A flexible kinetic assay efficiently sorts prospective biocatalysts for PET plastic subunit hydrolysis

Beech, Jessica Lusty; Clare, Rita; Kincannon, William M.; Erickson, Erika; McGeehan, J.E.; Beckham, Gregg T.; DuBois, Jennifer L.

doi:10.1039/d2ra00612j

Cited by 9 publications

(7 citation statements)

References 67 publications

(71 reference statements)

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“…The sequence of TM1022 shares 28% identity with EstBs from B. subtilis 168 (CAB15367), 27% with CE1Bp from Bacillus subtilis ARA (HQ441253), and 25% with Est30 from Geobacillus stearothermophilus , (OAO77298.1). TM1022 contained a highly conserved region, Gly-Lys-Ser-Xaa-Gly; the catalytic amino acid 98 Ser residue is part of the conserved domain polypeptide Gly-Xaa-Ser-Xaa-Gly.…”

Section: Resultsmentioning

confidence: 99%

Characterization of Thermotoga maritima Esterase Capable of Hydrolyzing Bis(2-hydroxyethyl) Terephthalate

Feng,

Xue,

Xie

et al. 2024

J. Agric. Food Chem.

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The gene-encoding carboxylesterase (TM1022) from the hyperthermophilic bacterium Thermotoga maritima (T. maritima) was cloned and expressed in Escherichia coli Top10 and BL21 (DE3). Recombinant TM1022 showed the best activity at pH 8.0 and 85 °C and retained 57% activity after 8 h cultivation at 90 °C. TM1022 exhibited good stability at pH 6.0−9.0, maintaining 53% activity after incubation at pH 10.0 and 37 °C for 6 h. The esterase TM1022 exhibited the optimum thermo-alkali stability and k cat /K m (598.57 ± 19.97 s −1 mM −1 ) for pN−C4. TM1022 hydrolyzed poly(ethylene terephthalate) (PET) degradation intermediates, such as bis(2-hydroxyethyl) terephthalate (BHET) and mono(2-hydroxyethyl) terephthalate (MHET). The K m , k cat , and k cat /K m values for BHET were 0.82 ± 0.01 mM, 2.20 ± 0.02 s −1 , and 2.67 ± 0.02 mM −1 s −1 , respectively; those for MHET were 2.43 ± 0.07 mM, 0.04 ± 0.001 s −1 , and 0.02 ± 0.001 mM −1 s −1 , respectively. When purified TM1022 was added to the cutinase BhrPETase, hydrolysis of PET from drinking water bottle tops produced pure terephthalic acids (TPA) with 166% higher yield than those obtained after 72 h of incubation with BhrPETase alone as control. The above findings demonstrate that the esterase TM1022 from T. maritima has substantial potential for depolymerizing PET into monomers for reuse.

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Section: Resultsmentioning

confidence: 99%

Characterization of Thermotoga maritima Esterase Capable of Hydrolyzing Bis(2-hydroxyethyl) Terephthalate

Feng,

Xue,

Xie

et al. 2024

J. Agric. Food Chem.

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“…Namely, a major limitation of microplate format directed evolution and enzyme engineering campaigns is accumulation of the terephthalic acid product, which limits the extents of PET conversion at industrially relevant solid loadings (19). For example, phenol red dye-based indicator assays have been successfully applied for pH-sensitive monitoring of the soluble terephthalate and acidic oligomer products released by PETase activity, yet these approaches are also hampered by a lack of pH control (22).…”

Section: Resultsmentioning

confidence: 99%

Integration of pH control into Chi.Bio reactors and demonstration with small-scale enzymatic poly(ethylene terephthalate) hydrolysis

Denton,

Murphy,

Norton-Baker

et al. 2024

Preprint

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Small-scale bioreactors that are affordable and accessible would be of major benefit to the research community. In previous work, an open-source, automated bioreactor system was designed to operate up to the 30 mL scale with online optical monitoring, stirring, and temperature control, and this system, dubbed Chi.Bio, is now commercially available at a cost that is typically 1-2 orders of magnitude less than commercial bioreactors. In this work, we further expand the capabilities of the Chi.Bio system by enabling continuous pH monitoring and control through hardware and software modifications. For hardware modifications, we sourced low-cost, commercial pH circuits and made straightforward modifications to the Chi.Bio head plate to enable continuous pH monitoring. For software integration, we introduced closed-loop feedback control of the pH measured inside the Chi.Bio reactors and integrated a pH-control module into the existing Chi.Bio user interface. We demonstrated the utility of pH control through the small-scale depolymerization of the synthetic polyester, poly(ethylene terephthalate) (PET), using a benchmark cutinase enzyme, and compared this to 250 mL bioreactor hydrolysis reactions. The results in terms of PET conversion and rate, measured both by base addition and product release profiles, are statistically equivalent, with the Chi.Bio system allowing for a 20-fold reduction of purified enzyme required relative to the 250 mL bioreactor setup. Through inexpensive modifications, the ability to conduct pH control in Chi.Bio reactors widens the potential slate of biochemical reactions and biological cultivations for study in this system, and may also be adapted for use in other bioreactor platforms.

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“…Factors that affect reaction rate are temperature, pressure, proton or hydroxyl concentration, PET particle size, and the extent of surface wetting with the reaction medium. [193][194][195] F I G U R E 6 Aminolysis of polyethylene terephthalate.…”

Section: Hydrolysis: Pet Depolymerization Technique Without Applying ...mentioning

confidence: 99%

“…So, hydrolysis is a heterogeneous reaction occurring at the surface of PET. Factors that affect reaction rate are temperature, pressure, proton or hydroxyl concentration, PET particle size, and the extent of surface wetting with the reaction medium 193–195 …”

Section: Hydrolysis: Pet Depolymerization Technique Without Applying ...mentioning

confidence: 99%

A focused review on recycling and hydrolysis techniques of polyethylene terephthalate

Abedsoltan

2023

Polymer Engineering & Sci

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Polyethylene terephthalate (PET) is used in textile and packaging industries. The main source of PET production is fossil fuels with limited capacity. Also, PET products are single use that transform into high volumes of wastes, causing ecosystem problems. Recycling is proposed to confront this challenge. The four major PET recycling techniques are mechanical, chemical, pyrolysis, and enzymatic. Mechanical, pyrolysis, and enzymatic techniques have constrained capabilities to manage PET waste. Chemical recycling is the potential path to expanding recycling PET waste with possibility of upcycling and addressing dirty waste streams. Several chemical methods are introduced and discussed in literature. The five major chemical recycling techniques are glycolysis, alcoholysis, aminolysis, ammonolysis, and hydrolysis. This review describes PET depolymerization via these techniques and introduces hydrolysis as the one that can depolymerize PET in an organic‐free solvent environment. Hydrolysis tolerates PET mixed wastes streams including copolymers. It helps avoid challenges attributed to using organic solvents in reaction systems. Moreover, hydrolysis produces terephthalic acid, PET monomer, which has recently gained attention as the initiative monomer for PET production. The review focuses on three forms of hydrolysis—alkaline, neutral, and acid, by presenting background studies, issued patents, and recent trends on application of hydrolysis.

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A flexible kinetic assay efficiently sorts prospective biocatalysts for PET plastic subunit hydrolysis

Abstract: We have developed a colorimetric assay for the high throughput detection of enzymatic ester hydrolysis of PET plastic monomers, applied in a microplate format.

Cited by 9 publications

References 67 publications

Characterization of Thermotoga maritima Esterase Capable of Hydrolyzing Bis(2-hydroxyethyl) Terephthalate

Characterization of Thermotoga maritima Esterase Capable of Hydrolyzing Bis(2-hydroxyethyl) Terephthalate

Integration of pH control into Chi.Bio reactors and demonstration with small-scale enzymatic poly(ethylene terephthalate) hydrolysis

A focused review on recycling and hydrolysis techniques of polyethylene terephthalate

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