Title: Efficient polyethylene terephthalate degradation at moderate temperature: a protein engineering study of LC-cutinase highlights the key role of residue 243 Pirillo V, Orlando M, Battaglia C, Pollegioni L, Molla G Ref: Febs J, :, 2023 : PubMed
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 (deltaLCC), single and double variants possessing a higher activity on PET were isolated. The single-point F243T deltaLCC variant partially (~67%) depolymerized amorphous PET film producing ~21.9 mM of products after 27 hours of reaction at 72 degreesC. The S101N/F243T deltaLCC 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 degreesC. 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 degreesC, the conversion of amorphous PET into a more crystalline polymer, resistant to enzymatic hydrolysis, is favored. The evolved S101N/F243T deltaLCC variant is able to depolymerize fully 1.3 g of untreated post-consumer PET waste in >=3 days at 55 degreesC (using 1.25 mg of enzyme only), this making PET enzymatic degradation by engineering LCC a more ecofriendly and sustainable process.
Pirillo V, Orlando M, Battaglia C, Pollegioni L, Molla G (2023) Efficient polyethylene terephthalate degradation at moderate temperature: a protein engineering study of LC-cutinase highlights the key role of residue 243 Febs J
Pirillo V, Orlando M, Battaglia C, Pollegioni L, Molla G (2023) Febs J