An Efficient Protein Evolution Workflow for the Improvement of Bacterial PET Hydrolyzing Enzymes
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 s...
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| Format: | Article |
| Language: | English |
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MDPI AG
2021-12-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/23/1/264 |
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| author | Valentina Pirillo Marco Orlando Davide Tessaro Loredano Pollegioni Gianluca Molla |
| author_facet | Valentina Pirillo Marco Orlando Davide Tessaro Loredano Pollegioni Gianluca Molla |
| author_sort | Valentina Pirillo |
| collection | DOAJ |
| description | 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 <i>Ideonella sakaiensis</i> 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<sup>−1</sup> of TS-ΔIsPET, ~10.6 mM of degradation products were produced in 2 days from 9 mg mL<sup>−1</sup> 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<sup>−1</sup>. 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. |
| first_indexed | 2024-03-10T03:39:53Z |
| format | Article |
| id | doaj.art-b2d67844ea5b4906845f00138fba171e |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-10T03:39:53Z |
| publishDate | 2021-12-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-b2d67844ea5b4906845f00138fba171e2023-11-23T11:37:21ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672021-12-0123126410.3390/ijms23010264An Efficient Protein Evolution Workflow for the Improvement of Bacterial PET Hydrolyzing EnzymesValentina Pirillo0Marco Orlando1Davide Tessaro2Loredano Pollegioni3Gianluca Molla4“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“The Protein Factory 2.0”, Dipartimento di Biotecnologie e Scienze della Vita, Università degli Studi dell’Insubria, Via J.H. Dunant 3, 21100 Varese, ItalyDipartimento di Chimica, Materiali e Ingegneria Chimica “Giulio Natta”, Politecnico di Milano, p.za L. da Vinci 32, 20133 Milano, Italy“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“The Protein Factory 2.0”, Dipartimento di Biotecnologie e Scienze della Vita, Università degli Studi dell’Insubria, Via J.H. Dunant 3, 21100 Varese, ItalyEnzymatic 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 <i>Ideonella sakaiensis</i> 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<sup>−1</sup> of TS-ΔIsPET, ~10.6 mM of degradation products were produced in 2 days from 9 mg mL<sup>−1</sup> 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<sup>−1</sup>. 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.https://www.mdpi.com/1422-0067/23/1/264biocatalysisbiodegradationprotein engineeringpolyethylene terephthalatehydrolases |
| spellingShingle | Valentina Pirillo Marco Orlando Davide Tessaro Loredano Pollegioni Gianluca Molla An Efficient Protein Evolution Workflow for the Improvement of Bacterial PET Hydrolyzing Enzymes International Journal of Molecular Sciences biocatalysis biodegradation protein engineering polyethylene terephthalate hydrolases |
| title | An Efficient Protein Evolution Workflow for the Improvement of Bacterial PET Hydrolyzing Enzymes |
| title_full | An Efficient Protein Evolution Workflow for the Improvement of Bacterial PET Hydrolyzing Enzymes |
| title_fullStr | An Efficient Protein Evolution Workflow for the Improvement of Bacterial PET Hydrolyzing Enzymes |
| title_full_unstemmed | An Efficient Protein Evolution Workflow for the Improvement of Bacterial PET Hydrolyzing Enzymes |
| title_short | An Efficient Protein Evolution Workflow for the Improvement of Bacterial PET Hydrolyzing Enzymes |
| title_sort | efficient protein evolution workflow for the improvement of bacterial pet hydrolyzing enzymes |
| topic | biocatalysis biodegradation protein engineering polyethylene terephthalate hydrolases |
| url | https://www.mdpi.com/1422-0067/23/1/264 |
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