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 (Delta LCC), single and double variants possessing a higher activity on PET were isolated. The single-point F243T Delta LCC variant partially (similar to 67%) depolymerized amorphous PET film producing similar to 21.9 mm of products after 27 h of reaction at 72 degrees C. The S101N/F243T Delta LCC 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 degrees 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 degrees C, the conversion of amorphous PET into a more crystalline polymer, resistant to enzymatic hydrolysis, is favoured. The evolved S101N/F243T Delta LCC variant is able to depolymerize fully 1.3 g of untreated postconsumer PET waste in <= 3 days at 55 degrees C (using 1.25 mg of enzyme only), this making PET enzymatic degradation by engineering LCC a more ecofriendly and sustainable process.

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

Pirillo, Valentina
Primo
;
Orlando, Marco;Battaglia, Caren;Pollegioni, Loredano;Molla, Gianluca
Ultimo
2023-01-01

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 (Delta LCC), single and double variants possessing a higher activity on PET were isolated. The single-point F243T Delta LCC variant partially (similar to 67%) depolymerized amorphous PET film producing similar to 21.9 mm of products after 27 h of reaction at 72 degrees C. The S101N/F243T Delta LCC 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 degrees 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 degrees C, the conversion of amorphous PET into a more crystalline polymer, resistant to enzymatic hydrolysis, is favoured. The evolved S101N/F243T Delta LCC variant is able to depolymerize fully 1.3 g of untreated postconsumer PET waste in <= 3 days at 55 degrees C (using 1.25 mg of enzyme only), this making PET enzymatic degradation by engineering LCC a more ecofriendly and sustainable process.
2023
2023
green technology; improved LCC; polyethylene terephthalate biodegradation; rational design; structure-function relationships
Pirillo, Valentina; Orlando, Marco; Battaglia, Caren; Pollegioni, Loredano; Molla, Gianluca
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11383/2167989
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