Mechanistic investigation of enzymatic degradation of polyethylene terephthalate by nuclear magnetic resonance

Falkenstein, Patricia; Wei, Ren; Matysik, Jörg; Song, Chen

doi:10.1016/bs.mie.2020.11.002

Cited by 16 publications

(10 citation statements)

References 60 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…While the structures of LCC ICCG S165A (PDB code: 7VVE; Figure H) and Is PETase R103G/S131A (PDB code: 5XH3; Figure I) exhibited productive ligand conformations with the ester carbonyl carbons interacting with the oxygen of the catalytic serine residue, the PES-H structures revealed multiple noncatalytic intermediate binding modes (IBMs) with the ester bonds (or amide bond in MHETA) positioned too far from S130 for nucleophilic attack. This supports the previous hypothesis that PET hydrolysis may involve dynamic reorientation of polymer chains in the active sites of PET hydrolases. , MHET and BHET have frequently been found to inhibit enzymatic PET depolymerization. , The multiple IBMs suggest an enhanced residence likelihood of these inhibitory degradation intermediates in the substrate-binding groove, where they may prevent productive binding of polyester segments.…”

Section: Resultssupporting

confidence: 87%

“…This supports the previous hypothesis that PET hydrolysis may involve dynamic reorientation of polymer chains in the active sites of PET hydrolases. 15 , 35 MHET and BHET have frequently been found to inhibit enzymatic PET depolymerization. 36 , 37 The multiple IBMs suggest an enhanced residence likelihood of these inhibitory degradation intermediates in the substrate-binding groove, where they may prevent productive binding of polyester segments.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Multiple Substrate Binding Mode-Guided Engineering of a Thermophilic PET Hydrolase

et al. 2022

Self Cite

View full text Add to dashboard Cite

Thermophilic polyester hydrolases (PES-H) have recently enabled biocatalytic recycling of the mass-produced synthetic polyester polyethylene terephthalate (PET), which has found widespread use in the packaging and textile industries. The growing demand for efficient PET hydrolases prompted us to solve high-resolution crystal structures of two metagenome-derived enzymes (PES-H1 and PES-H2) and notably also in complex with various PET substrate analogues. Structural analyses and computational modeling using molecular dynamics simulations provided an understanding of how product inhibition and multiple substrate binding modes influence key mechanistic steps of enzymatic PET hydrolysis. Key residues involved in substrate-binding and those identified previously as mutational hotspots in homologous enzymes were subjected to mutagenesis. At 72 °C, the L92F/Q94Y variant of PES-H1 exhibited 2.3-fold and 3.4-fold improved hydrolytic activity against amorphous PET films and pretreated real-world PET waste, respectively. The R204C/S250C variant of PES-H1 had a 6.4 °C higher melting temperature than the wild-type enzyme but retained similar hydrolytic activity. Under optimal reaction conditions, the L92F/Q94Y variant of PES-H1 hydrolyzed low-crystallinity PET materials 2.2-fold more efficiently than LCC ICCG, which was previously the most active PET hydrolase reported in the literature. This property makes the L92F/Q94Y variant of PES-H1 a good candidate for future applications in industrial plastic recycling processes.

show abstract

Section: Resultssupporting

confidence: 87%

Section: Resultsmentioning

confidence: 99%

Multiple Substrate Binding Mode-Guided Engineering of a Thermophilic PET Hydrolase

et al. 2022

Self Cite

View full text Add to dashboard Cite

show abstract

“…By substituting S238 to F, which is highly conserved in homologous cutinase-like PET hydrolases, increased hydrolytic activity on PET was determined when accompanied by the mutation W159H, although the binding cleft width appeared to be narrowed. This phenomenon suggests that the polymer binding in the surface groove is more likely a dynamic rather than static process, as supported by an NMR analysis of PET chain mobility in the context of enzymatic hydrolysis. , The residue equivalent of the Is PETase S238 is F in Tf Cut2 and LCC. , By substitution to A, I, and W, significantly increased PET hydrolysis activity was reported. , These findings suggest that further systematic iterative engineering of all variable residues in the PET hydrolase binding pocket may be beneficial for gaining a thorough understanding of the rate-limiting interactions with aromatic polyesters and, as a result, improving the overall degradation performance.…”

Section: Understanding Interfacial Enzymatic Pet Hydrolysis From a St...mentioning

confidence: 88%

Mechanism-Based Design of Efficient PET Hydrolases

et al. 2022

Self Cite

View full text Add to dashboard Cite

Polyethylene terephthalate (PET) is the most widespread synthetic polyester, having been utilized in textile fibers and packaging materials for beverages and food, contributing considerably to the global solid waste stream and environmental plastic pollution. While enzymatic PET recycling and upcycling have recently emerged as viable disposal methods for a circular plastic economy, only a handful of benchmark enzymes have been thoroughly described and subjected to protein engineering for improved properties over the last 16 years. By analyzing the specific material properties of PET and the reaction mechanisms in the context of interfacial biocatalysis, this Perspective identifies several limitations in current enzymatic PET degradation approaches. Unbalanced enzyme–substrate interactions, limited thermostability, and low catalytic efficiency at elevated reaction temperatures, and inhibition caused by oligomeric degradation intermediates still hamper industrial applications that require high catalytic efficiency. To overcome these limitations, successful protein engineering studies using innovative experimental and computational approaches have been published extensively in recent years in this thriving research field and are summarized and discussed in detail here. The acquired knowledge and experience will be applied in the near future to address plastic waste contributed by other mass-produced polymer types (e.g., polyamides and polyurethanes) that should also be properly disposed by biotechnological approaches.

show abstract

“…A higher flexibility of the active site often correlates with a higher catalytic rate at mild temperatures [ 24 , 25 ]. In our case, the enhanced flexibility of the active site could counteract the low degree of freedom of amorphous PET chain torsional angles under mild conditions [ 26 , 27 ], allowing alternative binding modes that result in a higher affinity for the substrate (i.e., a higher K A ).…”

Section: Resultsmentioning

confidence: 99%

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

Pirillo

Orlando

Tessaro

et al. 2021

IJMS

View full text Add to dashboard Cite

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.

show abstract

Mechanistic investigation of enzymatic degradation of polyethylene terephthalate by nuclear magnetic resonance

Cited by 16 publications

References 60 publications

Multiple Substrate Binding Mode-Guided Engineering of a Thermophilic PET Hydrolase

Multiple Substrate Binding Mode-Guided Engineering of a Thermophilic PET Hydrolase

Mechanism-Based Design of Efficient PET Hydrolases

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

Contact Info

Product

Resources

About