been improved through directed evolution, resulting in engineered variants such as HotPETase, FAST-PETase, and DuraPETase. − These variants exhibit greatly improved thermostability and PET-depolymerization kinetics relative to Is PETase.…”

“…In another example, in silico site-saturation mutagenesis was used to identify a double mutant of wild-type LCC with improved activity. 11 The PET-depolymerization activity of IsPETase has also been improved through directed evolution, resulting in engineered variants such as HotPETase, FAST-PETase, and DuraPETase. 12−17 These variants exhibit greatly improved thermostability and PET-depolymerization kinetics relative to IsPETase.…”

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

“…However, enzymatic incubation above 70 °C does not permit complete degradation of the amorphized PET due to an ageing process that results into polymer recrystallization after <10 h [ 139 , 140 ]. Recent studies [ 140 , 141 , 142 ] revealed relatively high degradation rates, even when performing the reaction at lower temperatures (50 to 60 °C), despite requiring more time (≥1 day). These milder conditions were also tested on untreated and non-micronized low-crystallinity post-consumer PET materials [ 92 , 93 ].…”

Mechanistic Insights into Cellular and Molecular Basis of Protein‐Nanoplastic Interactions

“…[ 205 ] Whereas, a novel S101N/F243T ΔLCC variant of PET‐hydrolyzing enzyme was found to depolymerize 1.3 g of PET waste in less than 3 days at 55 °C. [ 206 ] Although, the current plastic lifecycle is far from circular economy; an estimate by Ellen Macarthur foundation projected that with ideal application of circular economy has the potential to reduce the annual incorporation of plastic wastes into the sea by 80%. Lack of such projections in the bioenzymatic degradation of plastic makes it difficult to ascertain any superiority among the circular economy and bioenzymatic degradation approach of plastic waste management.…”