Fluorimetric high-throughput screening method for polyester hydrolase activity using polyethylene terephthalate nanoparticles

Pfaff, Lara; Breite, Daniel; Badenhorst, Christoffel P. S.; Bornscheuer, Uwe T.; Wei, Ren

doi:10.1016/bs.mie.2020.11.003

Cited by 23 publications

(23 citation statements)

References 42 publications

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“…In this equation, v 0 describes the initial reaction rate, k h the hydrolysis rate constant, [S 0 ] the initial substrate concentration, [E 0 ] the enzyme concentration, and K A the adsorption equilibrium constant. We used PET nanoparticles (PET-NP) prepared using previously published protocols 51 , 54 as the substrate to characterize the hydrolysis kinetics catalyzed by PES-H1, its variants, and LCC ICCG. The rates of turbidity change measured at 600 nm as a result of PET-NP degradation were determined in a microplate reader at 70 °C ( Figures S9 and S10 ).…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2022

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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.

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Section: Resultsmentioning

confidence: 99%

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

et al. 2022

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“…PET nanoparticles were prepared based on previous publications [ 39 , 40 ]. The degradation of PET nanoparticles (0.2 mg mL −1 ) was performed with an Is PETase concentration of 30 nM in 200 μL of 50 mM sodium phosphate buffer (pH 7.5).…”

Section: Methodsmentioning

confidence: 99%

Engineering and evaluation of thermostable IsPETase variants for PET degradation

Brott

Pfaff

Schuricht

et al. 2021

Engineering in Life Sciences

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Polyethylene terephthalate (PET) is a mass‐produced petroleum‐based synthetic polymer. Enzymatic PET degradation using, for example, Ideonella sakaiensis PETase (IsPETase) can be a more environmentally friendly and energy‐saving alternative to the chemical recycling of PET. However, IsPETase is a mesophilic enzyme with an optimal reaction temperature lower than the glass transition temperature (Tg) of PET, where the amorphous polymers can be readily accessed for enzymatic breakdown. In this study, we used error‐prone PCR to generate a mutant library based on a thermostable triple mutant (TM) of IsPETase. The library was screened against the commercially available polyester‐polyurethane Impranil DLN W 50 for more thermostable IsPETase variants, yielding four variants with higher melting points. The most promising IsPETaseTMK95N/F201I variant had a 5.0°C higher melting point than IsPETaseTM. Although this variant showed a slightly lower activity on PET at lower incubation temperatures, its increased thermostability makes it a more active PET hydrolase at higher reaction temperatures up to 60°C. Several other variants were compared and combined with selected previously published IsPETase mutants in terms of thermostability and hydrolytic activity against PET nanoparticles and amorphous PET films. Our findings indicate that thermostability is one of the most important characteristics of an effective PET hydrolase.

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“…For this reason, the PSP method can also be used to directly screen the activity of enzymes in crude extracts on PET nanoparticles. PSP based screening has the advantage in comparison with fluorescence-based methods that it is not affected by the reaction buffer (e.g., Tris-HCl) and, importantly, it can be used in a continuous mode while the reaction and the detection steps are separated in the fluorometric method [ 22 ].…”

Section: Resultsmentioning

confidence: 99%

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

Pirillo

Orlando

Tessaro

et al. 2021

IJMS

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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.

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Fluorimetric high-throughput screening method for polyester hydrolase activity using polyethylene terephthalate nanoparticles

Cited by 23 publications

References 42 publications

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

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

Engineering and evaluation of thermostable IsPETase variants for PET degradation

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

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