The polyester PET (poly(ethylene terephthalate)) plastic is chemically inert and remarkably persistent, posing relevant and global pollution concerns due to its accumulation in ecosystems across the globe. In past years, research focused on identifying bacteria active on PET and on the specific enzymes responsible for its degradation. Here, the enzymatic degradation of PET can be considered as an ‘erosion process’ that takes place on the surface of an insoluble material and results in an unusual, substrate‐limited kinetic condition. In this review, we report on the most suitable models to evaluate the kinetics of PET‐hydrolyzing enzymes, which takes into consideration the amount of enzyme adsorbed on the substrate, the enzyme‐accessible ester bonds, and the product inhibition effects. Careful kinetic analysis is especially relevant to compare enzymes from different sources and evolved variants generated by protein engineering studies as well. Furthermore, the analytical methods most suitable to screen natural bacteria and recombinant variant libraries generated by protein engineering have been also reported. These methods rely on different detection systems and are performed both on model compounds and on different PET samples (e.g., nanoparticles, microparticles, and waste products). All this meaningful information represents an optimal starting point and boosts the process of identifying systems able to biologically recycle PET waste products.

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.

Enzymatic degradation is a promising green approach to bioremediation and recycling of the polymer poly(ethylene terephthalate) (PET). In the past few years, several PET-hydrolysing enzymes (PHEs) have been discovered, and new variants have been evolved by protein engineering. Here, we report on a straightforward workflow employing semi-rational protein engineering combined to a high-throughput screening of variant libraries for their activity on PET nanoparticles. Using this approach, starting from the double variant W159H/S238F of Ideonella sakaiensis 201-F6 PETase, the W159H/F238A-ΔIsPET variant, possessing a higher hydrolytic activity on PET, was identified. This variant was stabilized by introducing two additional known substitutions (S121E and D186H) generating the TS-ΔIsPET variant. By using 0.1 mg mL⁻¹ of TS-ΔIsPET, ~10.6 mM of degradation products were produced in 2 days from 9 mg mL⁻¹ PET microparticles (~26% depolymerization yield). Indeed, TS-ΔIsPET allowed a massive degradation of PET nanoparticles (>80% depolymerization yield) in 1.5 h using only 20 μg of enzyme mL⁻¹. The rationale underlying the effect on the catalytic parameters due to the F238A substitution was studied by enzymatic investigation and molecular dynamics/docking analysis. The present workflow is a well-suited protocol for the evolution of PHEs to help generate an efficient enzymatic toolbox for polyester degradation.

Insects offer promising opportunities for organic waste bioconversion; however, they cannot efficiently degrade synthetic polymers such as polyethylene terephthalate (PET). Here, we generated transgenic Drosophila melanogaster lines to express in vitro -evolved variants of two PET-degrading enzymes with distinct biochemical properties: an engineered Ideonella sakaiensis PETase variant (TS-ΔIsPET) and a leaf-branch compost cutinase variant (TA-ΔLCC). Both enzymes, fused to a Drosophila gut-derived secretory signal, were produced and secreted by both Drosophila cultured S2R ⁺ cells and transgenic larvae. Both enzymes were glycosylated upon secretion, a post-translational modification that did not abolish their catalytic activity. Notably, TA-ΔLCC displayed ∼6-fold higher esterase activity than TS-ΔIsPET in larval extracts and TA-ΔLCC-containing extracts depolymerised PET nanoparticles in vitro under enzyme-favourable conditions. Transgenic flies showed normal development, fertility and survival. Morphological and biochemical analysis confirmed that TA-ΔLCC expression did not alter midgut structure and function. Together, these results establish Drosophila melanogaster as a model for functional expression and comparative evaluation of engineered PET-degrading enzymes and identify TA-ΔLCC as a promising candidate for exploitation in insect species relevant to plastic contaminated waste bioconversion. Highlights: Transgenic D. melanogaster enables in vivo study of engineered PET enzymes Engineered TS-ΔIsPET and TA-ΔLCC are functional in larval extracts TA-ΔLCC was selected for PET nanoparticle assays due to higher pNPA activity D. melanogaster model enables comparative evaluation of PET-degrading enzymes

Insects offer promising opportunities for organic waste bioconversion; however, they cannot efficiently degrade synthetic polymers such as polyethylene terephthalate (PET). Here, we generated transgenic Drosophila melanogaster lines to express in vitro-evolved variants of two PET-degrading enzymes with distinct biochemical properties: an engineered Ideonella sakaiensis PETase variant (TS-ΔIsPET) and a leaf-branch compost cutinase variant (TA-ΔLCC). Both enzymes, fused to a Drosophila gut-derived secretory signal, were produced and secreted by Drosophila cultured S2R+ cells and transgenic larvae. Upon secretion, TS-ΔIsPET and TA-ΔLCC enzymes were glycosylated, a post-translational modification that did not abolish their catalytic activity. Notably, TA-ΔLCC displayed ∼6-fold higher net esterase activity than TS-ΔIsPET in larval samples, and TA-ΔLCC-containing extracts depolymerised PET nanoparticles in vitro under enzyme-favourable conditions. Transgenic flies showed normal development, fertility and survival. Morphological and biochemical analyses confirmed that TA-ΔLCC expression did not alter midgut structure and function. Together, these results establish Drosophila melanogaster as a model for functional expression and comparative evaluation of engineered PET-degrading enzymes and identify TA-ΔLCC as a promising candidate for further evaluation in insect species relevant to plastic-contaminated waste bioconversion.