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Sourcing thermotolerant poly(ethylene terephthalate) hydrolase scaffolds from natural diversity.

ABSTRACT: Enzymatic deconstruction of poly(ethylene terephthalate) (PET) is under intense investigation, given the ability of hydrolase enzymes to depolymerize PET to its constituent monomers near the polymer glass transition temperature. To date, reported PET hydrolases have been sourced from a relatively narrow sequence space. Here, we identify additional PET-active biocatalysts from natural diversity by using bioinformatics and machine learning to mine 74 putative thermotolerant PET hydrolases. We successfully express, purify, and assay 51 enzymes from seven distinct phylogenetic groups; observing PET hydrolysis activity on amorphous PET film from 37 enzymes in reactions spanning pH from 4.5-9.0 and temperatures from 30-70 °C. We conduct PET hydrolysis time-course reactions with the best-performing enzymes, where we observe differences in substrate selectivity as function of PET morphology. We employed X-ray crystallography and AlphaFold to examine the enzyme architectures of all 74 candidates, revealing protein folds and accessory domains not previously associated with PET deconstruction. Overall, this study expands the number and diversity of thermotolerant scaffolds for enzymatic PET deconstruction.

SUBMITTER: Erickson E

PROVIDER: S-EPMC9772341 | biostudies-literature | 2022 Dec

REPOSITORIES: biostudies-literature

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Sourcing thermotolerant poly(ethylene terephthalate) hydrolase scaffolds from natural diversity.

Erickson Erika E Gado Japheth E JE Avilán Luisana L Bratti Felicia F Brizendine Richard K RK Cox Paul A PA Gill Raj R Graham Rosie R Kim Dong-Jin DJ König Gerhard G Michener William E WE Poudel Saroj S Ramirez Kelsey J KJ Shakespeare Thomas J TJ Zahn Michael M Boyd Eric S ES Payne Christina M CM DuBois Jennifer L JL Pickford Andrew R AR Beckham Gregg T GT McGeehan John E JE

Nature communications 20221221 1

Enzymatic deconstruction of poly(ethylene terephthalate) (PET) is under intense investigation, given the ability of hydrolase enzymes to depolymerize PET to its constituent monomers near the polymer glass transition temperature. To date, reported PET hydrolases have been sourced from a relatively narrow sequence space. Here, we identify additional PET-active biocatalysts from natural diversity by using bioinformatics and machine learning to mine 74 putative thermotolerant PET hydrolases. We succ ...[more]

PMID: 36543766

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Project description:Scaffolds from poly(ethylene oxide) and poly(butylene terephthalate), PEOT/PBT, with a PEO molecular weight of 1,000 and a PEOT content of 70 weight% (1000PEOT70PBT30) were prepared by leaching salt particles (425-500 microm). Scaffolds of 73.5, 80.6 and 85.0% porosity were treated with a CO(2) gas plasma and seeded with rat bone marrow stromal cells (BMSCs). After in vitro culture for 7 days (d) in an osteogenic medium the scaffolds were subcutaneously implanted for 4 weeks in nude mice. Poly(D, L-lactide) (PDLLA) and biphasic calcium phosphate (BCP) scaffolds were included as references. After 4 weeks (wks) all scaffolds showed ectopic formation of bone and bone marrow. For the scaffolds of different porosities, no significant differences were observed in the relative amounts of bone (7-9%) and bone marrow (6-11%) formed, even though micro computed tomography (mu-CT) data showed considerable differences in accessible pore volume and surface area. 1000PEOT70PBT30 scaffolds with a porosity of 85% could not maintain their original shape in vivo. Surprisingly, 1000PEOT70PBT30 scaffolds with a porosity of 73.5% showed cartilage formation. This cartilage formation is most likely due to poorly accessible pores in the scaffolds, as was observed in histological sections. mu-CT data showed a considerably smaller accessible pore volume (as a fraction of the total volume) than in 1000PEOT70PBT30 scaffolds of 80.6 and 85.0% porosity. BMSC seeded PDLLA (83.5% porosity) and BCP scaffolds (29% porosity) always showed considerably more bone and bone marrow formation (bone marrow formation is approximately 40%) and less fibrous tissue ingrowth than the 1000PEOT70PBT30 scaffolds. The scaffold material itself can be of great influence. In more hydrophobic and rigid scaffolds like the PDLLA or BCP scaffolds, the accessibility of the pore structure is more likely to be preserved under the prevailing physiological conditions than in the case of hydrophilic 1000PEOT70PBT30 scaffolds. Scaffolds prepared from other PEOT/PBT polymer compositions, might prove to be more suited.

Dataset Information

Sourcing thermotolerant poly(ethylene terephthalate) hydrolase scaffolds from natural diversity.

Publications

Sourcing thermotolerant poly(ethylene terephthalate) hydrolase scaffolds from natural diversity.

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