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Article

An Efficient Protein Evolution Workflow for the Improvement of Bacterial PET Hydrolyzing Enzymes

1
“The Protein Factory 2.0”, Dipartimento di Biotecnologie e Scienze della Vita, Università degli Studi dell’Insubria, Via J.H. Dunant 3, 21100 Varese, Italy
2
Dipartimento di Chimica, Materiali e Ingegneria Chimica “Giulio Natta”, Politecnico di Milano, p.za L. da Vinci 32, 20133 Milano, Italy
*
Authors to whom correspondence should be addressed.
Academic Editor: Antonio Trincone
Int. J. Mol. Sci. 2022, 23(1), 264; https://doi.org/10.3390/ijms23010264
Received: 10 December 2021 / Revised: 23 December 2021 / Accepted: 24 December 2021 / Published: 27 December 2021
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. View Full-Text
Keywords: biocatalysis; biodegradation; protein engineering; polyethylene terephthalate; hydrolases biocatalysis; biodegradation; protein engineering; polyethylene terephthalate; hydrolases
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MDPI and ACS Style

Pirillo, V.; Orlando, M.; Tessaro, D.; Pollegioni, L.; Molla, G. An Efficient Protein Evolution Workflow for the Improvement of Bacterial PET Hydrolyzing Enzymes. Int. J. Mol. Sci. 2022, 23, 264. https://doi.org/10.3390/ijms23010264

AMA Style

Pirillo V, Orlando M, Tessaro D, Pollegioni L, Molla G. An Efficient Protein Evolution Workflow for the Improvement of Bacterial PET Hydrolyzing Enzymes. International Journal of Molecular Sciences. 2022; 23(1):264. https://doi.org/10.3390/ijms23010264

Chicago/Turabian Style

Pirillo, Valentina, Marco Orlando, Davide Tessaro, Loredano Pollegioni, and Gianluca Molla. 2022. "An Efficient Protein Evolution Workflow for the Improvement of Bacterial PET Hydrolyzing Enzymes" International Journal of Molecular Sciences 23, no. 1: 264. https://doi.org/10.3390/ijms23010264

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