Particle Size Reduction of Poly(ethylene terephthalate) Increases the Rate of Enzymatic Depolymerization But Does Not Increase the Overall Conversion ExtentClick to copy article linkArticle link copied!
- Richard K. BrizendineRichard K. BrizendineRenewable Resources and Enabling Sciences Center, National Renewable Energy Laboratory, Golden, Colorado 80401, United StatesBOTTLE Consortium, Golden, Colorado 80401, United StatesMore by Richard K. Brizendine
- Erika EricksonErika EricksonRenewable Resources and Enabling Sciences Center, National Renewable Energy Laboratory, Golden, Colorado 80401, United StatesBOTTLE Consortium, Golden, Colorado 80401, United StatesMore by Erika Erickson
- Stefan J. HaugenStefan J. HaugenRenewable Resources and Enabling Sciences Center, National Renewable Energy Laboratory, Golden, Colorado 80401, United StatesMore by Stefan J. Haugen
- Kelsey J. RamirezKelsey J. RamirezRenewable Resources and Enabling Sciences Center, National Renewable Energy Laboratory, Golden, Colorado 80401, United StatesBOTTLE Consortium, Golden, Colorado 80401, United StatesMore by Kelsey J. Ramirez
- Joel MiscallJoel MiscallRenewable Resources and Enabling Sciences Center, National Renewable Energy Laboratory, Golden, Colorado 80401, United StatesBOTTLE Consortium, Golden, Colorado 80401, United StatesMore by Joel Miscall
- Davinia SalvachúaDavinia SalvachúaRenewable Resources and Enabling Sciences Center, National Renewable Energy Laboratory, Golden, Colorado 80401, United StatesMore by Davinia Salvachúa
- Andrew R. PickfordAndrew R. PickfordBOTTLE Consortium, Golden, Colorado 80401, United StatesCentre for Enzyme Innovation, School of Biological Sciences, Institute of Biological and Biomedical Sciences, University of Portsmouth, Portsmouth PO1 2DY, U.K.More by Andrew R. Pickford
- Margaret J. SobkowiczMargaret J. SobkowiczDepartment of Plastics Engineering, University of Massachusetts Lowell, Lowell, Massachusetts 01854, United StatesMore by Margaret J. Sobkowicz
- John E. McGeehan*John E. McGeehan*Email: [email protected]BOTTLE Consortium, Golden, Colorado 80401, United StatesCentre for Enzyme Innovation, School of Biological Sciences, Institute of Biological and Biomedical Sciences, University of Portsmouth, Portsmouth PO1 2DY, U.K.More by John E. McGeehan
- Gregg T. Beckham*Gregg T. Beckham*Email: [email protected]Renewable Resources and Enabling Sciences Center, National Renewable Energy Laboratory, Golden, Colorado 80401, United StatesBOTTLE Consortium, Golden, Colorado 80401, United StatesMore by Gregg T. Beckham
Abstract
Enzymatic depolymerization of poly(ethylene terephthalate) (PET) has emerged as a potential method for PET recycling, but extensive thermomechanical preprocessing to reduce both the crystallinity and particle size of PET is often conducted, which is costly and energy-intensive. In the current work, we use high-crystallinity PET (HC-PET) and low-crystallinity cryomilled PET (CM-PET) with three distinct particle size distributions to investigate the effect of PET particle size and crystallinity on the performance of a variant of the leaf compost-cutinase enzyme (LCC-ICCG). We show that LCC-ICCG hydrolyzes PET, resulting in the accumulation of terephthalic acid and, interestingly, also releases significant amount of mono(2-hydroxyethyl)terephthalate. Particle size reduction of PET increased the maximum rate of reaction for HC-PET, while the maximum hydrolysis rate for CM-PET was not significantly different across particle sizes. For both substrates, however, we show that particle size reduction has little effect on the overall conversion extent. Specifically, the CM-PET film was converted to 99 ± 0.2% mass loss within 48 h, while the HC-PET powder reached only 23.5 ± 0.0% conversion in 144 h. Overall, these results suggest that amorphization of PET is a necessary pretreatment step for enzymatic PET recycling using the LCC-ICCG enzyme but that particle size reduction may not be required.
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Attribution (BY): Credit must be given to the creator.
*Disclaimer
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License Summary*
You are free to share(copy and redistribute) this article in any medium or format and to adapt(remix, transform, and build upon) the material for any purpose, even commercially within the parameters below:
Creative Commons (CC): This is a Creative Commons license.
Attribution (BY): Credit must be given to the creator.
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Synopsis
PET particle size reduction increases the rate but not the extent of conversion by a PET-degrading enzyme.
Introduction
Materials and Methods
Reagents, Stocks, and Buffers
Preparation and Characterization of PET Particles

Expression and Purification of the LCC-ICCG Enzyme
Enzymatic Hydrolysis Experiments
Ultrahigh-Performance Liquid Chromatography Analysis of Reaction Timepoints
Determination of Initial Rates and Kinetic Analysis
Results and Discussion
Characterization of PET Substrates
Effect of Particle Size and Crystallinity on Enzymatic Degradation of PET
Figure 1
Figure 1. Size distributions of CM-PET and HC-PET particles. Histograms indicate the volume percentage as a function of Feret’s minimum diameter of CM-PET (blue) and HC-PET (red) substrates. (A,D) 250, (B,E) 125, and (C,F) sub125 μm sieve fractions. Inset graphs show the expanded view of the data.
PET substrate | source | sieve fraction (μm) | specific SA (m2 g–1) | Tg (°C) | % crystallinity | Mn (kDa) | Mw (kDa) |
---|---|---|---|---|---|---|---|
LC-PET | low-crystallinity PET film, Goodfellow ES301445 | N/A | N/A | 75.7 ± 0.6 | 4.2 ± 2 | 19.7 ± 8.3 | 33.8 ± 6.8 |
CM-PET | cryomilled LC-PET, from Goodfellow ES301445 | 250 | 0.01 | 79.0 ± 0.8 | 11.0 ± 1.8 | 20.9 ± 7.5 | 34.9 ± 6.7 |
125 | 0.02 | 74.2 ± 0.3 | 7.6 ± 1 | 19.3 ± 8.6 | 34.8 ± 6.5 | ||
sub125 | 0.12 | 69.4 ± 0.7 | 14.3 ± 2.3 | 20.3 ± 4.9 | 34.5 ± 6.4 | ||
HC-PET | semicrystalline powder, Goodfellow ES306031 | 250 | 0.012 | 76.1 ± 0.3 | 35.7 ± 3.8 | 19.9 ± 6.4 | 32.5 ± 7.1 |
125 | 0.022 | 76.6 ± 0.6 | 33.0 ± 2.1 | 19.2 ± 5.7 | 33.0 ± 6.3 | ||
sub125 | 0.06 | 76.8 ± 0.6 | 32.5 ± 1.7 | 20.1 ± 7.2 | 32.1 ± 6.9 | ||
WC-PET | fines supplied by Western Container Corporation | N/A | N/A | 70.0 ± 0.5 | 37.3 ± 2.5 | 26.7 ± 8.4 | 43.5 ± 6.6 |
Specific surface area calculated from stereomicroscopy, DSC data, and GPC data for all PET substrates. ± indicates SD, n = 3.
Figure 2
Figure 2. Monomer release as a function of time for 10 mg mL–1 PET particles with 1 μM LCC-ICCG. Monomers present in the reaction were analyzed by HPLC. (A) 250 μm CM-PET, (B) 125 μm CM-PET, (C) sub125 μm CM-PET, (D) 250 μm HC-PET, (E) 125 μm HC-PET, and (F) sub125 μm HC-PET. Reactions were performed in triplicate and errors bars represent the standard deviation. The data shown in this figure are provided in Dataset S1.
Figure 3
Figure 3. Total amount of monomers released from PET particles hydrolyzed with LCC-ICCG. The bars show the concentrations of all monomers released at the 72 h endpoint of the reaction with indicated [LCC-ICCG] and 10 mg mL–1 PET substrate. Reactions were performed in triplicate, and error bars represent standard deviation. The data shown in this figure are provided in Dataset S3.
Figure 4
Figure 4. InvMM kinetic analysis and total amount of monomers released from PET particles upon hydrolysis with LCC-ICCG. InvMM analysis of 10 mg mL–1 (A) CM-PET and (B) HC-PET hydrolysis rate as a function of LCC-ICCG concentration ([E]). Lines are fits of the invMM equation. Table 2 shows the fitted parameters. Reactions were performed in triplicate and error bars represent standard deviation. The data in this figure are provided in Dataset S5.
PET substrate | sieve fraction (μm) | invKm (μM) | invVmax (μmol g–1 s–1) |
---|---|---|---|
CM-PET | 250 | 3.4 ± 1.7 | 0.49 ± 0.14 |
125 | 2.7 ± 1.2 | 0.48 ± 0.11 | |
sub125 | 1.1 ± 0.3 | 0.40 ± 0.04 | |
HC-PET | 250 | 0.6 ± 0.1 | 0.08 ± 0.01 |
125 | 1.3 ± 0.3 | 0.14 ± 0.02 | |
sub125 | 0.7 ± 0.1 | 0.16 ± 0.01 |
Calculated from data shown in Figure 3. The error indicates the standard error of the fit.
Figure 5
Figure 5. Conversion time courses of 1 L scale reactions conducted in bioreactors. Results from the reaction with various PET substrates at 100 g L–1 and 3 mg g–1 PET LCC-ICCG enzyme. (A–C) Product release profiles as a function of time measured by HPLC for (A) LC-PET film cut into ∼1 × 1 cm squares, (B) HC-PET particles, and (C) WC-PET particles. (D–F) Percent conversion of total PET for (D) LC-PET film cut into ∼1 × 1 cm squares, (E) HC-PET particles, and (F) WC-PET particles. Reactions were performed in duplicate, data points show the mean, and error bars show the absolute difference between the duplicates. The data shown in this figure are provided in Dataset S6.
Conclusions
Supporting Information
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acssuschemeng.2c01961.
Additional characterization and kinetic data as well as datasets for all figures (PDF)
Terms & Conditions
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Acknowledgments
Funding was provided by the U.S. DOE, Office of Energy Efficiency and Renewable Energy, Advanced Manufacturing Office (AMO) and Bioenergy Technologies Office (BETO) under contract no. DE-FOA-0002029. This work was also performed as part of the Bio-Optimized Technologies to keep Thermoplastics out of Landfills and the Environment (BOTTLE) Consortium and was supported by AMO and BETO under contract no. DE-AC36-08GO28308 with NREL, operated by Alliance for Sustainable Energy, LLC. The BOTTLE Consortium includes members from the University of Portsmouth, funded under contract no. DE-AC36-08GO28308 with NREL and additionally supported by Research England (E3 scheme). The bioreactor experiments were funded by DARPA via cooperative agreement number IAG-21-17585. This document was approved by DARPA on January 24, 2022, for public release, distribution unlimited. We thank Western Container Corporation for providing PET fines in support of this work. The views expressed in the article do not necessarily represent the views of the DOE or the U.S. Government. The U.S. Government retains and the publisher by accepting the article for publication, acknowledges that the U.S. Government retains a nonexclusive, paid-up, irrevocable, worldwide license to publish or reproduce the published form of this work, or allow others to do so, for U.S. Government purposes.
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- 11Wei, R.; Zimmermann, W. Biocatalysis as a green route for recycling the recalcitrant plastic polyethylene terephthalate. Microbiol. Biotechnol. 2017, 10, 1302– 1307, DOI: 10.1111/1751-7915.12714Google ScholarThere is no corresponding record for this reference.
- 12Müller, R.-J.; Schrader, H.; Profe, J.; Dresler, K.; Deckwer, W.-D. Enzymatic Degradation of Poly(ethylene terephthalate): Rapid Hydrolyse using a Hydrolase from T. fusca. Macromol. Rapid Commun. 2005, 26, 1400– 1405, DOI: 10.1002/marc.200500410Google Scholar12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXhtVGiur7E&md5=f3a2691f34a2d3e363b80090bb265d2fEnzymatic degradation of poly(ethylene terephthalate): Rapid hydrolyse using a hydrolase from T. fuscaMueller, Rolf-Joachim; Schrader, Hedwig; Profe, Joern; Dresler, Karolin; Deckwer, Wolf-DieterMacromolecular Rapid Communications (2005), 26 (17), 1400-1405CODEN: MRCOE3; ISSN:1022-1336. (Wiley-VCH Verlag GmbH & Co. KGaA)It is demonstrated that PET, which is usually regarded as 'non-biodegradable', can effectively be depolymd. by a hydrolase from the actinomycete Thermobifida fusca. Erosion rates of 8 to 17 μm per wk were obtained upon incubation at 55°. Lipases from Pseudomonas sp. and Candida antarctica did not degrade PET under comparable conditions. The influences of crystallinity, m.p., and glass transition temp. on the enzymic attack on PET, PBT, and PHB are discussed.
- 13Alisch-Mark, M.; Herrmann, A.; Zimmermann, W. Increase of the hydrophilicity of polyethylene terephthalate fibres by hydrolases from Thermomonospora fusca and Fusarium solani f. sp. pisi. Biotechnol. Lett. 2006, 28, 681– 685, DOI: 10.1007/s10529-006-9041-7Google Scholar13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XmtVOqs7Y%253D&md5=0f59affe7b16b73a8d674480024d5621Increase of the hydrophilicity of polyethylene terephthalate fibres by hydrolases from Thermomonospora fusca and Fusarium solani f. sp. pisiAlisch-Mark, Mandy; Herrmann, Anne; Zimmermann, WolfgangBiotechnology Letters (2006), 28 (10), 681-685CODEN: BILED3; ISSN:0141-5492. (Springer)Treatment of polyethylene terephthalate fibers with hydrolase prepns. from Thermomonospora (Thermobifida) fusca and Fusarium solani f. sp. pisi resulted in an increase of the hydrophilicity of the fibers detd. by measurement of their dyeing behavior with reactive dyes and their water absorption ability. Reflectance spectrometry of treated fibers dyed with a reactive dye showed that the color became more intense corresponding to an increase of hydroxyl groups on the fiber surfaces and indicated a stepwise peeling of the fibers by the enzymes comparable to the effects obtained by alk. treatments. The synthetic fibers treated with the hydrolase from T. fusca also showed enhanced water absorption ability further confirming the increased surface hydrophilicity caused by the enzyme.
- 14Eberl, A.; Heumann, S.; Brückner, T.; Araujo, R.; Cavaco-Paulo, A.; Kaufmann, F.; Kroutil, W.; Guebitz, G. M. Enzymatic surface hydrolysis of poly(ethylene terephthalate) and bis(benzoyloxyethyl) terephthalate by lipase and cutinase in the presence of surface active molecules. J. Biotechnol. 2009, 143, 207– 212, DOI: 10.1016/j.jbiotec.2009.07.008Google Scholar14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhtVKksbfJ&md5=21046f062c67b8d8ec6ab8bc4518dfa9Enzymatic surface hydrolysis of poly(ethylene terephthalate) and bis(benzoyloxyethyl) terephthalate by lipase and cutinase in the presence of surface active moleculesEberl, Anita; Heumann, Sonja; Brueckner, Tina; Araujo, Rita; Cavaco-Paulo, Artur; Kaufmann, Franz; Kroutil, Wolfgang; Guebitz, Georg M.Journal of Biotechnology (2009), 143 (3), 207-212CODEN: JBITD4; ISSN:0168-1656. (Elsevier B.V.)A lipase from Thermomyces lanuginosus and cutinases from Thermobifida fusca and Fusarium solani hydrolyzed poly(ethylene terephthalate) (PET) fabrics and films and bis(benzoyloxyethyl) terephthalate (3PET) endo-wise as shown by MALDI-Tof-MS, LC-UVD/MS, cationic dyeing and XPS anal. Due to interfacial activation of the lipase in the presence of Triton X-100, a seven-fold increase of hydrolysis products released from 3PET was measured. In the presence of the plasticizer N,N-diethyl-2-phenylacetamide (DEPA), increased hydrolysis rates of semi-cryst. PET films and fabrics were measured both for lipase and cutinase. The formation of novel polar groups resulted in enhanced dye ability with addnl. increase in color depth by 130% and 300% for cutinase and lipase, resp., in the presence of plasticizer.
- 15Ronkvist, Å. M.; Xie, W.; Lu, W.; Gross, R. A. Cutinase-Catalyzed Hydrolysis of Poly(ethylene terephthalate). Macromolecules 2009, 42, 5128– 5138, DOI: 10.1021/ma9005318Google Scholar15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXotVGksb0%253D&md5=3efba9137ee43e2684de56c081549668Cutinase-Catalyzed Hydrolysis of Poly(ethylene terephthalate)Ronkvist, Asa M.; Xie, Wenchun; Lu, Wenhua; Gross, Richard A.Macromolecules (Washington, DC, United States) (2009), 42 (14), 5128-5138CODEN: MAMOBX; ISSN:0024-9297. (American Chemical Society)A detailed study and comparison was made on the catalytic activities of cutinases from Humilica insolens (HiC), Pseudomonas mendocina (PmC), and Fusarium solani (FsC) using low-crystallinity (lc) and biaxially oriented (bo) poly(ethylene terephthalate) (PET) films as model substrates. Cutinase activity for PET hydrolysis was assayed using a pH-stat to measure NaOH consumption vs. time, where initial activity was expressed as units of micromoles of NaOH added per h and per mL of reaction vol. HiC was found to have good thermostability with max. initial activity from 70 to 80°, whereas PmC and FsC performed best at 50°. Assays by pH-stat showed that the cutinases had about 10-fold higher activity for the lcPET (7% crystallinity) than for the boPET (35% crystallinity). Under optimal reaction conditions, initial activities of cutinases were successfully fit by a heterogeneous kinetic model. The hydrolysis rate const. k2 was 7-fold higher for HiC at 70° (0.62 μmol/Cm2/h) relative to PmC and FsC at 50 and 40°, resp. With respect to PET affinity, PmC had the highest affinity, while FsC had the lowest value. In a 96 h degrdn. study using lcPET films, incubation with PmC and FsC both resulted in a 5% film wt. loss at 50 and 40°, resp. In contrast, HiC-catalyzed lcPET film hydrolysis at 70° resulted in a 97±3% wt. loss in 96 h, corresponding to a loss in film thickness of 30 μm per day. As degrdn. of lcPET progressed, crystallinity of the remaining film increased to 27% due to preferential degrdn. of amorphous regions. Furthermore, for all three cutinases, anal. of aq. sol. degrdn. products showed that they consist exclusively of terephthalic acid and ethylene glycol.
- 16Ribitsch, D.; Heumann, S.; Trotscha, E.; Herrero Acero, E.; Greimel, K.; Leber, R.; Birner-Gruenberger, R.; Deller, S.; Eiteljoerg, I.; Remler, P. Hydrolysis of polyethyleneterephthalate by p-nitrobenzylesterase from Bacillus subtilis. Biotechnol. Prog. 2011, 27, 951– 960, DOI: 10.1002/btpr.610Google Scholar16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhtValt7jM&md5=c2c3355b13942877c10c2f060f22529fHydrolysis of polyethyleneterephthalate by p-nitrobenzylesterase from Bacillus subtilisRibitsch, Doris; Heumann, Sonja; Trotscha, Eva; Acero, Enrique Herrero; Greimel, Katrin; Leber, Regina; Birner-Gruenberger, Ruth; Deller, Sigrid; Eiteljoerg, Inge; Remler, Peter; Weber, Thomas; Siegert, Petra; Maurer, Karl-Heinz; Donelli, Ilaria; Freddi, Giuliano; Schwab, Helmut; Guebitz, Georg M.Biotechnology Progress (2011), 27 (4), 951-960CODEN: BIPRET; ISSN:1520-6033. (Wiley-Blackwell)From a screening on agar plates with bis(benzoyloxyethyl) terephthalate (3PET), a Bacillus subtilis p-nitrobenzylesterase (BsEstB) was isolated and demonstrated to hydrolyze polyethyleneterephthalate (PET). PET-hydrolase active strains produced clearing zones and led to the release of the 3PET hydrolysis products terephthalic acid (TA), benzoic acid (BA), 2-hydroxyethyl benzoate (HEB), and mono-(2-hydroxyethyl) terephthalate (MHET) in 3PET supplemented liq. cultures. The 3PET-hydrolase was isolated from non-denaturating polyacrylamide gels using fluorescein diacetate (FDA) and identified as BsEstB by LC-MS/MS anal. BsEstB was expressed in Escherichia coli with C-terminally fused StrepTag II for purifn. The tagged enzyme had a mol. mass of 55.2 kDa and a specific activity of 77 U/mg on p-nitrophenyl acetate and 108 U/mg on p-nitrophenyl butyrate. BsEstB was most active at 40°C and pH 7.0 and stable for several days at pH 7.0 and 37°C while the half-life times decreased to 3 days at 40°C and only 6 h at 45°C. From 3PET, BsEstB released TA, MHET, and BA, but neither bis(2-hydroxyethyl) terephthalate (BHET) nor hydroxyethylbenzoate (HEB). The kcat values decreased with increasing complexity of the substrate from 6 and 8 (s-1) for p-nitrophenyl-acetate (4NPA) and p-nitrophenyl-butyrate (4NPB), resp., to 0.14 (s-1) for bis(2-hydroxyethyl) terephthalate (BHET). The enzyme hydrolyzed PET films releasing TA and MHET with a concomitant decrease of the water-contact angle (WCA) from 68.2° ± 1.7° to 62.6° ± 1.1° due to formation of novel hydroxyl and carboxyl groups. These data correlated with a fluorescence emission intensity increase seen for the enzyme treated sample after derivatization with 2-(bromomethyl)naphthalene.
- 17Sulaiman, S.; Yamato, S.; Kanaya, E.; Kim, J.-J.; Koga, Y.; Takano, K.; Kanaya, S. Isolation of a novel cutinase homolog with polyethylene terephthalate-degrading activity from leaf-branch compost by using a metagenomic approach. Appl. Environ. Microbiol. 2012, 78, 1556– 1562, DOI: 10.1128/aem.06725-11Google Scholar17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XjtVSntLY%253D&md5=5f056dac0e9ab4a5b00c2185ee936e9bIsolation of a novel cutinase homolog with polyethylene terephthalate-degrading activity from leaf-branch compost by using a metagenomic approachSulaiman, Sintawee; Yamato, Saya; Kanaya, Eiko; Kim, Joong-Jae; Koga, Yuichi; Takano, Kazufumi; Kanaya, ShigenoriApplied and Environmental Microbiology (2012), 78 (5), 1556-1562CODEN: AEMIDF; ISSN:0099-2240. (American Society for Microbiology)The gene encoding a cutinase homolog, LC-cutinase, was cloned from a fosmid library of a leaf-branch compost metagenome by functional screening using tributyrin agar plates. LC-cutinase shows the highest amino acid sequence identity of 59.7% to Thermomonospora curvata lipase. It also shows the 57.4% identity to Thermobifida fusca cutinase. When LC-cutinase without a putative signal peptide was secreted to the periplasm of Escherichia coli cells with the assistance of the pelB leader sequence, more than 50% of the recombinant protein, termed LC-cutinase*, was excreted into the extracellular medium. It was purified and characterized. LC-cutinase* hydrolyzed various fatty acid monoesters with acyl chain lengths of 2 to 18, with a preference for short-chain substrates (C4 substrate at most) most optimally at pH 8.5 and 50°C, but could not hydrolyze olive oil. It lost activity with half-lives of 40 min at 70°C and 7 min at 80°C. LC-cutinase* had an ability to degrade poly(ε-caprolactone) and polyethylene terephthalate (PET). The specific PET-degrading activity of LC-cutinase* was detd. to be 12 mg/h/mg of enzyme (2.7 mg/h/μkat of pNP-butyrate-degrading activity) at pH 8.0 and 50°C. This activity is higher than those of the bacterial and fungal cutinases reported thus far, suggesting that LC-cutinase* not only serves as a good model for understanding the mol. mechanism of PET-degrading enzyme but also is potentially applicable for surface modification and degrdn. of PET.
- 18Roth, C.; Wei, R.; Oeser, T.; Then, J.; Föllner, C.; Zimmermann, W.; Sträter, N. Structural and functional studies on a thermostable polyethylene terephthalate degrading hydrolase from Thermobifida fusca. Appl. Microbiol. Biotechnol. 2014, 98, 7815– 7823, DOI: 10.1007/s00253-014-5672-0Google Scholar18https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXmtlWiu7Y%253D&md5=d5bf2d167198e40b3d4c79b24b347fedStructural and functional studies on a thermostable polyethylene terephthalate degrading hydrolase from Thermobifida fuscaRoth, Christian; Wei, Ren; Oeser, Thorsten; Then, Johannes; Foellner, Christina; Zimmermann, Wolfgang; Straeter, NorbertApplied Microbiology and Biotechnology (2014), 98 (18), 7815-7823CODEN: AMBIDG; ISSN:0175-7598. (Springer)Bacterial cutinases are promising catalysts for the modification and degrdn. of the widely used plastic polyethylene terephthalate (PET). The improvement of the enzyme for industrial purposes is limited due to the lack of structural information for cutinases of bacterial origin. Here, the authors report having crystd. and structurally characterized a cutinase from T. fusca KW3 (TfCut2) in free as well as in inhibitor-bound form. Together with an anal. of the thermostability and modeling studies, the authors suggest possible reasons for the outstanding thermostability in comparison to the less thermostable homolog from T. alba AHK119 and propose a model for the binding of the enzyme toward its polymeric substrate. The TfCut2 structure is the basis for the rational design of catalytically more efficient enzyme variants for the hydrolysis of PET and other synthetic polyesters.
- 19Sulaiman, S.; You, D.-J.; Kanaya, E.; Koga, Y.; Kanaya, S. Crystal Structure and Thermodynamic and Kinetic Stability of Metagenome-Derived LC-Cutinase. Biochemistry 2014, 53, 1858– 1869, DOI: 10.1021/bi401561pGoogle Scholar19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXjsVehs7w%253D&md5=6a3d102dc43578ef18b633c5e09c3f38Crystal Structure and Thermodynamic and Kinetic Stability of Metagenome-Derived LC-CutinaseSulaiman, Sintawee; You, Dong-Ju; Kanaya, Eiko; Koga, Yuichi; Kanaya, ShigenoriBiochemistry (2014), 53 (11), 1858-1869CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)The crystal structure of metagenome-derived LC-cutinase with polyethylene terephthalate (PET)-degrading activity was detd. at 1.5 Å resoln. The structure strongly resembles that of Thermobifida alba cutinase. Ser165, Asp210, and His242 form the catalytic triad. Thermal denaturation and guanidine hydrochloride (GdnHCl)-induced unfolding of LC-cutinase were analyzed at pH 8.0 by CD spectroscopy. The midpoint of the transition of the thermal denaturation curve, T1/2, and that of the GdnHCl-induced unfolding curve, Cm, at 30 °C were 86.2 °C and 4.02 M, resp. The free energy change of unfolding in the absence of GdnHCl, ΔG(H2O), was 41.8 kJ mol-1 at 30 °C. LC-cutinase unfolded very slowly in GdnHCl with an unfolding rate, ku(H2O), of 3.28 × 10-6 s-1 at 50 °C. These results indicate that LC-cutinase is a kinetically robust protein. Nevertheless, the optimal temp. for the activity of LC-cutinase toward p-nitrophenyl butyrate (50 °C) was considerably lower than the T1/2 value. It increased by 10 °C in the presence of 1% polyethylene glycol (PEG) 1000. It also increased by at least 20 °C when PET was used as a substrate. These results suggest that the active site is protected from a heat-induced local conformational change by binding of PEG or PET. LC-cutinase contains one disulfide bond between Cys275 and Cys292. To examine whether this disulfide bond contributes to the thermodn. and kinetic stability of LC-cutinase, C275/292A-cutinase without this disulfide bond was constructed. Thermal denaturation studies and equil. and kinetic studies of the GdnHCl-induced unfolding of C275/292A-cutinase indicate that this disulfide bond contributes not only to the thermodn. stability but also to the kinetic stability of LC-cutinase.
- 20Wei, R.; Oeser, T.; Zimmermann, W. Synthetic polyester-hydrolyzing enzymes from thermophilic actinomycetes. Adv. Appl. Microbiol. 2014, 89, 267– 305, DOI: 10.1016/B978-0-12-800259-9.00007-XGoogle Scholar20https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2M%252Fisl2huw%253D%253D&md5=e7be6a3e3c6769542d851ffd65c9280eSynthetic polyester-hydrolyzing enzymes from thermophilic actinomycetesWei Ren; Oeser Thorsten; Zimmermann WolfgangAdvances in applied microbiology (2014), 89 (), 267-305 ISSN:0065-2164.Thermophilic actinomycetes produce enzymes capable of hydrolyzing synthetic polyesters such as polyethylene terephthalate (PET). In addition to carboxylesterases, which have hydrolytic activity predominantly against PET oligomers, esterases related to cutinases also hydrolyze synthetic polymers. The production of these enzymes by actinomycetes as well as their recombinant expression in heterologous hosts is described and their catalytic activity against polyester substrates is compared. Assays to analyze the enzymatic hydrolysis of synthetic polyesters are evaluated, and a kinetic model describing the enzymatic heterogeneous hydrolysis process is discussed. Structure-function and structure-stability relationships of actinomycete polyester hydrolases are compared based on molecular dynamics simulations and recently solved protein structures. In addition, recent progress in enhancing their activity and thermal stability by random or site-directed mutagenesis is presented.
- 21Perz, V.; Bleymaier, K.; Sinkel, C.; Kueper, U.; Bonnekessel, M.; Ribitsch, D.; Guebitz, G. M. Substrate specificities of cutinases on aliphatic-aromatic polyesters and on their model substrates. New Biotechnol. 2016, 33, 295– 304, DOI: 10.1016/j.nbt.2015.11.004Google Scholar21https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhvVykurvE&md5=64681992221ed7dc6cbc62a2979580edSubstrate specificities of cutinases on aliphatic-aromatic polyesters and on their model substratesPerz, Veronika; Bleymaier, Klaus; Sinkel, Carsten; Kueper, Ulf; Bonnekessel, Melanie; Ribitsch, Doris; Guebitz, Georg M.New Biotechnology (2016), 33 (2), 295-304CODEN: NBEIBR; ISSN:1871-6784. (Elsevier B.V.)The enzymic hydrolysis of the biodegradable polyester ecoflex and of a variety of oligomeric and polymeric ecoflex model substrates was investigated. For this purpose, substrate specificities of two enzymes of typical compost inhabitants, namely a fungal cutinase from Humicola insolens (HiC) and a bacterial cutinase from Thermobifida cellulosilytica (Thc_Cut1) were compared. Model substrates were systematically designed with variations of the chain length of the alc. and the acid as well as with varying content of the arom. constituent terephthalic acid (Ta).HPLC/MS identification and quantification of the hydrolysis products terephthalic acid (Ta), benzoic acid (Ba), adipic acid (Ada), mono(4-hydroxybutyl) terephthalate (BTa), mono-(2-hydroxyethyl) terephthalate (ETa), mono-(6-hydroxyhexyl) terephthalate (HTa) and bis(4-hydroxybutyl) terephthalate (BTaB) indicated that these enzymes indeed hydrolyze the tested esters. Shorter terminal chain length acids but longer chain length alcs. in oligomeric model substrates were generally hydrolyzed more efficiently. Thc_Cut1 hydrolyzed arom. ester bonds more efficiently than HiC resulting in up to 3-fold higher concns. of the monomeric hydrolysis product Ta. Nevertheless, HiC exhibited a higher overall hydrolytic activity on the tested polyesters, resulting in 2-fold higher concn. of released mols. Thermogravimetry and differential scanning calorimetry (TG-DSC) of the polymeric model substrates revealed a general trend that a lower difference between melting temp. (Tm) and the temp. at which the enzymic degrdn. takes place resulted in higher susceptibility to enzymic hydrolysis.
- 22Yoshida, S.; Hiraga, K.; Takehana, T.; Taniguchi, I.; Yamaji, H.; Maeda, Y.; Toyohara, K.; Miyamoto, K.; Kimura, Y.; Oda, K. A bacterium that degrades and assimilates poly(ethylene terephthalate). Science 2016, 351, 1196– 1199, DOI: 10.1126/science.aad6359Google Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xjs12gtr4%253D&md5=2bd8b9ff8d09fcc55944c8dc65b9cd46A bacterium that degrades and assimilates poly(ethylene terephthalate)Yoshida, Shosuke; Hiraga, Kazumi; Takehana, Toshihiko; Taniguchi, Ikuo; Yamaji, Hironao; Maeda, Yasuhito; Toyohara, Kiyotsuna; Miyamoto, Kenji; Kimura, Yoshiharu; Oda, KoheiScience (Washington, DC, United States) (2016), 351 (6278), 1196-1199CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Poly(ethylene terephthalate) (PET) is used extensively worldwide in plastic products, and its accumulation in the environment has become a global concern. Because the ability to enzymically degrade PET has been thought to be limited to a few fungal species, biodegrdn. is not yet a viable remediation or recycling strategy. By screening natural microbial communities exposed to PET in the environment, the authors isolated a novel bacterium, Ideonella sakaiensis 201-F6, that is able to use PET as its major energy and carbon source. When grown on PET, this strain produces two enzymes capable of hydrolyzing PET and the reaction intermediate, mono(2-hydroxyethyl) terephthalic acid. Both enzymes are required to enzymically convert PET efficiently into its two environmentally benign monomers, terephthalic acid and ethylene glycol.
- 23Ribitsch, D.; Hromic, A.; Zitzenbacher, S.; Zartl, B.; Gamerith, C.; Pellis, A.; Jungbauer, A.; Łyskowski, A.; Steinkellner, G.; Gruber, K. Small cause, large effect: Structural characterization of cutinases from Thermobifida cellulosilytica. Biotechnol. Bioeng. 2017, 114, 2481– 2488, DOI: 10.1002/bit.26372Google Scholar23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtlart7vL&md5=69ee041cdeba69c3be8a4582e4a7d69dSmall cause, large effect: Structural characterization of cutinases from Thermobifida cellulosilyticaRibitsch, Doris; Hromic, Altijana; Zitzenbacher, Sabine; Zartl, Barbara; Gamerith, Caroline; Pellis, Alessandro; Jungbauer, Alois; Lyskowski, Andrzej; Steinkellner, Georg; Gruber, Karl; Tscheliessnig, Rupert; Herrero Acero, Enrique; Guebitz, Georg M.Biotechnology and Bioengineering (2017), 114 (11), 2481-2488CODEN: BIBIAU; ISSN:0006-3592. (John Wiley & Sons, Inc.)We have investigated the structures of two native cutinases from Thermobifida cellulosilytica, namely Thc_Cut1 and Thc_Cut2 as well as of two variants, Thc_Cut2_DM (Thc_Cut2_ Arg29Asn_Ala30Val) and Thc_Cut2_TM (Thc_Cut2_Arg19Ser_Arg29Asn_Ala30Val). The four enzymes showed different activities towards the aliph. polyester poly(lactic acid) (PLLA). The crystal structures of the four enzymes were successfully solved and in combination with Small Angle X-Ray Scattering (SAXS) the structural features responsible for the selectivity difference were elucidated. Anal. of the crystal structures did not indicate significant conformational differences among the different cutinases. However, the distinctive SAXS scattering data collected from the enzymes in soln. indicated a remarkable surface charge difference. The difference in the electrostatic and hydrophobic surface properties could explain potential alternative binding modes of the four cutinases on PLLA explaining their distinct activities. Biotechnol. Bioeng. 2017;9999: 1-8. © 2017 Wiley Periodicals, Inc.
- 24Kawai, F.; Kawabata, T.; Oda, M. Current State and Perspectives Related to the Polyethylene Terephthalate Hydrolases Available for Biorecycling. ACS Sustainable Chem. Eng. 2020, 8, 8894– 8908, DOI: 10.1021/acssuschemeng.0c01638Google Scholar24https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXpvFWqtLY%253D&md5=ddbd5aaa091f9b6600fdd8dec57e216dCurrent State and Perspectives Related to the Polyethylene Terephthalate Hydrolases Available for BiorecyclingKawai, Fusako; Kawabata, Takeshi; Oda, MasayukiACS Sustainable Chemistry & Engineering (2020), 8 (24), 8894-8908CODEN: ASCECG; ISSN:2168-0485. (American Chemical Society)A review. Polyethylene terephthalate (PET) hydrolase is a challenging target as PET is a commonly used plastic that is extremely resistant to enzymic attack. Since the discovery of a PET hydrolase from Thermobifida fusca in 2005, novel PET hydrolases and their availability toward waste PET have been investigated. At present, at least four thermophilic cutinases are known as PET hydrolases that could be used for the management of amorphous PET waste, such as packaging materials. Heat-labile PETase from Ideonella sakaiensis and its homologues from mesophilic bacteria exist in the environment. However, PET can be efficiently hydrolyzed with thermophilic hydrolases. This Review focuses on the current state of PET hydrolases and the potential of their application. Contrary to an amorphous PET, the enzymic hydrolysis of cryst. PET (particularly PET bottles) remains to be fully elucidated. It cannot be assured whether the biorecycling of general PET would be put into practice in the near future, but the plan is getting closer to the goal. PET hydrolases can be versatile polyesterases as they can hydrolyze not only PET but also other polyesters. Addnl., the thermostability of PET hydrolases is advantageous to their application in terms of reaction speed and durability. Enzymic recycling is more ecofriendly than hazardous chem. recycling, and thermophilic PET hydrolases are indispensable for the biorecycling of PET.
- 25Sonnendecker, C.; Oeser, J.; Richter, P. K.; Hille, P.; Zhao, Z.; Fischer, C.; Lippold, H.; Blazquez-Sanchez, P.; Engelberger, F.; Ramirez-Sarmiento, C. A. Low Carbon Footprint Recycling of Post-Consumer PET Plastic with a Metagenomic Polyester Hydrolase. ChemSusChem 2022, 15, e202101062 DOI: 10.1002/cssc.202101062Google Scholar25https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XjsFCltro%253D&md5=2e80ffb54c6bedd31d57fcd46a2e8a56Low Carbon Footprint Recycling of Post-Consumer PET Plastic with a Metagenomic Polyester HydrolaseSonnendecker, Christian; Oeser, Juliane; Richter, P. Konstantin; Hille, Patrick; Zhao, Ziyue; Fischer, Cornelius; Lippold, Holger; Blazquez-Sanchez, Paula; Engelberger, Felipe; Ramirez-Sarmiento, Cesar A.; Oeser, Thorsten; Lihanova, Yuliia; Frank, Ronny; Jahnke, Heinz-Georg; Billig, Susan; Abel, Bernd; Strater, Norbert; Matysik, Jorg; Zimmermann, WolfgangChemSusChem (2022), 15 (9), e202101062CODEN: CHEMIZ; ISSN:1864-5631. (Wiley-VCH Verlag GmbH & Co. KGaA)Earth is flooded with plastics and the need for sustainable recycling strategies for polymers has become increasingly urgent. Enzyme-based hydrolysis of post-consumer plastic is an emerging strategy for closed-loop recycling of polyethylene terephthalate (PET). The polyester hydrolase PHL7, isolated from a compost metagenome, completely hydrolyzes amorphous PET films, releasing 91 mg of terephthalic acid per h and mg of enzyme. Vertical scanning interferometry shows degrdn. rates of the PET film of 6.8μm h-1. Structural anal. indicates the importance of leucine at position 210 for the extraordinarily high PET-hydrolyzing activity of PHL7. Within 24 h, 0.6 mgenzyme gPET-1 completely degrades post-consumer thermoform PET packaging in an aq. buffer at 70°C without any energy-intensive pretreatments. Terephthalic acid recovered from the enzymic hydrolyzate is then used to synthesize virgin PET, demonstrating the potential of polyester hydrolases as catalysts in sustainable PET recycling processes with a low carbon footprint.
- 26Herrero Acero, E.; Ribitsch, D.; Dellacher, A.; Zitzenbacher, S.; Marold, A.; Steinkellner, G.; Gruber, K.; Schwab, H.; Guebitz, G. M. Surface engineering of a cutinase from Thermobifida cellulosilytica for improved polyester hydrolysis. Biotechnol. Bioeng. 2013, 110, 2581– 2590, DOI: 10.1002/bit.24930Google Scholar26https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXms1KksLY%253D&md5=dff744691be4ad5f0cb5bb219a9b2d32Surface engineering of a cutinase from Thermobifida cellulosilytica for improved polyester hydrolysisHerrero Acero, Enrique; Ribitsch, Doris; Dellacher, Anita; Zitzenbacher, Sabine; Marold, Annemarie; Steinkellner, Georg; Gruber, Karl; Schwab, Helmut; Guebitz, Georg M.Biotechnology and Bioengineering (2013), 110 (10), 2581-2590CODEN: BIBIAU; ISSN:0006-3592. (John Wiley & Sons, Inc.)Modeling and comparison of the structures of the two closely related cutinases Thc_Cut1 and Thc_Cut2 from Thermobifida cellulosilytica DSM44535 revealed that dissimilarities in their electrostatic and hydrophobic surface properties in the vicinity to the active site could be responsible for pronounced differences in hydrolysis efficiencies of polyester (i.e., PET, polyethyleneterephthalate). To investigate this hypothesis in more detail, selected amino acids of surface regions outside the active site of Thc_Cut2, which hydrolyzes PET much less efficiently than Thc_Cut1 were exchanged by site-directed mutagenesis. The mutants were expressed in E. coli BL21-Gold(DE3), purified and characterized regarding their specific activities and kinetic parameters on sol. substrates and their ability to hydrolyze PET and the PET model substrate bis(benzoyloxyethyl) terephthalate (3PET). Compared to Thc_Cut2, mutants carrying Arg29Asn and/or Ala30Val exchanges showed considerable higher specific activity and higher kcat/KM values on sol. substrates. Exchange of the pos. charged arginine (Arg19 and Arg29) located on the enzyme surface to the non-charged amino acids serine and asparagine strongly increased the hydrolysis activity for 3PET and PET. In contrast, exchange of the uncharged glutamine (Glu65) by the neg. charged glutamic acid lead to a complete loss of hydrolysis activity on PET films. These findings clearly demonstrate that surface properties (i.e., amino acids located outside the active site on the protein surface) play an important role in PET hydrolysis. Biotechnol. Bioeng. 2013;9999: XX-XX. © 2013 Wiley Periodicals, Inc.
- 27Ribitsch, D.; Yebra, A. O.; Zitzenbacher, S.; Wu, J.; Nowitsch, S.; Steinkellner, G.; Greimel, K.; Doliska, A.; Oberdorfer, G.; Gruber, C. C. Fusion of binding domains to Thermobifida cellulosilytica cutinase to tune sorption characteristics and enhancing PET hydrolysis. Biomacromolecules 2013, 14, 1769– 1776, DOI: 10.1021/bm400140uGoogle Scholar27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXotlegtb0%253D&md5=c60d9519827ffdc53aff6e24ecf36406Fusion of Binding Domains to Thermobifida cellulosilytica Cutinase to Tune Sorption Characteristics and Enhancing PET HydrolysisRibitsch, Doris; Yebra, Antonio Orcal; Zitzenbacher, Sabine; Wu, Jing; Nowitsch, Susanne; Steinkellner, Georg; Greimel, Katrin; Doliska, Ales; Oberdorfer, Gustav; Gruber, Christian C.; Gruber, Karl; Schwab, Helmut; Stana-Kleinschek, Karin; Acero, Enrique Herrero; Guebitz, Georg M.Biomacromolecules (2013), 14 (6), 1769-1776CODEN: BOMAF6; ISSN:1525-7797. (American Chemical Society)A cutinase from Thermomyces cellullosylitica (Thc_Cut1), hydrolyzing the synthetic polymer polyethylene terephthalate (PET), was fused with two different binding modules to improve sorption and thereby hydrolysis. The binding modules were from cellobiohydrolase I from Hypocrea jecorina (CBM) and from a polyhydroxyalkanoate depolymerase from Alcaligenes faecalis (PBM). Although both binding modules have a hydrophobic nature, it was possible to express the proteins in E. coli. Both fusion enzymes and the native one had comparable kcat values in the range of 311 to 342 s-1 on pNP-butyrate, while the catalytic efficiencies kcat/Km decreased from 0.41 s-1/ μM (native enzyme) to 0.21 and 0.33 s-1/μM for Thc_Cut1+PBM and Thc_Cut1+CBM, resp. The fusion enzymes were active both on the insol. PET model substrate bis(benzoyloxyethyl) terephthalate (3PET) and on PET although the hydrolysis pattern was differed when compared to Thc_Cut1. Enhanced adsorption of the fusion enzymes was visible by chemiluminescence after incubation with a 6xHisTag specific horseradish peroxidase (HRP) labeled probe. Increased adsorption to PET by the fusion enzymes was confirmed with Quarz Crystal Microbalance (QCM-D) anal. and indeed resulted in enhanced hydrolysis activity (3.8× for Thc_Cut1+CBM) on PET, as quantified, based on released mono/oligomers.
- 28Ribitsch, D.; Herrero Acero, E.; Przylucka, A.; Zitzenbacher, S.; Marold, A.; Gamerith, C.; Tscheliessnig, R.; Jungbauer, A.; Rennhofer, H.; Lichtenegger, H. Enhanced cutinase-catalyzed hydrolysis of polyethylene terephthalate by covalent fusion to hydrophobins. Appl. Environ. Microbiol. 2015, 81, 3586– 3592, DOI: 10.1128/AEM.04111-14Google Scholar28https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXosV2msrw%253D&md5=28bcd2b900e693c753321739f6a3b811Enhanced cutinase-catalyzed hydrolysis of polyethylene terephthalate by covalent fusion to hydrophobinsRibitsch, Doris; Acero, Enrique Herrero; Przylucka, Agnieszka; Zitzenbacher, Sabine; Marold, Annemarie; Gamerith, Caroline; Tscheliessnig, Rupert; Jungbauer, Alois; Rennhofer, Harald; Lichtenegger, Helga; Amenitsch, Heinz; Bonazza, Klaus; Kubicek, Christian P.; Druzhinina, Irina S.; Guebitz, Georg M.Applied and Environmental Microbiology (2015), 81 (11), 3586-3592CODEN: AEMIDF; ISSN:1098-5336. (American Society for Microbiology)Cutinases have shown potential for hydrolysis of the recalcitrant synthetic polymer polyethylene terephthalate (PET). We have shown previously that the rate of this hydrolysis can be enhanced by the addn. of hydrophobins, small fungal proteins that can alter the physicochem. properties of surfaces. Here we have investigated whether the PET-hydrolyzing activity of a bacterial cutinase from Thermobifida cellulosilytica (Thc_Cut1) would be further enhanced by fusion to one of three Trichoderma hydrophobins, i.e., the class II hydrophobins HFB4 and HFB7 and the pseudo-class I hydrophobin HFB9b. The fusion enzymes exhibited decreased kcat values on sol. substrates (p-nitrophenyl acetate and p-nitrophenyl butyrate) and strongly decreased the hydrophilicity of glass but caused only small changes in the hydrophobicity of PET. When the enzyme was fused to HFB4 or HFB7, the hydrolysis of PET was enhanced >16-fold over the level with the free enzyme, while a mixt. of the enzyme and the hydrophobins led only to a 4-fold increase at most. Fusion with the non-class II hydrophobin HFB9b did not increase the rate of hydrolysis over that of the enzyme-hydrophobin mixt., but HFB9b performed best when PET was preincubated with the hydrophobins before enzyme treatment. The pattern of hydrolysis by the fusion enzymes differed from that of Thc_Cut1 as the concn. of the product mono(2-hydroxyethyl) terephthalate relative to that of the main product, terephthalic acid, increased. Small-angle X-ray scattering (SAXS) anal. revealed an increased scattering contrast of the fusion proteins over that of the free proteins, suggesting a change in conformation or enhanced protein aggregation. Our data show that the level of hydrolysis of PET by cutinase can be significantly increased by fusion to hydrophobins. The data further suggest that this likely involves binding of the hydrophobins to the cutinase and changes in the conformation of its active center.
- 29Then, J.; Wei, R.; Oeser, T.; Barth, M.; Belisário-Ferrari, M. R.; Schmidt, J.; Zimmermann, W. Ca2+ and Mg2+ binding site engineering increases the degradation of polyethylene terephthalate films by polyester hydrolases from Thermobifida fusca. Biotechnol. J. 2015, 10, 592– 598, DOI: 10.1002/biot.201400620Google Scholar29https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhtFKgtbc%253D&md5=0dc9a61f83e7024b4ae1c47d93708cc6Ca2+ and Mg2+ binding site engineering increases the degradation of polyethylene terephthalate films by polyester hydrolases from Thermobifida fuscaThen, Johannes; Wei, Ren; Oeser, Thorsten; Barth, Markus; Belisario-Ferrari, Matheus R.; Schmidt, Juliane; Zimmermann, WolfgangBiotechnology Journal (2015), 10 (4), 592-598CODEN: BJIOAM; ISSN:1860-6768. (Wiley-VCH Verlag GmbH & Co. KGaA)Several bacterial polyester hydrolases are able to hydrolyze the synthetic polyester, polyethylene terephthalate (PET). For an efficient enzymic degrdn. of PET, reaction temps. close to the glass transition temp. of the polymer need to be applied. Esterases TfH, BTA2, Tfu_0882, TfCut1, and TfCut2 produced by the thermophilic actinomycete, Thermobifida fusca exhibit PET-hydrolyzing activity. However, these enzymes are not sufficiently stable in this temp. range for an efficient degrdn. of post-consumer PET materials. The addn. of Ca2+ or Mg2+ cations to the enzymes resulted in an increase of their m.ps. between 10.8 and 14.1° detd. by CD spectroscopy. The thermostability of the polyester hydrolases was sufficient to degrade semi-cryst. PET films at 65° in the presence of 10 mM Ca2+ and 10 mM Mg2+ resulting in wt. losses of up to 12.9% after a reaction time of 48 h. Residues Asp-174, Asp-204, and Glu-253 were identified by mol. dynamics simulations as potential binding residues for the 2 cations in TfCut2. This was confirmed by their substitution with Arg residues, resulting in a higher thermostability of the corresponding enzyme variants. The generated variants of TfCut2 represented stabilized catalysts suitable for PET hydrolysis reactions performed in the absence of Ca2+ or Mg2+.
- 30Wei, R.; Oeser, T.; Schmidt, J.; Meier, R.; Barth, M.; Then, J.; Zimmermann, W. Engineered bacterial polyester hydrolases efficiently degrade polyethylene terephthalate due to relieved product inhibition. Biotechnol. Bioeng. 2016, 113, 1658– 1665, DOI: 10.1002/bit.25941Google Scholar30https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhvFSlsbw%253D&md5=8f856fb7770bd715660fcb7954228d6aEngineered bacterial polyester hydrolases efficiently degrade polyethylene terephthalate due to relieved product inhibitionWei, Ren; Oeser, Thorsten; Schmidt, Juliane; Meier, Rene; Barth, Markus; Then, Johannes; Zimmermann, WolfgangBiotechnology and Bioengineering (2016), 113 (8), 1658-1665CODEN: BIBIAU; ISSN:0006-3592. (John Wiley & Sons, Inc.)Recent studies on the enzymic degrdn. of synthetic polyesters have shown the potential of polyester hydrolases from thermophilic actinomycetes for modifying or degrading polyethylene terephthalate (PET). TfCut2 from Thermobifida fusca KW3 and LC-cutinase (LCC) isolated from a compost metagenome are remarkably active polyester hydrolases with high sequence and structural similarity. Both enzymes exhibit an exposed active site in a substrate binding groove located at the protein surface. By exchanging selected amino acid residues of TfCut2 involved in substrate binding with those present in LCC, enzyme variants with increased PET hydrolytic activity at 65°C were obtained. The highest activity in hydrolyzing PET films and fibers were detected with the single variant G62A and the double variant G62A/I213S. Both variants caused a wt. loss of PET films of more than 42% after 50 h of hydrolysis, corresponding to a 2.7-fold increase compared to the wild type enzyme. Kinetic anal. based on the released PET hydrolysis products confirmed the superior hydrolytic activity of G62A with a fourfold higher hydrolysis rate const. and a 1.5-fold lower substrate binding const. than those of the wild type enzyme. Mono-(2-hydroxyethyl) terephthalate is a strong inhibitor of TfCut2. A detn. of the Rosetta binding energy suggested a reduced interaction of G62A with 2PET, a dimer of the PET monomer ethylene terephthalate. Indeed, G62A revealed a 5.5-fold lower binding const. to the inhibitor than the wild type enzyme indicating that its increased PET hydrolysis activity is the result of a relieved product inhibition by mono-(2-hydroxyethyl) terephthalate. Biotechnol. Bioeng. 2016;9999: 1-8. © 2016 Wiley Periodicals, Inc.
- 31Shirke, A. N.; Basore, D.; Butterfoss, G. L.; Bonneau, R.; Bystroff, C.; Gross, R. A. Toward rational thermostabilization of Aspergillus oryzae cutinase: Insights into catalytic and structural stability. Proteins 2016, 84, 60– 72, DOI: 10.1002/prot.24955Google Scholar31https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhvFWksrrM&md5=e6bf9ca17fb4888afa9e34dddd198daaToward rational thermostabilization of Aspergillus oryzae cutinase: Insights into catalytic and structural stabilityShirke, Abhijit N.; Basore, Danielle; Butterfoss, Glenn L.; Bonneau, Richard; Bystroff, Christopher; Gross, Richard A.Proteins: Structure, Function, and Bioinformatics (2016), 84 (1), 60-72CODEN: PSFBAF; ISSN:1097-0134. (Wiley-Blackwell)Cutinases are powerful hydrolases that can cleave ester bonds of polyesters such as poly(ethylene terephthalate) (PET), opening up new options for enzymic routes for polymer recycling and surface modification reactions. Cutinase from Aspergillus oryzae (AoC) is promising in this respect, due to the presence of an extended groove near the catalytic triad which is important for the orientation of polymeric chains. However, the catalytic efficiency of AoC on rigid polymers like PET is limited by its low thermostability; as it is essential to work at or over the glass transition temp. (Tg) of PET, i.e., 70°C. Consequently, in this study we worked toward the thermostabilization of AoC. Use of Rosetta computational protein design software in conjunction with rational design led to a 6°C improvement in the thermal unfolding temp. (Tm) and a 10-fold increase in the half-life of the enzyme activity at 60°C. Surprisingly, thermostabilization did not improve the rate or temp. optimum of enzyme activity. Three notable findings are presented as steps toward designing more thermophilic cutinase: (a) surface salt bridge optimization produced enthalpic stabilization, (b) mutations to proline reduced the entropy loss upon folding, and (c) the lack of a correlative increase in the temp. optimum of catalytic activity with thermodn. stability suggests that the active site is locally denatured at a temp. below the Tm of the global structure. Proteins 2015. © 2015 Wiley Periodicals, Inc.
- 32Biundo, A.; Ribitsch, D.; Steinkellner, G.; Gruber, K.; Guebitz, G. M. Polyester hydrolysis is enhanced by a truncated esterase: Less is more. Biotechnol. J. 2017, 12, DOI: 10.1002/biot.201600450Google ScholarThere is no corresponding record for this reference.
- 33Shirke, A. N.; Butterfoss, G. L.; Saikia, R.; Basu, A.; Maria, L.; Svendsen, A.; Gross, R. A. Engineered Humicola insolens cutinase for efficient cellulose acetate deacetylation. Biotechnol. J. 2017, 12, 1700188, DOI: 10.1002/biot.201700188Google ScholarThere is no corresponding record for this reference.
- 34Austin, H. P.; Allen, M. D.; Donohoe, B. S.; Rorrer, N. A.; Kearns, F. L.; Silveira, R. L.; Pollard, B. C.; Dominick, G.; Duman, R.; El Omari, K. Characterization and engineering of a plastic-degrading aromatic polyesterase. Proc. Natl. Acad. Sci. U.S.A. 2018, 115, E4350– E4357, DOI: 10.1073/pnas.1718804115Google Scholar34https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhvVGhtLnF&md5=b8e8d841af1bb06fa2d1fddb6b9d6460Characterization and engineering of a plastic-degrading aromatic polyesteraseAustin, Harry P.; Allen, Mark D.; Donohoe, Bryon S.; Rorrer, Nicholas A.; Kearns, Fiona L.; Silveira, Rodrigo L.; Pollard, Benjamin C.; Dominick, Graham; Duman, Ramona; El Omari, Kamel; Mykhaylyk, Vitaliy; Wagner, Armin; Michener, William E.; Amore, Antonella; Skaf, Munir S.; Crowley, Michael F.; Thorne, Alan W.; Johnson, Christopher W.; Woodcock, H. Lee; McGeehan, John E.; Beckham, Gregg T.Proceedings of the National Academy of Sciences of the United States of America (2018), 115 (19), E4350-E4357CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Poly(ethylene terephthalate) (PET) is one of the most abundantly produced synthetic polymers and is accumulating in the environment at a staggering rate as discarded packaging and textiles. The properties that make PET so useful also endow it with an alarming resistance to biodegrdn., likely lasting centuries in the environment. Our collective reliance on PET and other plastics means that this buildup will continue unless solns. are found. Recently, a newly discovered bacterium, Ideonella sakaiensis 201-F6, was shown to exhibit the rare ability to grow on PET as a major carbon and energy source. Central to its PET biodegrdn. capability is a secreted PETase (PET-digesting enzyme). Here, we present a 0.92 Å resoln. X-ray crystal structure of PETase, which reveals features common to both cutinases and lipases. PETase retains the ancestral α/β-hydrolase fold but exhibits a more open active-site cleft than homologous cutinases. By narrowing the binding cleft via mutation of two active-site residues to conserved amino acids in cutinases, we surprisingly observe improved PET degrdn., suggesting that PETase is not fully optimized for cryst. PET degrdn., despite presumably evolving in a PET-rich environment. Addnl., we show that PETase degrades another semiarom. polyester, polyethylene-2,5-furandicarboxylate (PEF), which is an emerging, bioderived PET replacement with improved barrier properties. In contrast, PETase does not degrade aliph. polyesters, suggesting that it is generally an arom. polyesterase. These findings suggest that addnl. protein engineering to increase PETase performance is realistic and highlight the need for further developments of structure/activity relationships for biodegrdn. of synthetic polyesters.
- 35Biundo, A.; Ribitsch, D.; Guebitz, G. M. Surface engineering of polyester-degrading enzymes to improve efficiency and tune specificity. Appl. Microbiol. Biotechnol. 2018, 102, 3551– 3559, DOI: 10.1007/s00253-018-8850-7Google Scholar35https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXjvFWqsL4%253D&md5=0858dea4ffd7da9f898f38a1508c99ccSurface engineering of polyester-degrading enzymes to improve efficiency and tune specificityBiundo, Antonino; Ribitsch, Doris; Guebitz, Georg M.Applied Microbiology and Biotechnology (2018), 102 (8), 3551-3559CODEN: AMBIDG; ISSN:0175-7598. (Springer)Certain members of the carboxylesterase superfamily can act at the interface between water and water-insol. substrates. However, nonnatural bulky polyesters usually are not efficiently hydrolyzed. In the recent years, the potential of enzyme engineering to improve hydrolysis of synthetic polyesters has been demonstrated. Regions on the enzyme surface have been modified by using site-directed mutagenesis in order to tune sorption processes through increased hydrophobicity of the enzyme surface. Such modifications can involve specific amino acid substitutions, addn. of binding modules, or truncation of entire domains improving sorption properties and/or dynamics of the enzyme. In this review, we provide a comprehensive overview on different strategies developed in the recent years for enzyme surface engineering to improve the activity of polyester-hydrolyzing enzymes.
- 36Shirke, A. N.; White, C.; Englaender, J. A.; Zwarycz, A.; Butterfoss, G. L.; Linhardt, R. J.; Gross, R. A. Stabilizing Leaf and Branch Compost Cutinase (LCC) with Glycosylation: Mechanism and Effect on PET Hydrolysis. Biochemistry 2018, 57, 1190– 1200, DOI: 10.1021/acs.biochem.7b01189Google Scholar36https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXntleqsQ%253D%253D&md5=7a58568be5fdbdbcd8f7340ea74d761dStabilizing Leaf and Branch Compost Cutinase (LCC) with Glycosylation: Mechanism and Effect on PET HydrolysisShirke, Abhijit N.; White, Christine; Englaender, Jacob A.; Zwarycz, Allison; Butterfoss, Glenn L.; Linhardt, Robert J.; Gross, Richard A.Biochemistry (2018), 57 (7), 1190-1200CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)Cutinases are polyester hydrolases that show a remarkable capability to hydrolyze polyethylene terephthalate (PET) to its monomeric units. This revelation has stimulated research aimed at developing sustainable and green cutinase-catalyzed PET recycling methods. Leaf and branch compost cutinase (LCC) is particularly suited toward these ends given its relatively high PET hydrolysis activity and thermostability. Any practical enzymic PET recycling application will require that the protein have kinetic stability at or above the PET glass transition temp. (Tg, i.e., 70 °C). This paper elucidates the thermodn. and kinetics of LCC conformational and colloidal stability. Aggregation emerged as a major contributor that reduces LCC kinetic stability. In its native state, LCC is prone to aggregation owing to electrostatic interactions. Further, with increasing temp., perturbation of LCC's tertiary structure and corresponding exposure of hydrophobic domains leads to rapid aggregation. Glycosylation was employed in an attempt to impede LCC aggregation. Owing to the presence of three putative N-glycosylation sites, expression of native LCC in Pichia pastoris resulted in the prodn. of glycosylated LCC (LCC-G). LCC-G showed improved stability to native state aggregation while increasing the temp. for thermal induced aggregation by 10 °C. Furthermore, stabilization against thermal aggregation resulted in improved catalytic PET hydrolysis both at its optimum temp. and concn.
- 37Son, H. F.; Cho, I. J.; Joo, S.; Seo, H.; Sagong, H.-Y.; Choi, S. Y.; Lee, S. Y.; Kim, K.-J. Rational Protein Engineering of Thermo-Stable PETase from Ideonella sakaiensis for Highly Efficient PET Degradation. ACS Catal. 2019, 9, 3519– 3526, DOI: 10.1021/acscatal.9b00568Google Scholar37https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXksFegurY%253D&md5=ebe4cdfb118f20eba6e05a731717f8fbRational protein engineering of thermo-stable PETase from Ideonella sakaiensis for highly efficient PET degradationSon, Hyeoncheol Francis; Cho, In Jin; Joo, Seongjoon; Seo, Hogyun; Sagong, Hye-Young; Choi, So Young; Lee, Sang Yup; Kim, Kyung-JinACS Catalysis (2019), 9 (4), 3519-3526CODEN: ACCACS; ISSN:2155-5435. (American Chemical Society)Widespread utilization of polyethylene terephthalate (PET) has caused a variety of environmental and health problems; thus, the enzymic degrdn. of PET can be a promising soln. Although PETase from Ideonalla sakaiensis (IsPETase) has been reported to have the highest PET degrdn. activity under mild conditions of all PET-degrading enzymes reported to date, its low thermal stability limits its ability for efficient and practical enzymic degrdn. of PET. Using the structural information on IsPETase, we developed a rational protein engineering strategy using several IsPETase variants that were screened for high thermal stability to improve PET degrdn. activity. In particular, the IsPETaseS121E/D186H/R280A variant, which was designed to have a stabilized β6-β7 connecting loop and extended subsite IIc, had a Tm value that was increased by 8.81° and PET degrdn. activity was enhanced by 14-fold at 40 °C in comparison with IsPETaseWT. The designed structural modifications were further verified through structure detn. of the variants, and high thermal stability was further confirmed by a heat-inactivation expt. The proposed strategy and developed variants represent an important advancement for achieving the complete biodegrdn. of PET under mild conditions.
- 38Furukawa, M.; Kawakami, N.; Tomizawa, A.; Miyamoto, K. Efficient Degradation of Poly(ethylene terephthalate) with Thermobifida fusca Cutinase Exhibiting Improved Catalytic Activity Generated using Mutagenesis and Additive-based Approaches. Sci. Rep. 2019, 9, 16038, DOI: 10.1038/s41598-019-52379-zGoogle Scholar38https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3MjlsFKgtA%253D%253D&md5=1bce8289f05c2abdc9f937c15c3a61a3Efficient Degradation of Poly(ethylene terephthalate) with Thermobifida fusca Cutinase Exhibiting Improved Catalytic Activity Generated using Mutagenesis and Additive-based ApproachesFurukawa Makoto; Kawakami Norifumi; Tomizawa Atsushi; Miyamoto KenjiScientific reports (2019), 9 (1), 16038 ISSN:.Cutinases are promising agents for poly(ethylene terephthalate) (PET) bio-recycling because of their ability to produce the PET monomer terephthalic acid with high efficiency under mild reaction conditions. In this study, we found that the low-crystallinity PET (lcPET) hydrolysis activity of thermostable cutinase from Thermobifida fusca (TfCut2), was increased by the addition of cationic surfactant that attracts enzymes near the lcPET film surface via electrostatic interactions. This approach was applicable to the mutant TfCut2 G62A/F209A, which was designed based on a sequence comparison with PETase from Ideonella sakaiensis. As a result, the degradation rate of the mutant in the presence of cationic surfactant increased to 31 ± 0.1 nmol min(-1) cm(-2), 12.7 times higher than that of wild-type TfCut2 in the absence of surfactant. The long-duration reaction showed that lcPET film (200 μm) was 97 ± 1.8% within 30 h, the fastest biodegradation rate of lcPET film thus far. We therefore believe that our approach would expand the possibility of enzyme utilization in industrial PET biodegradation.
- 39Tournier, V.; Topham, C. M.; Gilles, A.; David, B.; Folgoas, C.; Moya-Leclair, E.; Kamionka, E.; Desrousseaux, M.-L.; Texier, H.; Gavalda, S. An engineered PET depolymerase to break down and recycle plastic bottles. Nature 2020, 580, 216– 219, DOI: 10.1038/s41586-020-2149-4Google Scholar39https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXmvVSht70%253D&md5=1bad7e606018f024e6fad5fbf7b082e4An engineered PET depolymerase to break down and recycle plastic bottlesTournier, V.; Topham, C. M.; Gilles, A.; David, B.; Folgoas, C.; Moya-Leclair, E.; Kamionka, E.; Desrousseaux, M.-L.; Texier, H.; Gavalda, S.; Cot, M.; Guemard, E.; Dalibey, M.; Nomme, J.; Cioci, G.; Barbe, S.; Chateau, M.; Andre, I.; Duquesne, S.; Marty, A.Nature (London, United Kingdom) (2020), 580 (7802), 216-219CODEN: NATUAS; ISSN:0028-0836. (Nature Research)Present ests. suggest that of the 359 million tons of plastics produced annually worldwide1, 150-200 million tons accumulate in landfill or in the natural environment2. Poly(ethylene terephthalate) (PET) is the most abundant polyester plastic, with almost 70 million tons manufd. annually worldwide for use in textiles and packaging3. The main recycling process for PET, via thermomech. means, results in a loss of mech. properties4. Consequently, de novo synthesis is preferred and PET waste continues to accumulate. With a high ratio of arom. terephthalate units-which reduce chain mobility-PET is a polyester that is extremely difficult to hydrolyze5. Several PET hydrolase enzymes have been reported, but show limited productivity6,7. Here we describe an improved PET hydrolase that ultimately achieves, over 10 h, a min. of 90 per cent PET depolymn. into monomers, with a productivity of 16.7 g of terephthalate per L per h (200 g per kg of PET suspension, with an enzyme concn. of 3 mg per g of PET). This highly efficient, optimized enzyme outperforms all PET hydrolases reported so far, including an enzyme8,9 from the bacterium Ideonella sakaiensis strain 201-F6 (even assisted by a secondary enzyme10) and related improved variants11-14 that have attracted recent interest. We also show that biol. recycled PET exhibiting the same properties as petrochem. PET can be produced from enzymically depolymd. PET waste, before being processed into bottles, thereby contributing towards the concept of a circular PET economy.
- 40Bell, E.; Smithson, R.; Kilbride, S.; Foster, J.; Hardy, F.; Ramachandran, S.; Tedstone, A.; Haigh, S.; Garforth, A.; Day, P. Directed Evolution of an Efficient and Thermostable PET Depolymerase. ChemRxiv 2021, DOI: 10.26434/chemrxiv-2021-mcjh6Google ScholarThere is no corresponding record for this reference.
- 41Cui, Y.; Chen, Y.; Liu, X.; Dong, S.; Tian, Y. e.; Qiao, Y.; Mitra, R.; Han, J.; Li, C.; Han, X. Computational Redesign of a PETase for Plastic Biodegradation under Ambient Condition by the GRAPE Strategy. ACS Catal. 2021, 11, 1340– 1350, DOI: 10.1021/acscatal.0c05126Google Scholar41https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXpt1CqtQ%253D%253D&md5=9cf0ade24ab9ab5beb726fe620b284f7Computational Redesign of a PETase for Plastic Biodegradation under Ambient Condition by the GRAPE StrategyCui, Yinglu; Chen, Yanchun; Liu, Xinyue; Dong, Saijun; Tian, Yu'e; Qiao, Yuxin; Mitra, Ruchira; Han, Jing; Li, Chunli; Han, Xu; Liu, Weidong; Chen, Quan; Wei, Wangqing; Wang, Xin; Du, Wenbin; Tang, Shuangyan; Xiang, Hua; Liu, Haiyan; Liang, Yong; Houk, Kendall N.; Wu, BianACS Catalysis (2021), 11 (3), 1340-1350CODEN: ACCACS; ISSN:2155-5435. (American Chemical Society)Nature has provided a fantastic array of enzymes that are responsible for essential biochem. functions but not usually suitable for technol. applications. Not content with the natural repertoire, protein engineering holds promise to extend the applications of improved enzymes with tailored properties. However, engineering of robust proteins remains a difficult task since the pos. mutation library may not cooperate to reach the target function in most cases owing to the ubiquity of epistatic effects. The main demand lies in identifying an efficient path of accumulated mutations. Herein, we devised a computational strategy (greedy accumulated strategy for protein engineering, GRAPE) to improve the robustness of a PETase from Ideonella sakaiensis. A systematic clustering anal. combined with greedy accumulation of beneficial mutations in a computationally derived library enabled the redesign of a variant, DuraPETase, which exhibits an apparent melting temp. that is drastically elevated by 31°C and a strikingly enhanced degrdn. toward semicryst. poly(ethylene terephthalate) (PET) films (30%) at mild temps. (over 300-fold). Complete biodegrdn. of 2 g/L microplastics to water-sol. products under mild conditions is also achieved, opening up opportunities to steer the biol. degrdn. of uncollectable PET waste and further conversion of the resulting monomers to high-value mols. The crystal structure revealed the individual mutation match with the design model. Concurrently, synergistic effects are captured, while epistatic interactions are alleviated during the accumulation process. We anticipate that our design strategy will provide a broadly applicable strategy for global optimization of enzyme performance.
- 42Nakamura, A.; Kobayashi, N.; Koga, N.; Iino, R. Positive Charge Introduction on the Surface of Thermostabilized PET Hydrolase Facilitates PET Binding and Degradation. ACS Catal. 2021, 11, 8550– 8564, DOI: 10.1021/acscatal.1c01204Google Scholar42https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhsVWrtL3E&md5=570df73ee6a1e63529e4517c76b21282Pos. charge introduction on the surface of thermostabilized PET hydrolase facilitates PET binding and degradationNakamura, Akihiko; Kobayashi, Naoya; Koga, Nobuyasu; Iino, RyotaACS Catalysis (2021), 11 (14), 8550-8564CODEN: ACCACS; ISSN:2155-5435. (American Chemical Society)A thermostable enzyme PET2, found in a metagenome library, has been engineered to improve its hydrolytic activity against polyethylene terephthalate (PET). The PET2 wild-type (WT) showed a melting temp. of 69.0°C and produced water-sol. reaction products at a rate of 0.40 min-1 (2.4μM products from 0.1μM enzyme after 60 min reaction) from an amorphous PET film at 60°C. Mutations for surface charge modification, backbone stabilization, and formation of addnl. disulfide bond were introduced into the PET2 WT, and the best mutant (PET2 7M) showed a melting temp. of 75.7°C and hydrolytic activity of 1.3 min-1 (7.8μM products from 0.1μM enzyme after 60 min reaction at 60°C). X-ray crystal structures of PET2 mutants showed that introduced arginine and lysine residues oriented to the solvent, similar to a PET hydrolase from Ideonella sakaiensis 201-F6. Single-mol. fluorescence imaging revealed that these pos. charged surface residues increased binding rate const. of PET2 7M to PET surface 2.7 times, compared with PET2 WT, and resulted in higher activity. Optimal temp. for amorphous PET hydrolysis by PET2 7M (68°C) was 8°C higher than that by PET2 WT (60°C), and hydrolytic activity of PET2 7M at the optimal temp. (2.7 min-1, 16.2μM products from 0.1μM enzyme after 60 min reaction) was 6.8 times higher than that of PET2 WT (0.40 min-1). Furthermore, PET2 7M generated reaction products with a const. rate for at least 24 h at 68°C, indicating long-term thermal stability at the optimal temp.
- 43Guo, B.; Vanga, S. R.; Lopez-Lorenzo, X.; Saenz-Mendez, P.; Ericsson, S. R.; Fang, Y.; Ye, X.; Schriever, K.; Bäckström, E.; Biundo, A. Conformational Selection in Biocatalytic Plastic Degradation by PETase. ACS Catal. 2022, 12, 3397– 3409, DOI: 10.1021/acscatal.1c05548Google Scholar43https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XltFehs78%253D&md5=0732543ed139b9fd36a771245bfd3db6Conformational Selection in Biocatalytic Plastic Degradation by PETaseGuo, Boyang; Vanga, Sudarsana Reddy; Lopez-Lorenzo, Ximena; Saenz-Mendez, Patricia; Ericsson, Sara Roennblad; Fang, Yuan; Ye, Xinchen; Schriever, Karen; Baeckstroem, Eva; Biundo, Antonino; Zubarev, Roman A.; Furo, Istvan; Hakkarainen, Minna; Syren, Per-OlofACS Catalysis (2022), 12 (6), 3397-3409CODEN: ACCACS; ISSN:2155-5435. (American Chemical Society)Due to the steric effects imposed by bulky polymers, the formation of catalytically competent enzyme and substrate conformations is crit. in the biodegrdn. of plastics. In poly(ethylene terephthalate) (PET), the backbone adopts different conformations, gauche and trans, coexisting to different extents in amorphous and cryst. regions. However, which conformation is susceptible to biodegrdn. and the extent of enzyme and substrate conformational changes required for expedient catalysis remain poorly understood. To overcome this obstacle, we utilized mol. dynamics simulations, docking, and enzyme engineering in concert with high-resoln. microscopy imaging and solid-state NMR to demonstrate the importance of conformational selection in biocatalytic plastic hydrolysis. Our results demonstrate how single-amino acid substitutions in Ideonella sakaiensis PETase can alter its conformational landscape, significantly affecting the relative abundance of productive ground-state structures ready to bind discrete substrate conformers. We exptl. show how an enzyme binds to plastic and provide a model for key residues involved in the recognition of gauche and trans conformations supported by in silico simulations. We demonstrate how enzyme engineering can be used to create a trans-selective variant, resulting in higher activity when combined with an all-trans PET-derived oligomeric substrate, stemming from both increased accessibility and conformational preference. Our work cements the importance of matching enzyme and substrate conformations in plastic hydrolysis, and we show that also the noncanonical trans conformation in PET is conducive for degrdn. Understanding the contribution of enzyme and substrate conformations to biocatalytic plastic degrdn. could facilitate the generation of designer enzymes with increased performance.
- 44Lu, H.; Diaz, D. J.; Czarnecki, N. J.; Zhu, C.; Kim, W.; Shroff, R.; Acosta, D. J.; Alexander, B. R.; Cole, H. O.; Zhang, Y. Machine learning-aided engineering of hydrolases for PET depolymerization. Nature 2022, 604, 662– 667, DOI: 10.1038/s41586-022-04599-zGoogle Scholar44https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XhtFKrtLfI&md5=737bb3fe419c581be70e0ae37fc20085Machine learning-aided engineering of hydrolases for PET depolymerizationLu, Hongyuan; Diaz, Daniel J.; Czarnecki, Natalie J.; Zhu, Congzhi; Kim, Wantae; Shroff, Raghav; Acosta, Daniel J.; Alexander, Bradley R.; Cole, Hannah O.; Zhang, Yan; Lynd, Nathaniel A.; Ellington, Andrew D.; Alper, Hal S.Nature (London, United Kingdom) (2022), 604 (7907), 662-667CODEN: NATUAS; ISSN:1476-4687. (Nature Portfolio)Plastic waste poses an ecol. challenge1-3 and enzymic degrdn. offers one, potentially green and scalable, route for polyesters waste recycling4. Poly(ethylene terephthalate) (PET) accounts for 12% of global solid waste5, and a circular carbon economy for PET is theor. attainable through rapid enzymic depolymn. followed by repolymn. or conversion/valorization into other products6-10. Application of PET hydrolases, however, has been hampered by their lack of robustness to pH and temp. ranges, slow reaction rates and inability to directly use untreated postconsumer plastics11. Here, we use a structure-based, machine learning algorithm to engineer a robust and active PET hydrolase. Our mutant and scaffold combination (FAST-PETase: functional, active, stable and tolerant PETase) contains five mutations compared to wild-type PETase (N233K/R224Q/S121E from prediction and D186H/R280A from scaffold) and shows superior PET-hydrolytic activity relative to both wild-type and engineered alternatives12 between 30 and 50°C and a range of pH levels. We demonstrate that untreated, postconsumer-PET from 51 different thermoformed products can all be almost completely degraded by FAST-PETase in 1 wk. FAST-PETase can also depolymerize untreated, amorphous portions of a com. water bottle and an entire thermally pretreated water bottle at 50°C. Finally, we demonstrate a closed-loop PET recycling process by using FAST-PETase and resynthesizing PET from the recovered monomers. Collectively, our results demonstrate a viable route for enzymic plastic recycling at the industrial scale.
- 45Wei, R.; von Haugwitz, G.; Pfaff, L.; Mican, J.; Badenhorst, C. P. S.; Liu, W.; Weber, G.; Austin, H. P.; Bednar, D.; Damborsky, J. Mechanism-Based Design of Efficient PET Hydrolases. ACS Catal. 2022, 12, 3382– 3396, DOI: 10.1021/acscatal.1c05856Google Scholar45https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XkvF2gsrs%253D&md5=d19ff0a8a98b6c9569e1d1e6a7ac7884Mechanism-Based Design of Efficient PET HydrolasesWei, Ren; von Haugwitz, Gerlis; Pfaff, Lara; Mican, Jan; Badenhorst, Christoffel P. S.; Liu, Weidong; Weber, Gert; Austin, Harry P.; Bednar, David; Damborsky, Jiri; Bornscheuer, Uwe T.ACS Catalysis (2022), 12 (6), 3382-3396CODEN: ACCACS; ISSN:2155-5435. (American Chemical Society)A review. 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 enzymic 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 enzymic PET degrdn. approaches. Unbalanced enzyme-substrate interactions, limited thermostability, and low catalytic efficiency at elevated reaction temps., and inhibition caused by oligomeric degrdn. intermediates still hamper industrial applications that require high catalytic efficiency. To overcome these limitations, successful protein engineering studies using innovative exptl. 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 biotechnol. approaches.
- 46Zeng, W.; Li, X.; Yang, Y.; Min, J.; Huang, J.-W.; Liu, W.; Niu, D.; Yang, X.; Han, X.; Zhang, L. Substrate-Binding Mode of a Thermophilic PET Hydrolase and Engineering the Enzyme to Enhance the Hydrolytic Efficacy. ACS Catal. 2022, 12, 3033– 3040, DOI: 10.1021/acscatal.1c05800Google Scholar46https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XjvFyjsbw%253D&md5=8e485295c20775ce47acb77886b134b1Substrate-Binding Mode of a Thermophilic PET Hydrolase and Engineering the Enzyme to Enhance the Hydrolytic EfficacyZeng, Wei; Li, Xiuqin; Yang, Yunyun; Min, Jian; Huang, Jian-Wen; Liu, Weidong; Niu, Du; Yang, Xuechun; Han, Xu; Zhang, Lilan; Dai, Longhai; Chen, Chun-Chi; Guo, Rey-TingACS Catalysis (2022), 12 (5), 3033-3040CODEN: ACCACS; ISSN:2155-5435. (American Chemical Society)Polyethylene terephthalate (PET) is among the most extensively produced plastics, but huge amts. of PET wastes that have accumulated in the environment have become a serious threat to the ecosystem. Applying PET hydrolytic enzymes to depolymerize PET is an attractive measure to manage PET pollution, and searching for more effective enzymes is a prerequisite to achieve this goal. A thermostable cutinase that originates from the leaf-branch compost termed ICCG is the most effective PET hydrolase reported so far. Here, we illustrated the crystal structure of ICCG in complex with the PET analog, mono(2-hydroxyethyl)terephthalic acid, to reveal the enzyme-substrate interaction network. Furthermore, we applied structure-based engineering to modify ICCG and screened for variants that exhibit higher efficacy than the parental enzyme. As a result, several variants with the measured melting temp. approaching 99°C and elevated PET hydrolytic activity were obtained. Finally, crystallog. analyses were performed to reveal the structural stabilization effects mediated by the introduced mutations. These results are of importance in the context of understanding the mechanism of action of the thermostable PET hydrolytic enzyme and shall be beneficial to the development of PET biodegrdn. platforms.
- 47Singh, A.; Rorrer, N. A.; Nicholson, S. R.; Erickson, E.; DesVeaux, J. S.; Avelino, A. F. T.; Lamers, P.; Bhatt, A.; Zhang, Y.; Avery, G. Techno-economic, life-cycle, and socioeconomic impact analysis of enzymatic recycling of poly(ethylene terephthalate). Joule 2021, 5, 2479– 2503, DOI: 10.1016/j.joule.2021.06.015Google Scholar47https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhs1Sks7jL&md5=e5f88c523cbb0bb29e7fe294dca4ec5dTechno-economic, life-cycle, and socioeconomic impact analysis of enzymatic recycling of poly(ethylene terephthalate)Singh, Avantika; Rorrer, Nicholas A.; Nicholson, Scott R.; Erickson, Erika; DesVeaux, Jason S.; Avelino, Andre F. T.; Lamers, Patrick; Bhatt, Arpit; Zhang, Yimin; Avery, Greg; Tao, Ling; Pickford, Andrew R.; Carpenter, Alberta C.; McGeehan, John E.; Beckham, Gregg T.Joule (2021), 5 (9), 2479-2503CODEN: JOULBR; ISSN:2542-4351. (Cell Press)Esterases have emerged as important biocatalysts for enzyme-based polyester recycling of poly(ethylene terephthalate) (PET) to terephthalic acid (TPA) and ethylene glycol (EG). Here, we present process modeling, techno-economic, life-cycle, and socioeconomic impact analyses for an enzymic PET depolymn.-based recycling process, which we compare with virgin TPA manufg. We predict that enzymically recycled TPA (rTPA) can be cost-competitive and highlight key areas to achieve this. In addn. to favorable long-term socioeconomic benefits, rTPA can reduce total supply chain energy use by 69%-83% and greenhouse gas emissions by 17%-43% per kg of TPA. An economy-wide assessment for the US ests. that the TPA recycling process can reduce environmental impacts by up to 95% while generating up to 45% more socioeconomic benefits, also relative to virgin TPA prodn. Sensitivity analyses highlight impactful research opportunities to pursue toward realizing biol. PET recycling and upcycling.
- 48Wei, R.; Breite, D.; Song, C.; Gräsing, D.; Ploss, T.; Hille, P.; Schwerdtfeger, R.; Matysik, J.; Schulze, A.; Zimmermann, W. Biocatalytic Degradation Efficiency of Postconsumer Polyethylene Terephthalate Packaging Determined by Their Polymer Microstructures. Adv. Sci. 2019, 6, 1900491, DOI: 10.1002/advs.201900491Google Scholar48https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3MvkslGqsA%253D%253D&md5=1fb8e90521767a6e6125016bb02c07adBiocatalytic Degradation Efficiency of Postconsumer Polyethylene Terephthalate Packaging Determined by Their Polymer MicrostructuresWei Ren; Hille Patrick; Zimmermann Wolfgang; Breite Daniel; Schulze Agnes; Song Chen; Grasing Daniel; Matysik Jorg; Ploss Tina; Schwerdtfeger RuthAdvanced science (Weinheim, Baden-Wurttemberg, Germany) (2019), 6 (14), 1900491 ISSN:2198-3844.Polyethylene terephthalate (PET) is the most important mass-produced thermoplastic polyester used as a packaging material. Recently, thermophilic polyester hydrolases such as TfCut2 from Thermobifida fusca have emerged as promising biocatalysts for an eco-friendly PET recycling process. In this study, postconsumer PET food packaging containers are treated with TfCut2 and show weight losses of more than 50% after 96 h of incubation at 70 °C. Differential scanning calorimetry analysis indicates that the high linear degradation rates observed in the first 72 h of incubation is due to the high hydrolysis susceptibility of the mobile amorphous fraction (MAF) of PET. The physical aging process of PET occurring at 70 °C is shown to gradually convert MAF to polymer microstructures with limited accessibility to enzymatic hydrolysis. Analysis of the chain-length distribution of degraded PET by nuclear magnetic resonance spectroscopy reveals that MAF is rapidly hydrolyzed via a combinatorial exo- and endo-type degradation mechanism whereas the remaining PET microstructures are slowly degraded only by endo-type chain scission causing no detectable weight loss. Hence, efficient thermostable biocatalysts are required to overcome the competitive physical aging process for the complete degradation of postconsumer PET materials close to the glass transition temperature of PET.
- 49Knott, B. C.; Erickson, E.; Allen, M. D.; Gado, J. E.; Graham, R.; Kearns, F. L.; Pardo, I.; Topuzlu, E.; Anderson, J. J.; Austin, H. P. Characterization and engineering of a two-enzyme system for plastics depolymerization. Proc. Natl. Acad. Sci. U.S.A. 2020, 117, 25476– 25485, DOI: 10.1073/pnas.2006753117Google Scholar49https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXitVKktL3P&md5=1c00658c41832cce8e99caa02a769836Characterization and engineering of a two-enzyme system for plastics depolymerizationKnott, Brandon C.; Erickson, Erika; Allen, Mark D.; Gado, Japheth E.; Graham, Rosie; Kearns, Fiona L.; Pardo, Isabel; Topuzlu, Ece; Anderson, Jared J.; Austin, Harry P.; Dominick, Graham; Johnson, Christopher W.; Rorrer, Nicholas A.; Szostkiewicz, Caralyn J.; Copie, Valerie; Payne, Christina M.; Woodcock, H. Lee; Donohoe, Bryon S.; Beckham, Gregg T.; McGeehan, John E.Proceedings of the National Academy of Sciences of the United States of America (2020), 117 (41), 25476-25485CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Plastics pollution represents a global environmental crisis. In response, microbes are evolving the capacity to utilize synthetic polymers as carbon and energy sources. Recently, Ideonella sakaiensis was reported to secrete a two-enzyme system to deconstruct polyethylene terephthalate (PET) to its constituent monomers. Specifically, the I. sakaiensis PETase depolymerizes PET, liberating sol. products, including mono(2-hydroxyethyl) terephthalate (MHET), which is cleaved to terephthalic acid and ethylene glycol by MHETase. Here, we report a 1.6 Å resoln. MHETase structure, illustrating that the MHETase core domain is similar to PETase, capped by a lid domain. Simulations of the catalytic itinerary predict that MHETase follows the canonical two-step serine hydrolase mechanism. Bioinformatics anal. suggests that MHETase evolved from ferulic acid esterases, and two homologous enzymes are shown to exhibit MHET turnover. Anal. of the two homologous enzymes and the MHETase S131G mutant demonstrates the importance of this residue for accommodation of MHET in the active site. We also demonstrate that the MHETase lid is crucial for hydrolysis of MHET and, furthermore, that MHETase does not turnover mono(2-hydroxyethyl)-furanoate or mono(2-hydroxyethyl)-isophthalate. A highly synergistic relationship between PETase and MHETase was obsd. for the conversion of amorphous PET film to monomers across all nonzero MHETase concns. tested. Finally, we compare the performance of MHETase:PETase chimeric proteins of varying linker lengths, which all exhibit improved PET and MHET turnover relative to the free enzymes. Together, these results offer insights into the two-enzyme PET depolymn. system and will inform future efforts in the biol. deconstruction and upcycling of mixed plastics.
- 50Erickson, E.; Shakespeare, T. J.; Bratti, F.; Buss, B. L.; Graham, R.; Hawkins, M. A.; König, G.; Michener, W. E.; Miscall, J.; Ramirez, K. J. Comparative performance of PETase as a function of reaction conditions, substrate properties, and product accumulation. ChemSusChem 2022, 15, e202101932 DOI: 10.1002/cssc.202101932Google Scholar50https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXisVehtrzP&md5=bc1cd0c2fa069ca6d834a99f5ec38cf2Comparative Performance of PETase as a Function of Reaction Conditions, Substrate Properties, and Product AccumulationErickson, Erika; Shakespeare, Thomas J.; Bratti, Felicia; Buss, Bonnie L.; Graham, Rosie; Hawkins, McKenzie A.; Konig, Gerhard; Michener, William E.; Miscall, Joel; Ramirez, Kelsey J.; Rorrer, Nicholas A.; Zahn, Michael; Pickford, Andrew R.; McGeehan, John E.; Beckham, Gregg T.ChemSusChem (2022), 15 (1), e202101932CODEN: CHEMIZ; ISSN:1864-5631. (Wiley-VCH Verlag GmbH & Co. KGaA)There is keen interest to develop new technologies to recycle the plastic poly(ethylene terephthalate) (PET). To this end, the use of PET-hydrolyzing enzymes has shown promise for PET deconstruction to its monomers, terephthalate (TPA) and ethylene glycol (EG). Here, the Ideonella sakaiensis PETase wild-type enzyme was compared to a previously reported improved variant (W159H/S238F). The thermostability of each enzyme was compared and a 1.45 S resoln. structure of the mutant was described, highlighting changes in the substrate binding cleft compared to the wild-type enzyme. Subsequently, the performance of the wild-type and variant enzyme was compared as a function of temp., substrate morphol., and reaction mixt. compn. These studies showed that reaction temp. had the strongest influence on performance between the two enzymes. It was also shown that both enzymes achieved higher levels of PET conversion for substrates with moderate crystallinity relative to amorphous substrates. Finally, the impact of product accumulation on reaction progress was assessed for the hydrolysis of both PET and bis(2-hydroxyethyl) terephthalate (BHET). Each enzyme displayed different inhibition profiles to mono(2-hydroxyethyl) terephthalate (MHET) and TPA, while both were sensitive to inhibition by EG. Overall, this study highlights the importance of reaction conditions, substrate selection, and product accumulation for catalytic performance of PET-hydrolyzing enzymes, which have implications for enzyme screening in the development of enzyme-based polyester recycling.
- 51Lu, H.; Diaz, D. J.; Czarnecki, N. J.; Zhu, C.; Kim, W.; Shroff, R.; Acosta, D. J.; Alexander, B.; Cole, H.; Zhang, Y. J. Deep learning redesign of PETase for practical PET degrading applications. bioRxiv 2021, DOI: 10.1101/2021.10.10.463845Google ScholarThere is no corresponding record for this reference.
- 52de Castro, A. M.; Carniel, A.; Nicomedes Junior, J.; da Conceição Gomes, A.; Valoni, É. Screening of commercial enzymes for poly(ethylene terephthalate) (PET) hydrolysis and synergy studies on different substrate sources. J. Ind. Microbiol. Biotechnol. 2017, 44, 835– 844, DOI: 10.1007/s10295-017-1942-zGoogle Scholar52https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1cvptlGkug%253D%253D&md5=894461cc3c0f4dcfaae0d80dc91a3b0aScreening of commercial enzymes for poly(ethylene terephthalate) (PET) hydrolysis and synergy studies on different substrate sourcesde Castro Aline Machado; Nicomedes Junior Jose; da Conceicao Gomes Absai; Valoni Erika; Carniel AdrianoJournal of industrial microbiology & biotechnology (2017), 44 (6), 835-844 ISSN:.Poly(ethylene terephthalate) (PET) is one of the most consumed plastics in the world. The development of efficient technologies for its depolymerization for monomers reuse is highly encouraged, since current recycling rates are still very low. In this study, 16 commercial lipases and cutinases were evaluated for their abilities to catalyze the hydrolysis of two PET samples. Humicola insolens cutinase showed the best performance and was then used in reactions on other PET sources, solely or in combination with the efficient mono(hydroxyethyl terephthalate)-converting lipase from Candida antarctica. Synergy degrees of the final titers of up to 2.2 (i.e., more than double of the concentration when both enzymes were used, as compared to their use alone) were found, with increased terephthalic acid formation rates, reaching a maximum of 59,989 μmol/L (9.36 g/L). These findings open up new possibilities for the conversion of post-consumer PET packages into their minimal monomers, which can be used as drop in at existing industrial facilities.
- 53Gamerith, C.; Zartl, B.; Pellis, A.; Guillamot, F.; Marty, A.; Acero, E. H.; Guebitz, G. M. Enzymatic recovery of polyester building blocks from polymer blends. Process Biochem. 2017, 59, 58– 64, DOI: 10.1016/j.procbio.2017.01.004Google Scholar53https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtVChu7s%253D&md5=443c19eaea24bd703646878c11313174Enzymatic recovery of polyester building blocks from polymer blendsGamerith, Caroline; Zartl, Barbara; Pellis, Alessandro; Guillamot, Frederique; Marty, Alain; Acero, Enrique Herrero; Guebitz, Georg M.Process Biochemistry (Oxford, United Kingdom) (2017), 59 (Part_A), 58-64CODEN: PBCHE5; ISSN:1359-5113. (Elsevier Ltd.)In this study we investigated the ability of a cutinase from Thermobifida cellulosilytica (Thc_Cut1) to hydrolyze poly(ethylene terephthalate) (PET) moieties in different polymer blends. The compn. of various materials including com. available bottles and packaging was detd. using Fourier Transform IR spectroscopy (FT-IR) and Differential Scanning Calorimetry (DSC). When incubated with PET blended with polyethylene (PE) or polyamide (PA) from packaging and bottles without prior sepn., Thc_Cut1 selectively hydrolyzed the PET moieties releasing terephthalic acid (TPA) and mono(2-hydroxyethyl) terephthalate (MHET). Polymer blends were hydrolyzed in an up to 9 times higher extent compared to higher cryst. pure PET. The influence of various parameters like temp., particle size, crystallinity and product inhibition on hydrolysis of PET moieties by Thc_Cut1 was investigated. The amt. of products released was up to 10 times higher when the incubation temp. was increased from 40 °C to 60 °C. The smaller the particle size the higher the hydrolysis rates were. Interestingly, semi-cryst. (24%) PET from bottles was hydrolyzed faster than powder from amorphous PET films (12%). An inhibitory effect of bis(2-hydroxyethyl) terephthalate (BHET) on hydrolysis of PET by Thc_Cut1 was obsd.
- 54Castro, A. M. d.; Carniel, A.; Stahelin, D.; Chinelatto Junior, L. S.; Honorato, H. d. A.; de Menezes, S. M. C. High-fold improvement of assorted post-consumer poly(ethylene terephthalate) (PET) packages hydrolysis using Humicola insolens cutinase as a single biocatalyst. Process Biochem. 2019, 81, 85– 91, DOI: 10.1016/j.procbio.2019.03.006Google Scholar54https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXkvVegurs%253D&md5=fe9b8a35851a08c00e68134feb16483cHigh-fold improvement of assorted post-consumer poly(ethylene terephthalate) (PET) packages hydrolysis using Humicola insolens cutinase as a single biocatalystCastro, Aline Machado de; Carniel, Adriano; Stahelin, Diego; Chinelatto Junior, Luiz Silvino; Honorato, Hercilio de Angeli; de Menezes, Sonia Maria CabralProcess Biochemistry (Oxford, United Kingdom) (2019), 81 (), 85-91CODEN: PBCHE5; ISSN:1359-5113. (Elsevier Ltd.)The dissemination of technologies for poly(ethylene terephthalate) (PET) recycling is of paramount importance in the context of the plastics circular economy. One of the most promising alternatives is to use enzymes as catalysts for PET depolymn. to its monomers, but this route still needs improvement, esp. regarding titer and productivity. In the present work, a sequential approach comprised of fractional factorial and central composite rotatable designs, the path of steepest ascent and one-way evaluation of variable effect, was performed to address these limitations, during assorted post-consumer PET (PC-PET) hydrolysis catalyzed by Humicola insolens cutinase. The highest terephthalic acid concn. and productivity during PC-PET hydrolysis were 100.9 mM (16.8 g/L) and 14.4 mM/day, corresponding to overall improvements of 10-fold and 20-fold, resp. These data are among the best results described so far for enzyme-catalyzed hydrolysis of used PET packages. Also, the use of a single enzyme system, instead of multiple biocatalysts to achieve final conversion of PET to its monomers, lowers the process complexity and costs.
- 55Kari, J.; Andersen, M.; Borch, K.; Westh, P. An Inverse Michaelis–Menten Approach for Interfacial Enzyme Kinetics. ACS Catal. 2017, 7, 4904– 4914, DOI: 10.1021/acscatal.7b00838Google Scholar55https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtVSjtrjK&md5=414b5391cab840baf87cfdbef2f4a2cbAn inverse Michaelis-Menten approach for interfacial enzyme kineticsKari, Jeppe; Andersen, Morten; Borch, Kim; Westh, PeterACS Catalysis (2017), 7 (7), 4904-4914CODEN: ACCACS; ISSN:2155-5435. (American Chemical Society)Interfacial enzyme reactions are ubiquitous both in vivo and in tech. applications, but anal. of their kinetics remains controversial. In particular, it is unclear whether conventional Michaelis-Menten theory, which requires a large excess of substrate, can be applied. Here, an extensive exptl. study of the enzymic hydrolysis of insol. cellulose by cellobiohydrolase Cel7A and cellulase/endoglucanase Cel12A indeed showed that the conventional approach had a limited applicability. Instead, we argue that, unlike bulk reactions, interfacial enzyme catalysis may reach a steady-state condition in the opposite exptl. limit, where the concn. of enzyme far exceeded the molar concn. of accessible surface sites. Under this condition, an "inverse Michaelis-Menten equation", where the roles of enzyme and substrate had been swapped, proved to be readily applicable. We suggest that this inverted approach provides a general tool for kinetic analyses of interfacial enzyme reactions and that its analogy to established theory provides a bridge to the accumulated understanding of steady-state enzyme kinetics. Finally, we show that the ratio of parameters from conventional and inverted Michaelis-Menten anal. reveals the d. of enzyme attack sites on the substrate surface as probed by one specific enzyme. This d., which is an analog to a molar substrate concn. for interfacial reactions, was shown to vary strongly even among related enzymes. This difference reflected how the enzyme discriminates between local differences in surface structure on the substrate.
- 56Andersen, M.; Kari, J.; Borch, K.; Westh, P. Michaelis-Menten equation for degradation of insoluble substrate. Math. Biosci. 2018, 296, 93– 97, DOI: 10.1016/j.mbs.2017.11.011Google Scholar56https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXntVCitQ%253D%253D&md5=863c7ae829e6ced4495ee36206ac2500Michaelis-Menten equation for degradation of insoluble substrateAndersen, Morten; Kari, Jeppe; Borch, Kim; Westh, PeterMathematical Biosciences (2018), 296 (), 93-97CODEN: MABIAR; ISSN:0025-5564. (Elsevier Inc.)Kinetic studies of homogeneous enzyme reactions where both the substrate and enzyme are sol. have been well described by the Michaelis-Menten (MM) equation for more than a century. However, many reactions are taking place at the interface of a solid substrate and enzyme in soln. Such heterogeneous reactions are abundant both in vivo and in industrial application of enzymes but it is not clear whether traditional enzyme kinetic theory developed for homogeneous catalysis can be applied. Since the molar concn. of surface accessible sites (attack-sites) often is unknown for a solid substrate it is difficult to assess whether the requirement of the MM equation is met. In this paper we study a simple kinetic model, where removal of attack sites expose new ones which preserve the total accessible substrate, and denote this approach the substrate conserving model. The kinetic equations are solved in closed form, both steady states and progress curves, for any admissible values of initial conditions and rate consts. The model is shown to merge with the MM equation and the reverse MM equation when these are valid. The relation between available molar concn. of attack sites and mass load of substrate is analyzed and this introduces an extra parameter to the equations. Various exptl. setups to practically and reliably est. all parameters are discussed.
- 57Bååth, J. A.; Borch, K.; Jensen, K.; Brask, J.; Westh, P. Comparative Biochemistry of Four Polyester (PET) Hydrolases. ChemBioChem 2021, 22, 1627– 1637, DOI: 10.1002/cbic.202000793Google Scholar57https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXjsFKit7o%253D&md5=f005b3f1062a71972d3445ab85a36fcbComparative Biochemistry of Four Polyester (PET) Hydrolases**Baath, Jenny Arnling; Borch, Kim; Jensen, Kenneth; Brask, Jesper; Westh, PeterChemBioChem (2021), 22 (9), 1627-1637CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)The potential of bioprocessing in a circular plastic economy has strongly stimulated research into the enzymic degrdn. of different synthetic polymers. Particular interest has been devoted to the commonly used polyester, poly(ethylene terephthalate) (PET), and a no. of PET hydrolases have been described. However, a kinetic framework for comparisons of PET hydrolases (or other plastic-degrading enzymes) acting on the insol. substrate has not been established. Herein, we propose such a framework, which we have tested against kinetic measurements for four PET hydrolases. The anal. provided values of kcat and KM, as well as an apparent specificity const. in the conventional units of M-1s-1. These parameters, together with exptl. values for the no. of enzyme attack sites on the PET surface, enabled comparative analyses. A variant of the PET hydrolase from Ideonella sakaiensis was the most efficient enzyme at ambient conditions; it relied on a high kcat rather than a low KM. Moreover, both sol. and insol. PET fragments were consistently hydrolyzed much faster than intact PET. This suggests that interactions between polymer strands slow down PET degrdn., whereas the chem. steps of catalysis and the low accessibility assocd. with solid substrate were less important for the overall rate. Finally, the investigated enzymes showed a remarkable substrate affinity, and reached half the satn. rate on PET when the concn. of attack sites in the suspension was only about 50 nM. We propose that this is linked to nonspecific adsorption, which promotes the nearness of enzyme and attack sites.
- 58Werner, A. Z.; Clare, R.; Mand, T. D.; Pardo, I.; Ramirez, K. J.; Haugen, S. J.; Bratti, F.; Dexter, G. N.; Elmore, J. R.; Huenemann, J. D. Tandem chemical deconstruction and biological upcycling of poly(ethylene terephthalate) to beta-ketoadipic acid by Pseudomonas putida KT2440. Metab. Eng. 2021, 67, 250– 261, DOI: 10.1016/j.ymben.2021.07.005Google Scholar58https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhsFOktrrL&md5=b97b521b29dad963f5d5bd148889814aTandem chemical deconstruction and biological upcycling of poly(ethylene terephthalate) to β-ketoadipic acid by Pseudomonas putida KT2440Werner, Allison Z.; Clare, Rita; Mand, Thomas D.; Pardo, Isabel; Ramirez, Kelsey J.; Haugen, Stefan J.; Bratti, Felicia; Dexter, Gara N.; Elmore, Joshua R.; Huenemann, Jay D.; Peabody, George L. V.; Johnson, Christopher W.; Rorrer, Nicholas A.; Salvachua, Davinia; Guss, Adam M.; Beckham, Gregg T.Metabolic Engineering (2021), 67 (), 250-261CODEN: MEENFM; ISSN:1096-7176. (Elsevier B.V.)Poly(ethylene terephthalate) (PET) is the most abundantly consumed synthetic polyester and accordingly a major source of plastic waste. The development of chemocatalytic approaches for PET depolymn. to monomers offers new options for open-loop upcycling of PET, which can leverage biol. transformations to higher-value products. To that end, here we perform four sequential metabolic engineering efforts in Pseudomonas putida KT2440 to enable the conversion of PET glycolysis products via: (i) ethylene glycol utilization by constitutive expression of native genes, (ii) terephthalate (TPA) catabolism by expression of tphA2IIA3IIBIIA1II from Comamonas and tpaK from Rhodococcus jostii, (iii) bis(2-hydroxyethyl) terephthalate (BHET) hydrolysis to TPA by expression of PETase and MHETase from Ideonella sakaiensis, and (iv) BHET conversion to a performance-advantaged bioproduct, β-ketoadipic acid (βKA) by deletion of pcaIJ. Using this strain, we demonstrate prodn. of 15.1 g/L βKA from BHET at 76% molar yield in bioreactors and conversion of catalytically depolymd. PET to βKA. Overall, this work highlights the potential of tandem catalytic deconstruction and biol. conversion as a means to upcycle waste PET.
- 59Blundell, D. J.; MacKerron, D. H.; Fuller, W.; Mahendrasingam, A.; Martin, C.; Oldman, R. J.; Rule, R. J.; Riekel, C. Characterization of strain-induced crystallization of poly(ethylene terephthalate) at fast draw rates using synchrotron radiation. Polymer 1996, 37, 3303– 3311, DOI: 10.1016/0032-3861(96)88476-XGoogle Scholar59https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28Xks1Ghtb8%253D&md5=6b6455387aa6cbc8ca8c0bc5d9589530Characterization of strain-induced crystallization of poly(ethylene terephthalate) at fast draw rates using synchrotron radiationBlundell, D. J.; MacKerron, D. H.; Fuller, W.; Mahendrasingam, A.; Martin, C.; Oldman, R. J.; Rule, R. J.; Riekel, C.Polymer (1996), 37 (15), 3303-3311CODEN: POLMAG; ISSN:0032-3861. (Elsevier)Structural changes during fast drawing of poly(ethylene terephthalate) were studied by wide-angle X-ray scattering using synchrotron radiation. Drawing was studied at 80, 90, 100 or 110° to a final draw ratio of ∼4:1 at a draw rate of ∼ 10s-1. Simultaneous video recording of the sample enabled variation in the X-ray pattern to be correlated with local extension. Essentially all oriented crystn. occurred after final extension. Primary crystn. fits a first-order transformation with little change in the rate of crystn. obsd. over the 30° range of temp. These results show that it can be misleading to rely on crystallinity information obtained when samples from interrupted draw expts. are quenched.
- 60Blundell, D. J.; Mahendrasingam, A.; Martin, C.; Fuller, W.; MacKerron, D. H.; Harvie, J. L.; Oldman, R. J.; Riekel, C. Orientation prior to crystallisation during drawing of poly(ethylene terephthalate). Polymer 2000, 41, 7793– 7802, DOI: 10.1016/S0032-3861(00)00128-2Google Scholar60https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXltF2rtbo%253D&md5=d978de444c9c294ec68f246f934f37b9Orientation prior to crystallisation during drawing of poly(ethylene terephthalate)Blundell, D. J.; Mahendrasingam, A.; Martin, C.; Fuller, W.; MacKerron, D. H.; Harvie, J. L.; Oldman, R. J.; Riekel, C.Polymer (2000), 41 (21), 7793-7802CODEN: POLMAG; ISSN:0032-3861. (Elsevier Science Ltd.)Wide angle X-ray scattering data have been recorded during the drawing of poly(ethylene terephthalate) (PET) using a wide range of draw rates (0.05-12 s-1), temps. (90-120°C) and draw ratios. The data were analyzed to follow the development of mol. orientation and the onset of crystn. The mol. orientation prior to crystn. has been characterized in terms of the orientation order parameter <P2(cos θ)>. The rate of increase of <P2(cos θ)> with draw ratio decreases with both increasing temp. and decreasing draw rate. A superposition of all the data to a common ref. temp. of 90°C was obtained using a WLF shift factor to provide a master curve showing the dependence of the development of <P2(cos θ)> on draw rate. A comparison of the known chain relaxation motions of PET with the obsd. relation between draw rate and the onset of crystn. provides an explanation of a previous discrepancy in the literature concerning the point of onset of crystn. For draw rates faster than the rate of the chain retraction motion, the onset of crystn. is delayed until the end of the deformation process. For draw rates slower than the chain retraction motion, there is evidence of the onset of crystn. occurring before the end of the deformation process.
- 61Mahendrasingam, A.; Martin, C.; Fuller, W.; Blundell, D. J.; Oldman, R. J.; MacKerron, D. H.; Harvie, J. L.; Riekel, C. Observation of a transient structure prior to strain-induced crystallization in poly(ethylene terephthalate). Polymer 2000, 41, 1217– 1221, DOI: 10.1016/S0032-3861(99)00461-9Google Scholar61https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXns12jsL8%253D&md5=6292d5ec77c4441f956f70ff0d6d779cObservation of a transient structure prior to strain-induced crystallization in poly(ethylene terephthalate)Mahendrasingam, A.; Martin, C.; Fuller, W.; Blundell, D. J.; Oldman, R. J.; MacKerron, D. H.; Harvie, J. L.; Riekel, C.Polymer (1999), 41 (3), 1217-1221CODEN: POLMAG; ISSN:0032-3861. (Elsevier Science Ltd.)Using time-resolved X-ray diffraction at the European Synchrotron Radiation Facility we have obsd. a highly oriented weak transient diffraction peak which persists for about 0.2 s prior to strain-induced crystn. during the uniaxial drawing of poly(ethylene terephthalate) (PET) under industrial processing conditions. This structure may be identified with the mesophase structure proposed by a no. of workers to occur during drawing of PET, poly(ethylene naphthalate) (PEN) and random copolymers of PET and PEN. In our studies, the transient structure was not obsd. at draw temps. greater than 90°C nor when the draw conditions resulted in a degree of polymer orientation below a crit. level. The possibility that this transient structure is a precursor of strain-induced crystn. is suggested by our observation of a correlation between the decay of the diffraction assocd. with it and an increase in the intensity of diffraction peaks assocd. with the development of crystn.
- 62Forestier, E.; Combeaud, C.; Guigo, N.; Sbirrazzuoli, N.; Billon, N. Understanding of strain-induced crystallization developments scenarios for polyesters: Comparison of poly(ethylene furanoate), PEF, and poly(ethylene terephthalate), PET. Polymer 2020, 203, 122755, DOI: 10.1016/j.polymer.2020.122755Google Scholar62https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhsVWhs7rF&md5=40ec244b7664224feefd92d55e540a36Understanding of strain-induced crystallization developments scenarios for polyesters: Comparison of poly(ethylene furanoate), PEF, and poly(ethylene terephthalate), PETForestier, Emilie; Combeaud, Christelle; Guigo, Nathanael; Sbirrazzuoli, Nicolas; Billon, NoellePolymer (2020), 203 (), 122755CODEN: POLMAG; ISSN:0032-3861. (Elsevier Ltd.)Specific conditions of strain, stretching, strain rate and temp. are known to be necessary for the strain induced crystn. phenomenon (SIC) to occur. It leads to the formation of a crystal in different amorphous polymers, stretched above their glassy transition. This phenomenon was intensively documented in case of poly(ethylene terephthalate), PET. More recently, some studies focused on SIC development in biobased poly(ethylene furandicarboxylate), PEF. Comparison of these crystn. abilities and crystn. kinetics upon stretching in the two materials allows to describe main differences between them, and to enlighten the role of chain architecture on SIC. To achieve that point, different mech. tensile tests were conducted using well controlled loading paths to explore the different steps of the microstructural changes induced by the stretching and their correlation with mech. behavior. Several macroscopic equivalence in the effects of SIC were found, such as increase in modulus, appearance of organized phase, increase I n α-relaxation temp. despite some differences in chain architecture. Combining both loading-unloading tests and quenching protocols, it was found that inducing more or less strong interactions between constitutive units, and more or less stable cryst. phases, leads to differences in apparent strain induced crystn. kinetics:• PET stretching can induce, prior to main strain hardening step, the formation of re-enforcing intermediate phases (or imperfect crystal) being stable upon unloading and able to be improved upon relaxation or thermal treatments;• PEF stretching exhibits a more "simple" two-steps path with no intermediate phases stable upon unloading. This can be related with the weaker stability of PEF crystal compared to PET (PEF quiescent crystn. temp. and melting temp. are very close to each other), and to the more complex cryst. lattice in PEF (two units are needed instead of one due to furanic cycle). In addn., for PET, Young modulus increases more gradually during strain hardening than for PEF. The final microstructure after stretching is therefore more dependent on thermomech. treatments (annealing or relaxation steps) in PET in comparison to PEF.
- 63Bashir, Z.; Al-Aloush, I.; Al-Raqibah, I.; Ibrahim, M. Evaluation of three methods for the measurement of crystallinity of PET resins, preforms, and bottles. Polym. Eng. Sci. 2000, 40, 2442– 2455, DOI: 10.1002/pen.11376Google Scholar63https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXoslahsbw%253D&md5=a865125bc74ef411008e9c755788e386Evaluation of three methods for the measurement of crystallinity of PET resins, preforms, and bottlesBashir, Z.; Al-Aloush, I.; Al-Raqibah, I.; Ibrahim, M.Polymer Engineering and Science (2000), 40 (11), 2442-2455CODEN: PYESAZ; ISSN:0032-3888. (Society of Plastics Engineers)The control of crystn. is important at all processing stages of the PET bottle industry, from the manuf. of bottle resins to the fabrication of preforms and bottles. The authors evaluate critically 3 methods of crystallinity measurement. They have used d., Differential Scanning Calorimetry (DSC), and Modulated Differential Scanning Calorimetry (MDSC) to study the crystallinity of PET chips, preforms, and bottles. The accuracy, precision, and general validity of each technique and the problems of interpretation are discussed.
- 64Scandola, M.; Focarete, M. L.; Frisoni, G. Simple Kinetic Model for the Heterogeneous Enzymatic Hydrolysis of Natural Poly(3-hydroxybutyrate). Macromolecules 1998, 31, 3846– 3851, DOI: 10.1021/ma980137yGoogle Scholar64https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXjtlygu7w%253D&md5=a9333839795596c7ca94cc2a2ebe675fSimple Kinetic Model for the Heterogeneous Enzymic Hydrolysis of Natural Poly(3-hydroxybutyrate)Scandola, Mariastella; Focarete, Maria Letizia; Frisoni, GiovannaMacromolecules (1998), 31 (12), 3846-3851CODEN: MAMOBX; ISSN:0024-9297. (American Chemical Society)The kinetics of the enzymic degrdn. of bacterial poly(3-hydroxybutyrate) (PHB) is studied using PHB-depolymerase A from Pseudomonas lemoignei (Tris-HCl buffer, pH 8, 37°). Biodegrdn. expts. are performed on PHB in the form of both compression-molded films and a powder suspension. From WAXS and DSC measurements the two substrates show the same cryst. fraction. The rate of hydrolysis of PHB films is detd. by gravimetrical and also through spectrophotometric quantification of the hydrolysis products at λ = 210 nm. For the suspension of PHB particles, a turbidimetric detn. of the biodegrdn. rate is applied. A simple two-step kinetic model is proposed, which predicts that the hydrolysis rate per unit substrate surface area reaches a plateau at high enzyme concns. The model satisfactorily describes the enzymic degrdn. results of PHB film and PHB powder suspension, provided that the remarkable changes of the exposed area caused by enzymic attack to the latter substrate are taken into account. Anal. of the enzymic degrdn. results yields analogous hydrolysis rate consts. for film (1.48 μg cm-2 min-1) and powder suspension (1.42 μg cm-2 min-1).
- 65Thomsen, T. B.; Hunt, C. J.; Meyer, A. S. Influence of substrate crystallinity and glass transition temperature on enzymatic degradation of polyethylene terephthalate (PET). New Biotechnol. 2022, 69, 28– 35, DOI: 10.1016/j.nbt.2022.02.006Google Scholar65https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XntFCgu78%253D&md5=abdf6a78ca51b882a02a6fb99b03922fInfluence of substrate crystallinity and glass transition temperature on enzymatic degradation of polyethylene terephthalate (PET)Thomsen, Thore Bach; Hunt, Cameron J.; Meyer, Anne S.New Biotechnology (2022), 69 (), 28-35CODEN: NBEIBR; ISSN:1871-6784. (Elsevier B.V.)This work examines the significance of the degree of crystallinity (XC) of polyethylene terephthalate (PET) and the PET glass transition temp. (Tg) on enzymic degrdn. of PET at elevated temps. using two engineered, thermostable PET degrading enzymes: LCCICCG, a variant of the leaf-branch compost cutinase, and DuraPETase, evolved from the Ideonella sakaiensis PETase. The XC was systematically varied by thermal annealing of PET disks (O 6 mm, thickness 1 mm). The XC affected the enzymic product release rate that essentially ceased at XC 22-27% for the LCCICCG and at XC ∼17% for the DuraPETase. SEM revealed that enzymic treatment produced cavities on the PET surface when the XC was > 10% but resulted in a smooth surface on amorphous PET (XC ∼10%). The Tg of amorphous PET disks decreased from 75 °C to 60 °C during 24 h pre-soaking in water at 65 °C, while the XC remained unchanged. Enzymic reaction on pre-soaked disks at 68 °C, i.e. above the Tg, did not affect the enzymic product release rate catalyzed by LCCICCG. These findings improve the understanding of enzymic PET degrdn. and have implications for development of efficient enzymic PET upcycling processes.
- 66Barth, M.; Honak, A.; Oeser, T.; Wei, R.; Belisário-Ferrari, M. R.; Then, J.; Schmidt, J.; Zimmermann, W. A dual enzyme system composed of a polyester hydrolase and a carboxylesterase enhances the biocatalytic degradation of polyethylene terephthalate films. Biotechnol. J. 2016, 11, 1082– 1087, DOI: 10.1002/biot.201600008Google Scholar66https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtVWkt7rL&md5=37b71dbedbbcdc97e07df882075b6ffdA dual enzyme system composed of a polyester hydrolase and a carboxylesterase enhances the biocatalytic degradation of polyethylene terephthalate filmsBarth, Markus; Honak, Annett; Oeser, Thorsten; Wei, Ren; Belisario-Ferrari, Matheus R.; Then, Johannes; Schmidt, Juliane; Zimmermann, WolfgangBiotechnology Journal (2016), 11 (8), 1082-1087CODEN: BJIOAM; ISSN:1860-6768. (Wiley-VCH Verlag GmbH & Co. KGaA)TfCut2 from Thermobifida fusca KW3 and the metagenome-derived LC-cutinase are bacterial polyester hydrolases capable of efficiently degrading polyethylene terephthalate (PET) films. Since the enzymic PET hydrolysis is inhibited by the degrdn. intermediate mono-(2-hydroxyethyl) terephthalate (MHET), a dual enzyme system consisting of a polyester hydrolase and the immobilized carboxylesterase TfCa from Thermobifida fusca KW3 was employed for the hydrolysis of PET films at 60°C. HPLC anal. of the reaction products obtained after 24 h of hydrolysis showed an increased amt. of sol. products with a lower proportion of MHET in the presence of the immobilized TfCa. The results indicated a continuous hydrolysis of the inhibitory MHET by the immobilized TfCa and demonstrated its advantage as a second biocatalyst in combination with a polyester hydrolase for an efficient degrdn. oft PET films. The dual enzyme system with LC-cutinase produced a 2.4-fold higher amt. of degrdn. products compared to TfCut2 after a reaction time of 24 h confirming the superior activity of his polyester hydrolase against PET films.
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Abstract
Figure 1
Figure 1. Size distributions of CM-PET and HC-PET particles. Histograms indicate the volume percentage as a function of Feret’s minimum diameter of CM-PET (blue) and HC-PET (red) substrates. (A,D) 250, (B,E) 125, and (C,F) sub125 μm sieve fractions. Inset graphs show the expanded view of the data.
Figure 2
Figure 2. Monomer release as a function of time for 10 mg mL–1 PET particles with 1 μM LCC-ICCG. Monomers present in the reaction were analyzed by HPLC. (A) 250 μm CM-PET, (B) 125 μm CM-PET, (C) sub125 μm CM-PET, (D) 250 μm HC-PET, (E) 125 μm HC-PET, and (F) sub125 μm HC-PET. Reactions were performed in triplicate and errors bars represent the standard deviation. The data shown in this figure are provided in Dataset S1.
Figure 3
Figure 3. Total amount of monomers released from PET particles hydrolyzed with LCC-ICCG. The bars show the concentrations of all monomers released at the 72 h endpoint of the reaction with indicated [LCC-ICCG] and 10 mg mL–1 PET substrate. Reactions were performed in triplicate, and error bars represent standard deviation. The data shown in this figure are provided in Dataset S3.
Figure 4
Figure 4. InvMM kinetic analysis and total amount of monomers released from PET particles upon hydrolysis with LCC-ICCG. InvMM analysis of 10 mg mL–1 (A) CM-PET and (B) HC-PET hydrolysis rate as a function of LCC-ICCG concentration ([E]). Lines are fits of the invMM equation. Table 2 shows the fitted parameters. Reactions were performed in triplicate and error bars represent standard deviation. The data in this figure are provided in Dataset S5.
Figure 5
Figure 5. Conversion time courses of 1 L scale reactions conducted in bioreactors. Results from the reaction with various PET substrates at 100 g L–1 and 3 mg g–1 PET LCC-ICCG enzyme. (A–C) Product release profiles as a function of time measured by HPLC for (A) LC-PET film cut into ∼1 × 1 cm squares, (B) HC-PET particles, and (C) WC-PET particles. (D–F) Percent conversion of total PET for (D) LC-PET film cut into ∼1 × 1 cm squares, (E) HC-PET particles, and (F) WC-PET particles. Reactions were performed in duplicate, data points show the mean, and error bars show the absolute difference between the duplicates. The data shown in this figure are provided in Dataset S6.
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- 12Müller, R.-J.; Schrader, H.; Profe, J.; Dresler, K.; Deckwer, W.-D. Enzymatic Degradation of Poly(ethylene terephthalate): Rapid Hydrolyse using a Hydrolase from T. fusca. Macromol. Rapid Commun. 2005, 26, 1400– 1405, DOI: 10.1002/marc.20050041012https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXhtVGiur7E&md5=f3a2691f34a2d3e363b80090bb265d2fEnzymatic degradation of poly(ethylene terephthalate): Rapid hydrolyse using a hydrolase from T. fuscaMueller, Rolf-Joachim; Schrader, Hedwig; Profe, Joern; Dresler, Karolin; Deckwer, Wolf-DieterMacromolecular Rapid Communications (2005), 26 (17), 1400-1405CODEN: MRCOE3; ISSN:1022-1336. (Wiley-VCH Verlag GmbH & Co. KGaA)It is demonstrated that PET, which is usually regarded as 'non-biodegradable', can effectively be depolymd. by a hydrolase from the actinomycete Thermobifida fusca. Erosion rates of 8 to 17 μm per wk were obtained upon incubation at 55°. Lipases from Pseudomonas sp. and Candida antarctica did not degrade PET under comparable conditions. The influences of crystallinity, m.p., and glass transition temp. on the enzymic attack on PET, PBT, and PHB are discussed.
- 13Alisch-Mark, M.; Herrmann, A.; Zimmermann, W. Increase of the hydrophilicity of polyethylene terephthalate fibres by hydrolases from Thermomonospora fusca and Fusarium solani f. sp. pisi. Biotechnol. Lett. 2006, 28, 681– 685, DOI: 10.1007/s10529-006-9041-713https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XmtVOqs7Y%253D&md5=0f59affe7b16b73a8d674480024d5621Increase of the hydrophilicity of polyethylene terephthalate fibres by hydrolases from Thermomonospora fusca and Fusarium solani f. sp. pisiAlisch-Mark, Mandy; Herrmann, Anne; Zimmermann, WolfgangBiotechnology Letters (2006), 28 (10), 681-685CODEN: BILED3; ISSN:0141-5492. (Springer)Treatment of polyethylene terephthalate fibers with hydrolase prepns. from Thermomonospora (Thermobifida) fusca and Fusarium solani f. sp. pisi resulted in an increase of the hydrophilicity of the fibers detd. by measurement of their dyeing behavior with reactive dyes and their water absorption ability. Reflectance spectrometry of treated fibers dyed with a reactive dye showed that the color became more intense corresponding to an increase of hydroxyl groups on the fiber surfaces and indicated a stepwise peeling of the fibers by the enzymes comparable to the effects obtained by alk. treatments. The synthetic fibers treated with the hydrolase from T. fusca also showed enhanced water absorption ability further confirming the increased surface hydrophilicity caused by the enzyme.
- 14Eberl, A.; Heumann, S.; Brückner, T.; Araujo, R.; Cavaco-Paulo, A.; Kaufmann, F.; Kroutil, W.; Guebitz, G. M. Enzymatic surface hydrolysis of poly(ethylene terephthalate) and bis(benzoyloxyethyl) terephthalate by lipase and cutinase in the presence of surface active molecules. J. Biotechnol. 2009, 143, 207– 212, DOI: 10.1016/j.jbiotec.2009.07.00814https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhtVKksbfJ&md5=21046f062c67b8d8ec6ab8bc4518dfa9Enzymatic surface hydrolysis of poly(ethylene terephthalate) and bis(benzoyloxyethyl) terephthalate by lipase and cutinase in the presence of surface active moleculesEberl, Anita; Heumann, Sonja; Brueckner, Tina; Araujo, Rita; Cavaco-Paulo, Artur; Kaufmann, Franz; Kroutil, Wolfgang; Guebitz, Georg M.Journal of Biotechnology (2009), 143 (3), 207-212CODEN: JBITD4; ISSN:0168-1656. (Elsevier B.V.)A lipase from Thermomyces lanuginosus and cutinases from Thermobifida fusca and Fusarium solani hydrolyzed poly(ethylene terephthalate) (PET) fabrics and films and bis(benzoyloxyethyl) terephthalate (3PET) endo-wise as shown by MALDI-Tof-MS, LC-UVD/MS, cationic dyeing and XPS anal. Due to interfacial activation of the lipase in the presence of Triton X-100, a seven-fold increase of hydrolysis products released from 3PET was measured. In the presence of the plasticizer N,N-diethyl-2-phenylacetamide (DEPA), increased hydrolysis rates of semi-cryst. PET films and fabrics were measured both for lipase and cutinase. The formation of novel polar groups resulted in enhanced dye ability with addnl. increase in color depth by 130% and 300% for cutinase and lipase, resp., in the presence of plasticizer.
- 15Ronkvist, Å. M.; Xie, W.; Lu, W.; Gross, R. A. Cutinase-Catalyzed Hydrolysis of Poly(ethylene terephthalate). Macromolecules 2009, 42, 5128– 5138, DOI: 10.1021/ma900531815https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXotVGksb0%253D&md5=3efba9137ee43e2684de56c081549668Cutinase-Catalyzed Hydrolysis of Poly(ethylene terephthalate)Ronkvist, Asa M.; Xie, Wenchun; Lu, Wenhua; Gross, Richard A.Macromolecules (Washington, DC, United States) (2009), 42 (14), 5128-5138CODEN: MAMOBX; ISSN:0024-9297. (American Chemical Society)A detailed study and comparison was made on the catalytic activities of cutinases from Humilica insolens (HiC), Pseudomonas mendocina (PmC), and Fusarium solani (FsC) using low-crystallinity (lc) and biaxially oriented (bo) poly(ethylene terephthalate) (PET) films as model substrates. Cutinase activity for PET hydrolysis was assayed using a pH-stat to measure NaOH consumption vs. time, where initial activity was expressed as units of micromoles of NaOH added per h and per mL of reaction vol. HiC was found to have good thermostability with max. initial activity from 70 to 80°, whereas PmC and FsC performed best at 50°. Assays by pH-stat showed that the cutinases had about 10-fold higher activity for the lcPET (7% crystallinity) than for the boPET (35% crystallinity). Under optimal reaction conditions, initial activities of cutinases were successfully fit by a heterogeneous kinetic model. The hydrolysis rate const. k2 was 7-fold higher for HiC at 70° (0.62 μmol/Cm2/h) relative to PmC and FsC at 50 and 40°, resp. With respect to PET affinity, PmC had the highest affinity, while FsC had the lowest value. In a 96 h degrdn. study using lcPET films, incubation with PmC and FsC both resulted in a 5% film wt. loss at 50 and 40°, resp. In contrast, HiC-catalyzed lcPET film hydrolysis at 70° resulted in a 97±3% wt. loss in 96 h, corresponding to a loss in film thickness of 30 μm per day. As degrdn. of lcPET progressed, crystallinity of the remaining film increased to 27% due to preferential degrdn. of amorphous regions. Furthermore, for all three cutinases, anal. of aq. sol. degrdn. products showed that they consist exclusively of terephthalic acid and ethylene glycol.
- 16Ribitsch, D.; Heumann, S.; Trotscha, E.; Herrero Acero, E.; Greimel, K.; Leber, R.; Birner-Gruenberger, R.; Deller, S.; Eiteljoerg, I.; Remler, P. Hydrolysis of polyethyleneterephthalate by p-nitrobenzylesterase from Bacillus subtilis. Biotechnol. Prog. 2011, 27, 951– 960, DOI: 10.1002/btpr.61016https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhtValt7jM&md5=c2c3355b13942877c10c2f060f22529fHydrolysis of polyethyleneterephthalate by p-nitrobenzylesterase from Bacillus subtilisRibitsch, Doris; Heumann, Sonja; Trotscha, Eva; Acero, Enrique Herrero; Greimel, Katrin; Leber, Regina; Birner-Gruenberger, Ruth; Deller, Sigrid; Eiteljoerg, Inge; Remler, Peter; Weber, Thomas; Siegert, Petra; Maurer, Karl-Heinz; Donelli, Ilaria; Freddi, Giuliano; Schwab, Helmut; Guebitz, Georg M.Biotechnology Progress (2011), 27 (4), 951-960CODEN: BIPRET; ISSN:1520-6033. (Wiley-Blackwell)From a screening on agar plates with bis(benzoyloxyethyl) terephthalate (3PET), a Bacillus subtilis p-nitrobenzylesterase (BsEstB) was isolated and demonstrated to hydrolyze polyethyleneterephthalate (PET). PET-hydrolase active strains produced clearing zones and led to the release of the 3PET hydrolysis products terephthalic acid (TA), benzoic acid (BA), 2-hydroxyethyl benzoate (HEB), and mono-(2-hydroxyethyl) terephthalate (MHET) in 3PET supplemented liq. cultures. The 3PET-hydrolase was isolated from non-denaturating polyacrylamide gels using fluorescein diacetate (FDA) and identified as BsEstB by LC-MS/MS anal. BsEstB was expressed in Escherichia coli with C-terminally fused StrepTag II for purifn. The tagged enzyme had a mol. mass of 55.2 kDa and a specific activity of 77 U/mg on p-nitrophenyl acetate and 108 U/mg on p-nitrophenyl butyrate. BsEstB was most active at 40°C and pH 7.0 and stable for several days at pH 7.0 and 37°C while the half-life times decreased to 3 days at 40°C and only 6 h at 45°C. From 3PET, BsEstB released TA, MHET, and BA, but neither bis(2-hydroxyethyl) terephthalate (BHET) nor hydroxyethylbenzoate (HEB). The kcat values decreased with increasing complexity of the substrate from 6 and 8 (s-1) for p-nitrophenyl-acetate (4NPA) and p-nitrophenyl-butyrate (4NPB), resp., to 0.14 (s-1) for bis(2-hydroxyethyl) terephthalate (BHET). The enzyme hydrolyzed PET films releasing TA and MHET with a concomitant decrease of the water-contact angle (WCA) from 68.2° ± 1.7° to 62.6° ± 1.1° due to formation of novel hydroxyl and carboxyl groups. These data correlated with a fluorescence emission intensity increase seen for the enzyme treated sample after derivatization with 2-(bromomethyl)naphthalene.
- 17Sulaiman, S.; Yamato, S.; Kanaya, E.; Kim, J.-J.; Koga, Y.; Takano, K.; Kanaya, S. Isolation of a novel cutinase homolog with polyethylene terephthalate-degrading activity from leaf-branch compost by using a metagenomic approach. Appl. Environ. Microbiol. 2012, 78, 1556– 1562, DOI: 10.1128/aem.06725-1117https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XjtVSntLY%253D&md5=5f056dac0e9ab4a5b00c2185ee936e9bIsolation of a novel cutinase homolog with polyethylene terephthalate-degrading activity from leaf-branch compost by using a metagenomic approachSulaiman, Sintawee; Yamato, Saya; Kanaya, Eiko; Kim, Joong-Jae; Koga, Yuichi; Takano, Kazufumi; Kanaya, ShigenoriApplied and Environmental Microbiology (2012), 78 (5), 1556-1562CODEN: AEMIDF; ISSN:0099-2240. (American Society for Microbiology)The gene encoding a cutinase homolog, LC-cutinase, was cloned from a fosmid library of a leaf-branch compost metagenome by functional screening using tributyrin agar plates. LC-cutinase shows the highest amino acid sequence identity of 59.7% to Thermomonospora curvata lipase. It also shows the 57.4% identity to Thermobifida fusca cutinase. When LC-cutinase without a putative signal peptide was secreted to the periplasm of Escherichia coli cells with the assistance of the pelB leader sequence, more than 50% of the recombinant protein, termed LC-cutinase*, was excreted into the extracellular medium. It was purified and characterized. LC-cutinase* hydrolyzed various fatty acid monoesters with acyl chain lengths of 2 to 18, with a preference for short-chain substrates (C4 substrate at most) most optimally at pH 8.5 and 50°C, but could not hydrolyze olive oil. It lost activity with half-lives of 40 min at 70°C and 7 min at 80°C. LC-cutinase* had an ability to degrade poly(ε-caprolactone) and polyethylene terephthalate (PET). The specific PET-degrading activity of LC-cutinase* was detd. to be 12 mg/h/mg of enzyme (2.7 mg/h/μkat of pNP-butyrate-degrading activity) at pH 8.0 and 50°C. This activity is higher than those of the bacterial and fungal cutinases reported thus far, suggesting that LC-cutinase* not only serves as a good model for understanding the mol. mechanism of PET-degrading enzyme but also is potentially applicable for surface modification and degrdn. of PET.
- 18Roth, C.; Wei, R.; Oeser, T.; Then, J.; Föllner, C.; Zimmermann, W.; Sträter, N. Structural and functional studies on a thermostable polyethylene terephthalate degrading hydrolase from Thermobifida fusca. Appl. Microbiol. Biotechnol. 2014, 98, 7815– 7823, DOI: 10.1007/s00253-014-5672-018https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXmtlWiu7Y%253D&md5=d5bf2d167198e40b3d4c79b24b347fedStructural and functional studies on a thermostable polyethylene terephthalate degrading hydrolase from Thermobifida fuscaRoth, Christian; Wei, Ren; Oeser, Thorsten; Then, Johannes; Foellner, Christina; Zimmermann, Wolfgang; Straeter, NorbertApplied Microbiology and Biotechnology (2014), 98 (18), 7815-7823CODEN: AMBIDG; ISSN:0175-7598. (Springer)Bacterial cutinases are promising catalysts for the modification and degrdn. of the widely used plastic polyethylene terephthalate (PET). The improvement of the enzyme for industrial purposes is limited due to the lack of structural information for cutinases of bacterial origin. Here, the authors report having crystd. and structurally characterized a cutinase from T. fusca KW3 (TfCut2) in free as well as in inhibitor-bound form. Together with an anal. of the thermostability and modeling studies, the authors suggest possible reasons for the outstanding thermostability in comparison to the less thermostable homolog from T. alba AHK119 and propose a model for the binding of the enzyme toward its polymeric substrate. The TfCut2 structure is the basis for the rational design of catalytically more efficient enzyme variants for the hydrolysis of PET and other synthetic polyesters.
- 19Sulaiman, S.; You, D.-J.; Kanaya, E.; Koga, Y.; Kanaya, S. Crystal Structure and Thermodynamic and Kinetic Stability of Metagenome-Derived LC-Cutinase. Biochemistry 2014, 53, 1858– 1869, DOI: 10.1021/bi401561p19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXjsVehs7w%253D&md5=6a3d102dc43578ef18b633c5e09c3f38Crystal Structure and Thermodynamic and Kinetic Stability of Metagenome-Derived LC-CutinaseSulaiman, Sintawee; You, Dong-Ju; Kanaya, Eiko; Koga, Yuichi; Kanaya, ShigenoriBiochemistry (2014), 53 (11), 1858-1869CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)The crystal structure of metagenome-derived LC-cutinase with polyethylene terephthalate (PET)-degrading activity was detd. at 1.5 Å resoln. The structure strongly resembles that of Thermobifida alba cutinase. Ser165, Asp210, and His242 form the catalytic triad. Thermal denaturation and guanidine hydrochloride (GdnHCl)-induced unfolding of LC-cutinase were analyzed at pH 8.0 by CD spectroscopy. The midpoint of the transition of the thermal denaturation curve, T1/2, and that of the GdnHCl-induced unfolding curve, Cm, at 30 °C were 86.2 °C and 4.02 M, resp. The free energy change of unfolding in the absence of GdnHCl, ΔG(H2O), was 41.8 kJ mol-1 at 30 °C. LC-cutinase unfolded very slowly in GdnHCl with an unfolding rate, ku(H2O), of 3.28 × 10-6 s-1 at 50 °C. These results indicate that LC-cutinase is a kinetically robust protein. Nevertheless, the optimal temp. for the activity of LC-cutinase toward p-nitrophenyl butyrate (50 °C) was considerably lower than the T1/2 value. It increased by 10 °C in the presence of 1% polyethylene glycol (PEG) 1000. It also increased by at least 20 °C when PET was used as a substrate. These results suggest that the active site is protected from a heat-induced local conformational change by binding of PEG or PET. LC-cutinase contains one disulfide bond between Cys275 and Cys292. To examine whether this disulfide bond contributes to the thermodn. and kinetic stability of LC-cutinase, C275/292A-cutinase without this disulfide bond was constructed. Thermal denaturation studies and equil. and kinetic studies of the GdnHCl-induced unfolding of C275/292A-cutinase indicate that this disulfide bond contributes not only to the thermodn. stability but also to the kinetic stability of LC-cutinase.
- 20Wei, R.; Oeser, T.; Zimmermann, W. Synthetic polyester-hydrolyzing enzymes from thermophilic actinomycetes. Adv. Appl. Microbiol. 2014, 89, 267– 305, DOI: 10.1016/B978-0-12-800259-9.00007-X20https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2M%252Fisl2huw%253D%253D&md5=e7be6a3e3c6769542d851ffd65c9280eSynthetic polyester-hydrolyzing enzymes from thermophilic actinomycetesWei Ren; Oeser Thorsten; Zimmermann WolfgangAdvances in applied microbiology (2014), 89 (), 267-305 ISSN:0065-2164.Thermophilic actinomycetes produce enzymes capable of hydrolyzing synthetic polyesters such as polyethylene terephthalate (PET). In addition to carboxylesterases, which have hydrolytic activity predominantly against PET oligomers, esterases related to cutinases also hydrolyze synthetic polymers. The production of these enzymes by actinomycetes as well as their recombinant expression in heterologous hosts is described and their catalytic activity against polyester substrates is compared. Assays to analyze the enzymatic hydrolysis of synthetic polyesters are evaluated, and a kinetic model describing the enzymatic heterogeneous hydrolysis process is discussed. Structure-function and structure-stability relationships of actinomycete polyester hydrolases are compared based on molecular dynamics simulations and recently solved protein structures. In addition, recent progress in enhancing their activity and thermal stability by random or site-directed mutagenesis is presented.
- 21Perz, V.; Bleymaier, K.; Sinkel, C.; Kueper, U.; Bonnekessel, M.; Ribitsch, D.; Guebitz, G. M. Substrate specificities of cutinases on aliphatic-aromatic polyesters and on their model substrates. New Biotechnol. 2016, 33, 295– 304, DOI: 10.1016/j.nbt.2015.11.00421https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhvVykurvE&md5=64681992221ed7dc6cbc62a2979580edSubstrate specificities of cutinases on aliphatic-aromatic polyesters and on their model substratesPerz, Veronika; Bleymaier, Klaus; Sinkel, Carsten; Kueper, Ulf; Bonnekessel, Melanie; Ribitsch, Doris; Guebitz, Georg M.New Biotechnology (2016), 33 (2), 295-304CODEN: NBEIBR; ISSN:1871-6784. (Elsevier B.V.)The enzymic hydrolysis of the biodegradable polyester ecoflex and of a variety of oligomeric and polymeric ecoflex model substrates was investigated. For this purpose, substrate specificities of two enzymes of typical compost inhabitants, namely a fungal cutinase from Humicola insolens (HiC) and a bacterial cutinase from Thermobifida cellulosilytica (Thc_Cut1) were compared. Model substrates were systematically designed with variations of the chain length of the alc. and the acid as well as with varying content of the arom. constituent terephthalic acid (Ta).HPLC/MS identification and quantification of the hydrolysis products terephthalic acid (Ta), benzoic acid (Ba), adipic acid (Ada), mono(4-hydroxybutyl) terephthalate (BTa), mono-(2-hydroxyethyl) terephthalate (ETa), mono-(6-hydroxyhexyl) terephthalate (HTa) and bis(4-hydroxybutyl) terephthalate (BTaB) indicated that these enzymes indeed hydrolyze the tested esters. Shorter terminal chain length acids but longer chain length alcs. in oligomeric model substrates were generally hydrolyzed more efficiently. Thc_Cut1 hydrolyzed arom. ester bonds more efficiently than HiC resulting in up to 3-fold higher concns. of the monomeric hydrolysis product Ta. Nevertheless, HiC exhibited a higher overall hydrolytic activity on the tested polyesters, resulting in 2-fold higher concn. of released mols. Thermogravimetry and differential scanning calorimetry (TG-DSC) of the polymeric model substrates revealed a general trend that a lower difference between melting temp. (Tm) and the temp. at which the enzymic degrdn. takes place resulted in higher susceptibility to enzymic hydrolysis.
- 22Yoshida, S.; Hiraga, K.; Takehana, T.; Taniguchi, I.; Yamaji, H.; Maeda, Y.; Toyohara, K.; Miyamoto, K.; Kimura, Y.; Oda, K. A bacterium that degrades and assimilates poly(ethylene terephthalate). Science 2016, 351, 1196– 1199, DOI: 10.1126/science.aad635922https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xjs12gtr4%253D&md5=2bd8b9ff8d09fcc55944c8dc65b9cd46A bacterium that degrades and assimilates poly(ethylene terephthalate)Yoshida, Shosuke; Hiraga, Kazumi; Takehana, Toshihiko; Taniguchi, Ikuo; Yamaji, Hironao; Maeda, Yasuhito; Toyohara, Kiyotsuna; Miyamoto, Kenji; Kimura, Yoshiharu; Oda, KoheiScience (Washington, DC, United States) (2016), 351 (6278), 1196-1199CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Poly(ethylene terephthalate) (PET) is used extensively worldwide in plastic products, and its accumulation in the environment has become a global concern. Because the ability to enzymically degrade PET has been thought to be limited to a few fungal species, biodegrdn. is not yet a viable remediation or recycling strategy. By screening natural microbial communities exposed to PET in the environment, the authors isolated a novel bacterium, Ideonella sakaiensis 201-F6, that is able to use PET as its major energy and carbon source. When grown on PET, this strain produces two enzymes capable of hydrolyzing PET and the reaction intermediate, mono(2-hydroxyethyl) terephthalic acid. Both enzymes are required to enzymically convert PET efficiently into its two environmentally benign monomers, terephthalic acid and ethylene glycol.
- 23Ribitsch, D.; Hromic, A.; Zitzenbacher, S.; Zartl, B.; Gamerith, C.; Pellis, A.; Jungbauer, A.; Łyskowski, A.; Steinkellner, G.; Gruber, K. Small cause, large effect: Structural characterization of cutinases from Thermobifida cellulosilytica. Biotechnol. Bioeng. 2017, 114, 2481– 2488, DOI: 10.1002/bit.2637223https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtlart7vL&md5=69ee041cdeba69c3be8a4582e4a7d69dSmall cause, large effect: Structural characterization of cutinases from Thermobifida cellulosilyticaRibitsch, Doris; Hromic, Altijana; Zitzenbacher, Sabine; Zartl, Barbara; Gamerith, Caroline; Pellis, Alessandro; Jungbauer, Alois; Lyskowski, Andrzej; Steinkellner, Georg; Gruber, Karl; Tscheliessnig, Rupert; Herrero Acero, Enrique; Guebitz, Georg M.Biotechnology and Bioengineering (2017), 114 (11), 2481-2488CODEN: BIBIAU; ISSN:0006-3592. (John Wiley & Sons, Inc.)We have investigated the structures of two native cutinases from Thermobifida cellulosilytica, namely Thc_Cut1 and Thc_Cut2 as well as of two variants, Thc_Cut2_DM (Thc_Cut2_ Arg29Asn_Ala30Val) and Thc_Cut2_TM (Thc_Cut2_Arg19Ser_Arg29Asn_Ala30Val). The four enzymes showed different activities towards the aliph. polyester poly(lactic acid) (PLLA). The crystal structures of the four enzymes were successfully solved and in combination with Small Angle X-Ray Scattering (SAXS) the structural features responsible for the selectivity difference were elucidated. Anal. of the crystal structures did not indicate significant conformational differences among the different cutinases. However, the distinctive SAXS scattering data collected from the enzymes in soln. indicated a remarkable surface charge difference. The difference in the electrostatic and hydrophobic surface properties could explain potential alternative binding modes of the four cutinases on PLLA explaining their distinct activities. Biotechnol. Bioeng. 2017;9999: 1-8. © 2017 Wiley Periodicals, Inc.
- 24Kawai, F.; Kawabata, T.; Oda, M. Current State and Perspectives Related to the Polyethylene Terephthalate Hydrolases Available for Biorecycling. ACS Sustainable Chem. Eng. 2020, 8, 8894– 8908, DOI: 10.1021/acssuschemeng.0c0163824https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXpvFWqtLY%253D&md5=ddbd5aaa091f9b6600fdd8dec57e216dCurrent State and Perspectives Related to the Polyethylene Terephthalate Hydrolases Available for BiorecyclingKawai, Fusako; Kawabata, Takeshi; Oda, MasayukiACS Sustainable Chemistry & Engineering (2020), 8 (24), 8894-8908CODEN: ASCECG; ISSN:2168-0485. (American Chemical Society)A review. Polyethylene terephthalate (PET) hydrolase is a challenging target as PET is a commonly used plastic that is extremely resistant to enzymic attack. Since the discovery of a PET hydrolase from Thermobifida fusca in 2005, novel PET hydrolases and their availability toward waste PET have been investigated. At present, at least four thermophilic cutinases are known as PET hydrolases that could be used for the management of amorphous PET waste, such as packaging materials. Heat-labile PETase from Ideonella sakaiensis and its homologues from mesophilic bacteria exist in the environment. However, PET can be efficiently hydrolyzed with thermophilic hydrolases. This Review focuses on the current state of PET hydrolases and the potential of their application. Contrary to an amorphous PET, the enzymic hydrolysis of cryst. PET (particularly PET bottles) remains to be fully elucidated. It cannot be assured whether the biorecycling of general PET would be put into practice in the near future, but the plan is getting closer to the goal. PET hydrolases can be versatile polyesterases as they can hydrolyze not only PET but also other polyesters. Addnl., the thermostability of PET hydrolases is advantageous to their application in terms of reaction speed and durability. Enzymic recycling is more ecofriendly than hazardous chem. recycling, and thermophilic PET hydrolases are indispensable for the biorecycling of PET.
- 25Sonnendecker, C.; Oeser, J.; Richter, P. K.; Hille, P.; Zhao, Z.; Fischer, C.; Lippold, H.; Blazquez-Sanchez, P.; Engelberger, F.; Ramirez-Sarmiento, C. A. Low Carbon Footprint Recycling of Post-Consumer PET Plastic with a Metagenomic Polyester Hydrolase. ChemSusChem 2022, 15, e202101062 DOI: 10.1002/cssc.20210106225https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XjsFCltro%253D&md5=2e80ffb54c6bedd31d57fcd46a2e8a56Low Carbon Footprint Recycling of Post-Consumer PET Plastic with a Metagenomic Polyester HydrolaseSonnendecker, Christian; Oeser, Juliane; Richter, P. Konstantin; Hille, Patrick; Zhao, Ziyue; Fischer, Cornelius; Lippold, Holger; Blazquez-Sanchez, Paula; Engelberger, Felipe; Ramirez-Sarmiento, Cesar A.; Oeser, Thorsten; Lihanova, Yuliia; Frank, Ronny; Jahnke, Heinz-Georg; Billig, Susan; Abel, Bernd; Strater, Norbert; Matysik, Jorg; Zimmermann, WolfgangChemSusChem (2022), 15 (9), e202101062CODEN: CHEMIZ; ISSN:1864-5631. (Wiley-VCH Verlag GmbH & Co. KGaA)Earth is flooded with plastics and the need for sustainable recycling strategies for polymers has become increasingly urgent. Enzyme-based hydrolysis of post-consumer plastic is an emerging strategy for closed-loop recycling of polyethylene terephthalate (PET). The polyester hydrolase PHL7, isolated from a compost metagenome, completely hydrolyzes amorphous PET films, releasing 91 mg of terephthalic acid per h and mg of enzyme. Vertical scanning interferometry shows degrdn. rates of the PET film of 6.8μm h-1. Structural anal. indicates the importance of leucine at position 210 for the extraordinarily high PET-hydrolyzing activity of PHL7. Within 24 h, 0.6 mgenzyme gPET-1 completely degrades post-consumer thermoform PET packaging in an aq. buffer at 70°C without any energy-intensive pretreatments. Terephthalic acid recovered from the enzymic hydrolyzate is then used to synthesize virgin PET, demonstrating the potential of polyester hydrolases as catalysts in sustainable PET recycling processes with a low carbon footprint.
- 26Herrero Acero, E.; Ribitsch, D.; Dellacher, A.; Zitzenbacher, S.; Marold, A.; Steinkellner, G.; Gruber, K.; Schwab, H.; Guebitz, G. M. Surface engineering of a cutinase from Thermobifida cellulosilytica for improved polyester hydrolysis. Biotechnol. Bioeng. 2013, 110, 2581– 2590, DOI: 10.1002/bit.2493026https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXms1KksLY%253D&md5=dff744691be4ad5f0cb5bb219a9b2d32Surface engineering of a cutinase from Thermobifida cellulosilytica for improved polyester hydrolysisHerrero Acero, Enrique; Ribitsch, Doris; Dellacher, Anita; Zitzenbacher, Sabine; Marold, Annemarie; Steinkellner, Georg; Gruber, Karl; Schwab, Helmut; Guebitz, Georg M.Biotechnology and Bioengineering (2013), 110 (10), 2581-2590CODEN: BIBIAU; ISSN:0006-3592. (John Wiley & Sons, Inc.)Modeling and comparison of the structures of the two closely related cutinases Thc_Cut1 and Thc_Cut2 from Thermobifida cellulosilytica DSM44535 revealed that dissimilarities in their electrostatic and hydrophobic surface properties in the vicinity to the active site could be responsible for pronounced differences in hydrolysis efficiencies of polyester (i.e., PET, polyethyleneterephthalate). To investigate this hypothesis in more detail, selected amino acids of surface regions outside the active site of Thc_Cut2, which hydrolyzes PET much less efficiently than Thc_Cut1 were exchanged by site-directed mutagenesis. The mutants were expressed in E. coli BL21-Gold(DE3), purified and characterized regarding their specific activities and kinetic parameters on sol. substrates and their ability to hydrolyze PET and the PET model substrate bis(benzoyloxyethyl) terephthalate (3PET). Compared to Thc_Cut2, mutants carrying Arg29Asn and/or Ala30Val exchanges showed considerable higher specific activity and higher kcat/KM values on sol. substrates. Exchange of the pos. charged arginine (Arg19 and Arg29) located on the enzyme surface to the non-charged amino acids serine and asparagine strongly increased the hydrolysis activity for 3PET and PET. In contrast, exchange of the uncharged glutamine (Glu65) by the neg. charged glutamic acid lead to a complete loss of hydrolysis activity on PET films. These findings clearly demonstrate that surface properties (i.e., amino acids located outside the active site on the protein surface) play an important role in PET hydrolysis. Biotechnol. Bioeng. 2013;9999: XX-XX. © 2013 Wiley Periodicals, Inc.
- 27Ribitsch, D.; Yebra, A. O.; Zitzenbacher, S.; Wu, J.; Nowitsch, S.; Steinkellner, G.; Greimel, K.; Doliska, A.; Oberdorfer, G.; Gruber, C. C. Fusion of binding domains to Thermobifida cellulosilytica cutinase to tune sorption characteristics and enhancing PET hydrolysis. Biomacromolecules 2013, 14, 1769– 1776, DOI: 10.1021/bm400140u27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXotlegtb0%253D&md5=c60d9519827ffdc53aff6e24ecf36406Fusion of Binding Domains to Thermobifida cellulosilytica Cutinase to Tune Sorption Characteristics and Enhancing PET HydrolysisRibitsch, Doris; Yebra, Antonio Orcal; Zitzenbacher, Sabine; Wu, Jing; Nowitsch, Susanne; Steinkellner, Georg; Greimel, Katrin; Doliska, Ales; Oberdorfer, Gustav; Gruber, Christian C.; Gruber, Karl; Schwab, Helmut; Stana-Kleinschek, Karin; Acero, Enrique Herrero; Guebitz, Georg M.Biomacromolecules (2013), 14 (6), 1769-1776CODEN: BOMAF6; ISSN:1525-7797. (American Chemical Society)A cutinase from Thermomyces cellullosylitica (Thc_Cut1), hydrolyzing the synthetic polymer polyethylene terephthalate (PET), was fused with two different binding modules to improve sorption and thereby hydrolysis. The binding modules were from cellobiohydrolase I from Hypocrea jecorina (CBM) and from a polyhydroxyalkanoate depolymerase from Alcaligenes faecalis (PBM). Although both binding modules have a hydrophobic nature, it was possible to express the proteins in E. coli. Both fusion enzymes and the native one had comparable kcat values in the range of 311 to 342 s-1 on pNP-butyrate, while the catalytic efficiencies kcat/Km decreased from 0.41 s-1/ μM (native enzyme) to 0.21 and 0.33 s-1/μM for Thc_Cut1+PBM and Thc_Cut1+CBM, resp. The fusion enzymes were active both on the insol. PET model substrate bis(benzoyloxyethyl) terephthalate (3PET) and on PET although the hydrolysis pattern was differed when compared to Thc_Cut1. Enhanced adsorption of the fusion enzymes was visible by chemiluminescence after incubation with a 6xHisTag specific horseradish peroxidase (HRP) labeled probe. Increased adsorption to PET by the fusion enzymes was confirmed with Quarz Crystal Microbalance (QCM-D) anal. and indeed resulted in enhanced hydrolysis activity (3.8× for Thc_Cut1+CBM) on PET, as quantified, based on released mono/oligomers.
- 28Ribitsch, D.; Herrero Acero, E.; Przylucka, A.; Zitzenbacher, S.; Marold, A.; Gamerith, C.; Tscheliessnig, R.; Jungbauer, A.; Rennhofer, H.; Lichtenegger, H. Enhanced cutinase-catalyzed hydrolysis of polyethylene terephthalate by covalent fusion to hydrophobins. Appl. Environ. Microbiol. 2015, 81, 3586– 3592, DOI: 10.1128/AEM.04111-1428https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXosV2msrw%253D&md5=28bcd2b900e693c753321739f6a3b811Enhanced cutinase-catalyzed hydrolysis of polyethylene terephthalate by covalent fusion to hydrophobinsRibitsch, Doris; Acero, Enrique Herrero; Przylucka, Agnieszka; Zitzenbacher, Sabine; Marold, Annemarie; Gamerith, Caroline; Tscheliessnig, Rupert; Jungbauer, Alois; Rennhofer, Harald; Lichtenegger, Helga; Amenitsch, Heinz; Bonazza, Klaus; Kubicek, Christian P.; Druzhinina, Irina S.; Guebitz, Georg M.Applied and Environmental Microbiology (2015), 81 (11), 3586-3592CODEN: AEMIDF; ISSN:1098-5336. (American Society for Microbiology)Cutinases have shown potential for hydrolysis of the recalcitrant synthetic polymer polyethylene terephthalate (PET). We have shown previously that the rate of this hydrolysis can be enhanced by the addn. of hydrophobins, small fungal proteins that can alter the physicochem. properties of surfaces. Here we have investigated whether the PET-hydrolyzing activity of a bacterial cutinase from Thermobifida cellulosilytica (Thc_Cut1) would be further enhanced by fusion to one of three Trichoderma hydrophobins, i.e., the class II hydrophobins HFB4 and HFB7 and the pseudo-class I hydrophobin HFB9b. The fusion enzymes exhibited decreased kcat values on sol. substrates (p-nitrophenyl acetate and p-nitrophenyl butyrate) and strongly decreased the hydrophilicity of glass but caused only small changes in the hydrophobicity of PET. When the enzyme was fused to HFB4 or HFB7, the hydrolysis of PET was enhanced >16-fold over the level with the free enzyme, while a mixt. of the enzyme and the hydrophobins led only to a 4-fold increase at most. Fusion with the non-class II hydrophobin HFB9b did not increase the rate of hydrolysis over that of the enzyme-hydrophobin mixt., but HFB9b performed best when PET was preincubated with the hydrophobins before enzyme treatment. The pattern of hydrolysis by the fusion enzymes differed from that of Thc_Cut1 as the concn. of the product mono(2-hydroxyethyl) terephthalate relative to that of the main product, terephthalic acid, increased. Small-angle X-ray scattering (SAXS) anal. revealed an increased scattering contrast of the fusion proteins over that of the free proteins, suggesting a change in conformation or enhanced protein aggregation. Our data show that the level of hydrolysis of PET by cutinase can be significantly increased by fusion to hydrophobins. The data further suggest that this likely involves binding of the hydrophobins to the cutinase and changes in the conformation of its active center.
- 29Then, J.; Wei, R.; Oeser, T.; Barth, M.; Belisário-Ferrari, M. R.; Schmidt, J.; Zimmermann, W. Ca2+ and Mg2+ binding site engineering increases the degradation of polyethylene terephthalate films by polyester hydrolases from Thermobifida fusca. Biotechnol. J. 2015, 10, 592– 598, DOI: 10.1002/biot.20140062029https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhtFKgtbc%253D&md5=0dc9a61f83e7024b4ae1c47d93708cc6Ca2+ and Mg2+ binding site engineering increases the degradation of polyethylene terephthalate films by polyester hydrolases from Thermobifida fuscaThen, Johannes; Wei, Ren; Oeser, Thorsten; Barth, Markus; Belisario-Ferrari, Matheus R.; Schmidt, Juliane; Zimmermann, WolfgangBiotechnology Journal (2015), 10 (4), 592-598CODEN: BJIOAM; ISSN:1860-6768. (Wiley-VCH Verlag GmbH & Co. KGaA)Several bacterial polyester hydrolases are able to hydrolyze the synthetic polyester, polyethylene terephthalate (PET). For an efficient enzymic degrdn. of PET, reaction temps. close to the glass transition temp. of the polymer need to be applied. Esterases TfH, BTA2, Tfu_0882, TfCut1, and TfCut2 produced by the thermophilic actinomycete, Thermobifida fusca exhibit PET-hydrolyzing activity. However, these enzymes are not sufficiently stable in this temp. range for an efficient degrdn. of post-consumer PET materials. The addn. of Ca2+ or Mg2+ cations to the enzymes resulted in an increase of their m.ps. between 10.8 and 14.1° detd. by CD spectroscopy. The thermostability of the polyester hydrolases was sufficient to degrade semi-cryst. PET films at 65° in the presence of 10 mM Ca2+ and 10 mM Mg2+ resulting in wt. losses of up to 12.9% after a reaction time of 48 h. Residues Asp-174, Asp-204, and Glu-253 were identified by mol. dynamics simulations as potential binding residues for the 2 cations in TfCut2. This was confirmed by their substitution with Arg residues, resulting in a higher thermostability of the corresponding enzyme variants. The generated variants of TfCut2 represented stabilized catalysts suitable for PET hydrolysis reactions performed in the absence of Ca2+ or Mg2+.
- 30Wei, R.; Oeser, T.; Schmidt, J.; Meier, R.; Barth, M.; Then, J.; Zimmermann, W. Engineered bacterial polyester hydrolases efficiently degrade polyethylene terephthalate due to relieved product inhibition. Biotechnol. Bioeng. 2016, 113, 1658– 1665, DOI: 10.1002/bit.2594130https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhvFSlsbw%253D&md5=8f856fb7770bd715660fcb7954228d6aEngineered bacterial polyester hydrolases efficiently degrade polyethylene terephthalate due to relieved product inhibitionWei, Ren; Oeser, Thorsten; Schmidt, Juliane; Meier, Rene; Barth, Markus; Then, Johannes; Zimmermann, WolfgangBiotechnology and Bioengineering (2016), 113 (8), 1658-1665CODEN: BIBIAU; ISSN:0006-3592. (John Wiley & Sons, Inc.)Recent studies on the enzymic degrdn. of synthetic polyesters have shown the potential of polyester hydrolases from thermophilic actinomycetes for modifying or degrading polyethylene terephthalate (PET). TfCut2 from Thermobifida fusca KW3 and LC-cutinase (LCC) isolated from a compost metagenome are remarkably active polyester hydrolases with high sequence and structural similarity. Both enzymes exhibit an exposed active site in a substrate binding groove located at the protein surface. By exchanging selected amino acid residues of TfCut2 involved in substrate binding with those present in LCC, enzyme variants with increased PET hydrolytic activity at 65°C were obtained. The highest activity in hydrolyzing PET films and fibers were detected with the single variant G62A and the double variant G62A/I213S. Both variants caused a wt. loss of PET films of more than 42% after 50 h of hydrolysis, corresponding to a 2.7-fold increase compared to the wild type enzyme. Kinetic anal. based on the released PET hydrolysis products confirmed the superior hydrolytic activity of G62A with a fourfold higher hydrolysis rate const. and a 1.5-fold lower substrate binding const. than those of the wild type enzyme. Mono-(2-hydroxyethyl) terephthalate is a strong inhibitor of TfCut2. A detn. of the Rosetta binding energy suggested a reduced interaction of G62A with 2PET, a dimer of the PET monomer ethylene terephthalate. Indeed, G62A revealed a 5.5-fold lower binding const. to the inhibitor than the wild type enzyme indicating that its increased PET hydrolysis activity is the result of a relieved product inhibition by mono-(2-hydroxyethyl) terephthalate. Biotechnol. Bioeng. 2016;9999: 1-8. © 2016 Wiley Periodicals, Inc.
- 31Shirke, A. N.; Basore, D.; Butterfoss, G. L.; Bonneau, R.; Bystroff, C.; Gross, R. A. Toward rational thermostabilization of Aspergillus oryzae cutinase: Insights into catalytic and structural stability. Proteins 2016, 84, 60– 72, DOI: 10.1002/prot.2495531https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhvFWksrrM&md5=e6bf9ca17fb4888afa9e34dddd198daaToward rational thermostabilization of Aspergillus oryzae cutinase: Insights into catalytic and structural stabilityShirke, Abhijit N.; Basore, Danielle; Butterfoss, Glenn L.; Bonneau, Richard; Bystroff, Christopher; Gross, Richard A.Proteins: Structure, Function, and Bioinformatics (2016), 84 (1), 60-72CODEN: PSFBAF; ISSN:1097-0134. (Wiley-Blackwell)Cutinases are powerful hydrolases that can cleave ester bonds of polyesters such as poly(ethylene terephthalate) (PET), opening up new options for enzymic routes for polymer recycling and surface modification reactions. Cutinase from Aspergillus oryzae (AoC) is promising in this respect, due to the presence of an extended groove near the catalytic triad which is important for the orientation of polymeric chains. However, the catalytic efficiency of AoC on rigid polymers like PET is limited by its low thermostability; as it is essential to work at or over the glass transition temp. (Tg) of PET, i.e., 70°C. Consequently, in this study we worked toward the thermostabilization of AoC. Use of Rosetta computational protein design software in conjunction with rational design led to a 6°C improvement in the thermal unfolding temp. (Tm) and a 10-fold increase in the half-life of the enzyme activity at 60°C. Surprisingly, thermostabilization did not improve the rate or temp. optimum of enzyme activity. Three notable findings are presented as steps toward designing more thermophilic cutinase: (a) surface salt bridge optimization produced enthalpic stabilization, (b) mutations to proline reduced the entropy loss upon folding, and (c) the lack of a correlative increase in the temp. optimum of catalytic activity with thermodn. stability suggests that the active site is locally denatured at a temp. below the Tm of the global structure. Proteins 2015. © 2015 Wiley Periodicals, Inc.
- 32Biundo, A.; Ribitsch, D.; Steinkellner, G.; Gruber, K.; Guebitz, G. M. Polyester hydrolysis is enhanced by a truncated esterase: Less is more. Biotechnol. J. 2017, 12, DOI: 10.1002/biot.201600450There is no corresponding record for this reference.
- 33Shirke, A. N.; Butterfoss, G. L.; Saikia, R.; Basu, A.; Maria, L.; Svendsen, A.; Gross, R. A. Engineered Humicola insolens cutinase for efficient cellulose acetate deacetylation. Biotechnol. J. 2017, 12, 1700188, DOI: 10.1002/biot.201700188There is no corresponding record for this reference.
- 34Austin, H. P.; Allen, M. D.; Donohoe, B. S.; Rorrer, N. A.; Kearns, F. L.; Silveira, R. L.; Pollard, B. C.; Dominick, G.; Duman, R.; El Omari, K. Characterization and engineering of a plastic-degrading aromatic polyesterase. Proc. Natl. Acad. Sci. U.S.A. 2018, 115, E4350– E4357, DOI: 10.1073/pnas.171880411534https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhvVGhtLnF&md5=b8e8d841af1bb06fa2d1fddb6b9d6460Characterization and engineering of a plastic-degrading aromatic polyesteraseAustin, Harry P.; Allen, Mark D.; Donohoe, Bryon S.; Rorrer, Nicholas A.; Kearns, Fiona L.; Silveira, Rodrigo L.; Pollard, Benjamin C.; Dominick, Graham; Duman, Ramona; El Omari, Kamel; Mykhaylyk, Vitaliy; Wagner, Armin; Michener, William E.; Amore, Antonella; Skaf, Munir S.; Crowley, Michael F.; Thorne, Alan W.; Johnson, Christopher W.; Woodcock, H. Lee; McGeehan, John E.; Beckham, Gregg T.Proceedings of the National Academy of Sciences of the United States of America (2018), 115 (19), E4350-E4357CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Poly(ethylene terephthalate) (PET) is one of the most abundantly produced synthetic polymers and is accumulating in the environment at a staggering rate as discarded packaging and textiles. The properties that make PET so useful also endow it with an alarming resistance to biodegrdn., likely lasting centuries in the environment. Our collective reliance on PET and other plastics means that this buildup will continue unless solns. are found. Recently, a newly discovered bacterium, Ideonella sakaiensis 201-F6, was shown to exhibit the rare ability to grow on PET as a major carbon and energy source. Central to its PET biodegrdn. capability is a secreted PETase (PET-digesting enzyme). Here, we present a 0.92 Å resoln. X-ray crystal structure of PETase, which reveals features common to both cutinases and lipases. PETase retains the ancestral α/β-hydrolase fold but exhibits a more open active-site cleft than homologous cutinases. By narrowing the binding cleft via mutation of two active-site residues to conserved amino acids in cutinases, we surprisingly observe improved PET degrdn., suggesting that PETase is not fully optimized for cryst. PET degrdn., despite presumably evolving in a PET-rich environment. Addnl., we show that PETase degrades another semiarom. polyester, polyethylene-2,5-furandicarboxylate (PEF), which is an emerging, bioderived PET replacement with improved barrier properties. In contrast, PETase does not degrade aliph. polyesters, suggesting that it is generally an arom. polyesterase. These findings suggest that addnl. protein engineering to increase PETase performance is realistic and highlight the need for further developments of structure/activity relationships for biodegrdn. of synthetic polyesters.
- 35Biundo, A.; Ribitsch, D.; Guebitz, G. M. Surface engineering of polyester-degrading enzymes to improve efficiency and tune specificity. Appl. Microbiol. Biotechnol. 2018, 102, 3551– 3559, DOI: 10.1007/s00253-018-8850-735https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXjvFWqsL4%253D&md5=0858dea4ffd7da9f898f38a1508c99ccSurface engineering of polyester-degrading enzymes to improve efficiency and tune specificityBiundo, Antonino; Ribitsch, Doris; Guebitz, Georg M.Applied Microbiology and Biotechnology (2018), 102 (8), 3551-3559CODEN: AMBIDG; ISSN:0175-7598. (Springer)Certain members of the carboxylesterase superfamily can act at the interface between water and water-insol. substrates. However, nonnatural bulky polyesters usually are not efficiently hydrolyzed. In the recent years, the potential of enzyme engineering to improve hydrolysis of synthetic polyesters has been demonstrated. Regions on the enzyme surface have been modified by using site-directed mutagenesis in order to tune sorption processes through increased hydrophobicity of the enzyme surface. Such modifications can involve specific amino acid substitutions, addn. of binding modules, or truncation of entire domains improving sorption properties and/or dynamics of the enzyme. In this review, we provide a comprehensive overview on different strategies developed in the recent years for enzyme surface engineering to improve the activity of polyester-hydrolyzing enzymes.
- 36Shirke, A. N.; White, C.; Englaender, J. A.; Zwarycz, A.; Butterfoss, G. L.; Linhardt, R. J.; Gross, R. A. Stabilizing Leaf and Branch Compost Cutinase (LCC) with Glycosylation: Mechanism and Effect on PET Hydrolysis. Biochemistry 2018, 57, 1190– 1200, DOI: 10.1021/acs.biochem.7b0118936https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXntleqsQ%253D%253D&md5=7a58568be5fdbdbcd8f7340ea74d761dStabilizing Leaf and Branch Compost Cutinase (LCC) with Glycosylation: Mechanism and Effect on PET HydrolysisShirke, Abhijit N.; White, Christine; Englaender, Jacob A.; Zwarycz, Allison; Butterfoss, Glenn L.; Linhardt, Robert J.; Gross, Richard A.Biochemistry (2018), 57 (7), 1190-1200CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)Cutinases are polyester hydrolases that show a remarkable capability to hydrolyze polyethylene terephthalate (PET) to its monomeric units. This revelation has stimulated research aimed at developing sustainable and green cutinase-catalyzed PET recycling methods. Leaf and branch compost cutinase (LCC) is particularly suited toward these ends given its relatively high PET hydrolysis activity and thermostability. Any practical enzymic PET recycling application will require that the protein have kinetic stability at or above the PET glass transition temp. (Tg, i.e., 70 °C). This paper elucidates the thermodn. and kinetics of LCC conformational and colloidal stability. Aggregation emerged as a major contributor that reduces LCC kinetic stability. In its native state, LCC is prone to aggregation owing to electrostatic interactions. Further, with increasing temp., perturbation of LCC's tertiary structure and corresponding exposure of hydrophobic domains leads to rapid aggregation. Glycosylation was employed in an attempt to impede LCC aggregation. Owing to the presence of three putative N-glycosylation sites, expression of native LCC in Pichia pastoris resulted in the prodn. of glycosylated LCC (LCC-G). LCC-G showed improved stability to native state aggregation while increasing the temp. for thermal induced aggregation by 10 °C. Furthermore, stabilization against thermal aggregation resulted in improved catalytic PET hydrolysis both at its optimum temp. and concn.
- 37Son, H. F.; Cho, I. J.; Joo, S.; Seo, H.; Sagong, H.-Y.; Choi, S. Y.; Lee, S. Y.; Kim, K.-J. Rational Protein Engineering of Thermo-Stable PETase from Ideonella sakaiensis for Highly Efficient PET Degradation. ACS Catal. 2019, 9, 3519– 3526, DOI: 10.1021/acscatal.9b0056837https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXksFegurY%253D&md5=ebe4cdfb118f20eba6e05a731717f8fbRational protein engineering of thermo-stable PETase from Ideonella sakaiensis for highly efficient PET degradationSon, Hyeoncheol Francis; Cho, In Jin; Joo, Seongjoon; Seo, Hogyun; Sagong, Hye-Young; Choi, So Young; Lee, Sang Yup; Kim, Kyung-JinACS Catalysis (2019), 9 (4), 3519-3526CODEN: ACCACS; ISSN:2155-5435. (American Chemical Society)Widespread utilization of polyethylene terephthalate (PET) has caused a variety of environmental and health problems; thus, the enzymic degrdn. of PET can be a promising soln. Although PETase from Ideonalla sakaiensis (IsPETase) has been reported to have the highest PET degrdn. activity under mild conditions of all PET-degrading enzymes reported to date, its low thermal stability limits its ability for efficient and practical enzymic degrdn. of PET. Using the structural information on IsPETase, we developed a rational protein engineering strategy using several IsPETase variants that were screened for high thermal stability to improve PET degrdn. activity. In particular, the IsPETaseS121E/D186H/R280A variant, which was designed to have a stabilized β6-β7 connecting loop and extended subsite IIc, had a Tm value that was increased by 8.81° and PET degrdn. activity was enhanced by 14-fold at 40 °C in comparison with IsPETaseWT. The designed structural modifications were further verified through structure detn. of the variants, and high thermal stability was further confirmed by a heat-inactivation expt. The proposed strategy and developed variants represent an important advancement for achieving the complete biodegrdn. of PET under mild conditions.
- 38Furukawa, M.; Kawakami, N.; Tomizawa, A.; Miyamoto, K. Efficient Degradation of Poly(ethylene terephthalate) with Thermobifida fusca Cutinase Exhibiting Improved Catalytic Activity Generated using Mutagenesis and Additive-based Approaches. Sci. Rep. 2019, 9, 16038, DOI: 10.1038/s41598-019-52379-z38https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3MjlsFKgtA%253D%253D&md5=1bce8289f05c2abdc9f937c15c3a61a3Efficient Degradation of Poly(ethylene terephthalate) with Thermobifida fusca Cutinase Exhibiting Improved Catalytic Activity Generated using Mutagenesis and Additive-based ApproachesFurukawa Makoto; Kawakami Norifumi; Tomizawa Atsushi; Miyamoto KenjiScientific reports (2019), 9 (1), 16038 ISSN:.Cutinases are promising agents for poly(ethylene terephthalate) (PET) bio-recycling because of their ability to produce the PET monomer terephthalic acid with high efficiency under mild reaction conditions. In this study, we found that the low-crystallinity PET (lcPET) hydrolysis activity of thermostable cutinase from Thermobifida fusca (TfCut2), was increased by the addition of cationic surfactant that attracts enzymes near the lcPET film surface via electrostatic interactions. This approach was applicable to the mutant TfCut2 G62A/F209A, which was designed based on a sequence comparison with PETase from Ideonella sakaiensis. As a result, the degradation rate of the mutant in the presence of cationic surfactant increased to 31 ± 0.1 nmol min(-1) cm(-2), 12.7 times higher than that of wild-type TfCut2 in the absence of surfactant. The long-duration reaction showed that lcPET film (200 μm) was 97 ± 1.8% within 30 h, the fastest biodegradation rate of lcPET film thus far. We therefore believe that our approach would expand the possibility of enzyme utilization in industrial PET biodegradation.
- 39Tournier, V.; Topham, C. M.; Gilles, A.; David, B.; Folgoas, C.; Moya-Leclair, E.; Kamionka, E.; Desrousseaux, M.-L.; Texier, H.; Gavalda, S. An engineered PET depolymerase to break down and recycle plastic bottles. Nature 2020, 580, 216– 219, DOI: 10.1038/s41586-020-2149-439https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXmvVSht70%253D&md5=1bad7e606018f024e6fad5fbf7b082e4An engineered PET depolymerase to break down and recycle plastic bottlesTournier, V.; Topham, C. M.; Gilles, A.; David, B.; Folgoas, C.; Moya-Leclair, E.; Kamionka, E.; Desrousseaux, M.-L.; Texier, H.; Gavalda, S.; Cot, M.; Guemard, E.; Dalibey, M.; Nomme, J.; Cioci, G.; Barbe, S.; Chateau, M.; Andre, I.; Duquesne, S.; Marty, A.Nature (London, United Kingdom) (2020), 580 (7802), 216-219CODEN: NATUAS; ISSN:0028-0836. (Nature Research)Present ests. suggest that of the 359 million tons of plastics produced annually worldwide1, 150-200 million tons accumulate in landfill or in the natural environment2. Poly(ethylene terephthalate) (PET) is the most abundant polyester plastic, with almost 70 million tons manufd. annually worldwide for use in textiles and packaging3. The main recycling process for PET, via thermomech. means, results in a loss of mech. properties4. Consequently, de novo synthesis is preferred and PET waste continues to accumulate. With a high ratio of arom. terephthalate units-which reduce chain mobility-PET is a polyester that is extremely difficult to hydrolyze5. Several PET hydrolase enzymes have been reported, but show limited productivity6,7. Here we describe an improved PET hydrolase that ultimately achieves, over 10 h, a min. of 90 per cent PET depolymn. into monomers, with a productivity of 16.7 g of terephthalate per L per h (200 g per kg of PET suspension, with an enzyme concn. of 3 mg per g of PET). This highly efficient, optimized enzyme outperforms all PET hydrolases reported so far, including an enzyme8,9 from the bacterium Ideonella sakaiensis strain 201-F6 (even assisted by a secondary enzyme10) and related improved variants11-14 that have attracted recent interest. We also show that biol. recycled PET exhibiting the same properties as petrochem. PET can be produced from enzymically depolymd. PET waste, before being processed into bottles, thereby contributing towards the concept of a circular PET economy.
- 40Bell, E.; Smithson, R.; Kilbride, S.; Foster, J.; Hardy, F.; Ramachandran, S.; Tedstone, A.; Haigh, S.; Garforth, A.; Day, P. Directed Evolution of an Efficient and Thermostable PET Depolymerase. ChemRxiv 2021, DOI: 10.26434/chemrxiv-2021-mcjh6There is no corresponding record for this reference.
- 41Cui, Y.; Chen, Y.; Liu, X.; Dong, S.; Tian, Y. e.; Qiao, Y.; Mitra, R.; Han, J.; Li, C.; Han, X. Computational Redesign of a PETase for Plastic Biodegradation under Ambient Condition by the GRAPE Strategy. ACS Catal. 2021, 11, 1340– 1350, DOI: 10.1021/acscatal.0c0512641https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXpt1CqtQ%253D%253D&md5=9cf0ade24ab9ab5beb726fe620b284f7Computational Redesign of a PETase for Plastic Biodegradation under Ambient Condition by the GRAPE StrategyCui, Yinglu; Chen, Yanchun; Liu, Xinyue; Dong, Saijun; Tian, Yu'e; Qiao, Yuxin; Mitra, Ruchira; Han, Jing; Li, Chunli; Han, Xu; Liu, Weidong; Chen, Quan; Wei, Wangqing; Wang, Xin; Du, Wenbin; Tang, Shuangyan; Xiang, Hua; Liu, Haiyan; Liang, Yong; Houk, Kendall N.; Wu, BianACS Catalysis (2021), 11 (3), 1340-1350CODEN: ACCACS; ISSN:2155-5435. (American Chemical Society)Nature has provided a fantastic array of enzymes that are responsible for essential biochem. functions but not usually suitable for technol. applications. Not content with the natural repertoire, protein engineering holds promise to extend the applications of improved enzymes with tailored properties. However, engineering of robust proteins remains a difficult task since the pos. mutation library may not cooperate to reach the target function in most cases owing to the ubiquity of epistatic effects. The main demand lies in identifying an efficient path of accumulated mutations. Herein, we devised a computational strategy (greedy accumulated strategy for protein engineering, GRAPE) to improve the robustness of a PETase from Ideonella sakaiensis. A systematic clustering anal. combined with greedy accumulation of beneficial mutations in a computationally derived library enabled the redesign of a variant, DuraPETase, which exhibits an apparent melting temp. that is drastically elevated by 31°C and a strikingly enhanced degrdn. toward semicryst. poly(ethylene terephthalate) (PET) films (30%) at mild temps. (over 300-fold). Complete biodegrdn. of 2 g/L microplastics to water-sol. products under mild conditions is also achieved, opening up opportunities to steer the biol. degrdn. of uncollectable PET waste and further conversion of the resulting monomers to high-value mols. The crystal structure revealed the individual mutation match with the design model. Concurrently, synergistic effects are captured, while epistatic interactions are alleviated during the accumulation process. We anticipate that our design strategy will provide a broadly applicable strategy for global optimization of enzyme performance.
- 42Nakamura, A.; Kobayashi, N.; Koga, N.; Iino, R. Positive Charge Introduction on the Surface of Thermostabilized PET Hydrolase Facilitates PET Binding and Degradation. ACS Catal. 2021, 11, 8550– 8564, DOI: 10.1021/acscatal.1c0120442https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhsVWrtL3E&md5=570df73ee6a1e63529e4517c76b21282Pos. charge introduction on the surface of thermostabilized PET hydrolase facilitates PET binding and degradationNakamura, Akihiko; Kobayashi, Naoya; Koga, Nobuyasu; Iino, RyotaACS Catalysis (2021), 11 (14), 8550-8564CODEN: ACCACS; ISSN:2155-5435. (American Chemical Society)A thermostable enzyme PET2, found in a metagenome library, has been engineered to improve its hydrolytic activity against polyethylene terephthalate (PET). The PET2 wild-type (WT) showed a melting temp. of 69.0°C and produced water-sol. reaction products at a rate of 0.40 min-1 (2.4μM products from 0.1μM enzyme after 60 min reaction) from an amorphous PET film at 60°C. Mutations for surface charge modification, backbone stabilization, and formation of addnl. disulfide bond were introduced into the PET2 WT, and the best mutant (PET2 7M) showed a melting temp. of 75.7°C and hydrolytic activity of 1.3 min-1 (7.8μM products from 0.1μM enzyme after 60 min reaction at 60°C). X-ray crystal structures of PET2 mutants showed that introduced arginine and lysine residues oriented to the solvent, similar to a PET hydrolase from Ideonella sakaiensis 201-F6. Single-mol. fluorescence imaging revealed that these pos. charged surface residues increased binding rate const. of PET2 7M to PET surface 2.7 times, compared with PET2 WT, and resulted in higher activity. Optimal temp. for amorphous PET hydrolysis by PET2 7M (68°C) was 8°C higher than that by PET2 WT (60°C), and hydrolytic activity of PET2 7M at the optimal temp. (2.7 min-1, 16.2μM products from 0.1μM enzyme after 60 min reaction) was 6.8 times higher than that of PET2 WT (0.40 min-1). Furthermore, PET2 7M generated reaction products with a const. rate for at least 24 h at 68°C, indicating long-term thermal stability at the optimal temp.
- 43Guo, B.; Vanga, S. R.; Lopez-Lorenzo, X.; Saenz-Mendez, P.; Ericsson, S. R.; Fang, Y.; Ye, X.; Schriever, K.; Bäckström, E.; Biundo, A. Conformational Selection in Biocatalytic Plastic Degradation by PETase. ACS Catal. 2022, 12, 3397– 3409, DOI: 10.1021/acscatal.1c0554843https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XltFehs78%253D&md5=0732543ed139b9fd36a771245bfd3db6Conformational Selection in Biocatalytic Plastic Degradation by PETaseGuo, Boyang; Vanga, Sudarsana Reddy; Lopez-Lorenzo, Ximena; Saenz-Mendez, Patricia; Ericsson, Sara Roennblad; Fang, Yuan; Ye, Xinchen; Schriever, Karen; Baeckstroem, Eva; Biundo, Antonino; Zubarev, Roman A.; Furo, Istvan; Hakkarainen, Minna; Syren, Per-OlofACS Catalysis (2022), 12 (6), 3397-3409CODEN: ACCACS; ISSN:2155-5435. (American Chemical Society)Due to the steric effects imposed by bulky polymers, the formation of catalytically competent enzyme and substrate conformations is crit. in the biodegrdn. of plastics. In poly(ethylene terephthalate) (PET), the backbone adopts different conformations, gauche and trans, coexisting to different extents in amorphous and cryst. regions. However, which conformation is susceptible to biodegrdn. and the extent of enzyme and substrate conformational changes required for expedient catalysis remain poorly understood. To overcome this obstacle, we utilized mol. dynamics simulations, docking, and enzyme engineering in concert with high-resoln. microscopy imaging and solid-state NMR to demonstrate the importance of conformational selection in biocatalytic plastic hydrolysis. Our results demonstrate how single-amino acid substitutions in Ideonella sakaiensis PETase can alter its conformational landscape, significantly affecting the relative abundance of productive ground-state structures ready to bind discrete substrate conformers. We exptl. show how an enzyme binds to plastic and provide a model for key residues involved in the recognition of gauche and trans conformations supported by in silico simulations. We demonstrate how enzyme engineering can be used to create a trans-selective variant, resulting in higher activity when combined with an all-trans PET-derived oligomeric substrate, stemming from both increased accessibility and conformational preference. Our work cements the importance of matching enzyme and substrate conformations in plastic hydrolysis, and we show that also the noncanonical trans conformation in PET is conducive for degrdn. Understanding the contribution of enzyme and substrate conformations to biocatalytic plastic degrdn. could facilitate the generation of designer enzymes with increased performance.
- 44Lu, H.; Diaz, D. J.; Czarnecki, N. J.; Zhu, C.; Kim, W.; Shroff, R.; Acosta, D. J.; Alexander, B. R.; Cole, H. O.; Zhang, Y. Machine learning-aided engineering of hydrolases for PET depolymerization. Nature 2022, 604, 662– 667, DOI: 10.1038/s41586-022-04599-z44https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XhtFKrtLfI&md5=737bb3fe419c581be70e0ae37fc20085Machine learning-aided engineering of hydrolases for PET depolymerizationLu, Hongyuan; Diaz, Daniel J.; Czarnecki, Natalie J.; Zhu, Congzhi; Kim, Wantae; Shroff, Raghav; Acosta, Daniel J.; Alexander, Bradley R.; Cole, Hannah O.; Zhang, Yan; Lynd, Nathaniel A.; Ellington, Andrew D.; Alper, Hal S.Nature (London, United Kingdom) (2022), 604 (7907), 662-667CODEN: NATUAS; ISSN:1476-4687. (Nature Portfolio)Plastic waste poses an ecol. challenge1-3 and enzymic degrdn. offers one, potentially green and scalable, route for polyesters waste recycling4. Poly(ethylene terephthalate) (PET) accounts for 12% of global solid waste5, and a circular carbon economy for PET is theor. attainable through rapid enzymic depolymn. followed by repolymn. or conversion/valorization into other products6-10. Application of PET hydrolases, however, has been hampered by their lack of robustness to pH and temp. ranges, slow reaction rates and inability to directly use untreated postconsumer plastics11. Here, we use a structure-based, machine learning algorithm to engineer a robust and active PET hydrolase. Our mutant and scaffold combination (FAST-PETase: functional, active, stable and tolerant PETase) contains five mutations compared to wild-type PETase (N233K/R224Q/S121E from prediction and D186H/R280A from scaffold) and shows superior PET-hydrolytic activity relative to both wild-type and engineered alternatives12 between 30 and 50°C and a range of pH levels. We demonstrate that untreated, postconsumer-PET from 51 different thermoformed products can all be almost completely degraded by FAST-PETase in 1 wk. FAST-PETase can also depolymerize untreated, amorphous portions of a com. water bottle and an entire thermally pretreated water bottle at 50°C. Finally, we demonstrate a closed-loop PET recycling process by using FAST-PETase and resynthesizing PET from the recovered monomers. Collectively, our results demonstrate a viable route for enzymic plastic recycling at the industrial scale.
- 45Wei, R.; von Haugwitz, G.; Pfaff, L.; Mican, J.; Badenhorst, C. P. S.; Liu, W.; Weber, G.; Austin, H. P.; Bednar, D.; Damborsky, J. Mechanism-Based Design of Efficient PET Hydrolases. ACS Catal. 2022, 12, 3382– 3396, DOI: 10.1021/acscatal.1c0585645https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XkvF2gsrs%253D&md5=d19ff0a8a98b6c9569e1d1e6a7ac7884Mechanism-Based Design of Efficient PET HydrolasesWei, Ren; von Haugwitz, Gerlis; Pfaff, Lara; Mican, Jan; Badenhorst, Christoffel P. S.; Liu, Weidong; Weber, Gert; Austin, Harry P.; Bednar, David; Damborsky, Jiri; Bornscheuer, Uwe T.ACS Catalysis (2022), 12 (6), 3382-3396CODEN: ACCACS; ISSN:2155-5435. (American Chemical Society)A review. 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 enzymic 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 enzymic PET degrdn. approaches. Unbalanced enzyme-substrate interactions, limited thermostability, and low catalytic efficiency at elevated reaction temps., and inhibition caused by oligomeric degrdn. intermediates still hamper industrial applications that require high catalytic efficiency. To overcome these limitations, successful protein engineering studies using innovative exptl. 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 biotechnol. approaches.
- 46Zeng, W.; Li, X.; Yang, Y.; Min, J.; Huang, J.-W.; Liu, W.; Niu, D.; Yang, X.; Han, X.; Zhang, L. Substrate-Binding Mode of a Thermophilic PET Hydrolase and Engineering the Enzyme to Enhance the Hydrolytic Efficacy. ACS Catal. 2022, 12, 3033– 3040, DOI: 10.1021/acscatal.1c0580046https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XjvFyjsbw%253D&md5=8e485295c20775ce47acb77886b134b1Substrate-Binding Mode of a Thermophilic PET Hydrolase and Engineering the Enzyme to Enhance the Hydrolytic EfficacyZeng, Wei; Li, Xiuqin; Yang, Yunyun; Min, Jian; Huang, Jian-Wen; Liu, Weidong; Niu, Du; Yang, Xuechun; Han, Xu; Zhang, Lilan; Dai, Longhai; Chen, Chun-Chi; Guo, Rey-TingACS Catalysis (2022), 12 (5), 3033-3040CODEN: ACCACS; ISSN:2155-5435. (American Chemical Society)Polyethylene terephthalate (PET) is among the most extensively produced plastics, but huge amts. of PET wastes that have accumulated in the environment have become a serious threat to the ecosystem. Applying PET hydrolytic enzymes to depolymerize PET is an attractive measure to manage PET pollution, and searching for more effective enzymes is a prerequisite to achieve this goal. A thermostable cutinase that originates from the leaf-branch compost termed ICCG is the most effective PET hydrolase reported so far. Here, we illustrated the crystal structure of ICCG in complex with the PET analog, mono(2-hydroxyethyl)terephthalic acid, to reveal the enzyme-substrate interaction network. Furthermore, we applied structure-based engineering to modify ICCG and screened for variants that exhibit higher efficacy than the parental enzyme. As a result, several variants with the measured melting temp. approaching 99°C and elevated PET hydrolytic activity were obtained. Finally, crystallog. analyses were performed to reveal the structural stabilization effects mediated by the introduced mutations. These results are of importance in the context of understanding the mechanism of action of the thermostable PET hydrolytic enzyme and shall be beneficial to the development of PET biodegrdn. platforms.
- 47Singh, A.; Rorrer, N. A.; Nicholson, S. R.; Erickson, E.; DesVeaux, J. S.; Avelino, A. F. T.; Lamers, P.; Bhatt, A.; Zhang, Y.; Avery, G. Techno-economic, life-cycle, and socioeconomic impact analysis of enzymatic recycling of poly(ethylene terephthalate). Joule 2021, 5, 2479– 2503, DOI: 10.1016/j.joule.2021.06.01547https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhs1Sks7jL&md5=e5f88c523cbb0bb29e7fe294dca4ec5dTechno-economic, life-cycle, and socioeconomic impact analysis of enzymatic recycling of poly(ethylene terephthalate)Singh, Avantika; Rorrer, Nicholas A.; Nicholson, Scott R.; Erickson, Erika; DesVeaux, Jason S.; Avelino, Andre F. T.; Lamers, Patrick; Bhatt, Arpit; Zhang, Yimin; Avery, Greg; Tao, Ling; Pickford, Andrew R.; Carpenter, Alberta C.; McGeehan, John E.; Beckham, Gregg T.Joule (2021), 5 (9), 2479-2503CODEN: JOULBR; ISSN:2542-4351. (Cell Press)Esterases have emerged as important biocatalysts for enzyme-based polyester recycling of poly(ethylene terephthalate) (PET) to terephthalic acid (TPA) and ethylene glycol (EG). Here, we present process modeling, techno-economic, life-cycle, and socioeconomic impact analyses for an enzymic PET depolymn.-based recycling process, which we compare with virgin TPA manufg. We predict that enzymically recycled TPA (rTPA) can be cost-competitive and highlight key areas to achieve this. In addn. to favorable long-term socioeconomic benefits, rTPA can reduce total supply chain energy use by 69%-83% and greenhouse gas emissions by 17%-43% per kg of TPA. An economy-wide assessment for the US ests. that the TPA recycling process can reduce environmental impacts by up to 95% while generating up to 45% more socioeconomic benefits, also relative to virgin TPA prodn. Sensitivity analyses highlight impactful research opportunities to pursue toward realizing biol. PET recycling and upcycling.
- 48Wei, R.; Breite, D.; Song, C.; Gräsing, D.; Ploss, T.; Hille, P.; Schwerdtfeger, R.; Matysik, J.; Schulze, A.; Zimmermann, W. Biocatalytic Degradation Efficiency of Postconsumer Polyethylene Terephthalate Packaging Determined by Their Polymer Microstructures. Adv. Sci. 2019, 6, 1900491, DOI: 10.1002/advs.20190049148https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3MvkslGqsA%253D%253D&md5=1fb8e90521767a6e6125016bb02c07adBiocatalytic Degradation Efficiency of Postconsumer Polyethylene Terephthalate Packaging Determined by Their Polymer MicrostructuresWei Ren; Hille Patrick; Zimmermann Wolfgang; Breite Daniel; Schulze Agnes; Song Chen; Grasing Daniel; Matysik Jorg; Ploss Tina; Schwerdtfeger RuthAdvanced science (Weinheim, Baden-Wurttemberg, Germany) (2019), 6 (14), 1900491 ISSN:2198-3844.Polyethylene terephthalate (PET) is the most important mass-produced thermoplastic polyester used as a packaging material. Recently, thermophilic polyester hydrolases such as TfCut2 from Thermobifida fusca have emerged as promising biocatalysts for an eco-friendly PET recycling process. In this study, postconsumer PET food packaging containers are treated with TfCut2 and show weight losses of more than 50% after 96 h of incubation at 70 °C. Differential scanning calorimetry analysis indicates that the high linear degradation rates observed in the first 72 h of incubation is due to the high hydrolysis susceptibility of the mobile amorphous fraction (MAF) of PET. The physical aging process of PET occurring at 70 °C is shown to gradually convert MAF to polymer microstructures with limited accessibility to enzymatic hydrolysis. Analysis of the chain-length distribution of degraded PET by nuclear magnetic resonance spectroscopy reveals that MAF is rapidly hydrolyzed via a combinatorial exo- and endo-type degradation mechanism whereas the remaining PET microstructures are slowly degraded only by endo-type chain scission causing no detectable weight loss. Hence, efficient thermostable biocatalysts are required to overcome the competitive physical aging process for the complete degradation of postconsumer PET materials close to the glass transition temperature of PET.
- 49Knott, B. C.; Erickson, E.; Allen, M. D.; Gado, J. E.; Graham, R.; Kearns, F. L.; Pardo, I.; Topuzlu, E.; Anderson, J. J.; Austin, H. P. Characterization and engineering of a two-enzyme system for plastics depolymerization. Proc. Natl. Acad. Sci. U.S.A. 2020, 117, 25476– 25485, DOI: 10.1073/pnas.200675311749https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXitVKktL3P&md5=1c00658c41832cce8e99caa02a769836Characterization and engineering of a two-enzyme system for plastics depolymerizationKnott, Brandon C.; Erickson, Erika; Allen, Mark D.; Gado, Japheth E.; Graham, Rosie; Kearns, Fiona L.; Pardo, Isabel; Topuzlu, Ece; Anderson, Jared J.; Austin, Harry P.; Dominick, Graham; Johnson, Christopher W.; Rorrer, Nicholas A.; Szostkiewicz, Caralyn J.; Copie, Valerie; Payne, Christina M.; Woodcock, H. Lee; Donohoe, Bryon S.; Beckham, Gregg T.; McGeehan, John E.Proceedings of the National Academy of Sciences of the United States of America (2020), 117 (41), 25476-25485CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Plastics pollution represents a global environmental crisis. In response, microbes are evolving the capacity to utilize synthetic polymers as carbon and energy sources. Recently, Ideonella sakaiensis was reported to secrete a two-enzyme system to deconstruct polyethylene terephthalate (PET) to its constituent monomers. Specifically, the I. sakaiensis PETase depolymerizes PET, liberating sol. products, including mono(2-hydroxyethyl) terephthalate (MHET), which is cleaved to terephthalic acid and ethylene glycol by MHETase. Here, we report a 1.6 Å resoln. MHETase structure, illustrating that the MHETase core domain is similar to PETase, capped by a lid domain. Simulations of the catalytic itinerary predict that MHETase follows the canonical two-step serine hydrolase mechanism. Bioinformatics anal. suggests that MHETase evolved from ferulic acid esterases, and two homologous enzymes are shown to exhibit MHET turnover. Anal. of the two homologous enzymes and the MHETase S131G mutant demonstrates the importance of this residue for accommodation of MHET in the active site. We also demonstrate that the MHETase lid is crucial for hydrolysis of MHET and, furthermore, that MHETase does not turnover mono(2-hydroxyethyl)-furanoate or mono(2-hydroxyethyl)-isophthalate. A highly synergistic relationship between PETase and MHETase was obsd. for the conversion of amorphous PET film to monomers across all nonzero MHETase concns. tested. Finally, we compare the performance of MHETase:PETase chimeric proteins of varying linker lengths, which all exhibit improved PET and MHET turnover relative to the free enzymes. Together, these results offer insights into the two-enzyme PET depolymn. system and will inform future efforts in the biol. deconstruction and upcycling of mixed plastics.
- 50Erickson, E.; Shakespeare, T. J.; Bratti, F.; Buss, B. L.; Graham, R.; Hawkins, M. A.; König, G.; Michener, W. E.; Miscall, J.; Ramirez, K. J. Comparative performance of PETase as a function of reaction conditions, substrate properties, and product accumulation. ChemSusChem 2022, 15, e202101932 DOI: 10.1002/cssc.20210193250https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXisVehtrzP&md5=bc1cd0c2fa069ca6d834a99f5ec38cf2Comparative Performance of PETase as a Function of Reaction Conditions, Substrate Properties, and Product AccumulationErickson, Erika; Shakespeare, Thomas J.; Bratti, Felicia; Buss, Bonnie L.; Graham, Rosie; Hawkins, McKenzie A.; Konig, Gerhard; Michener, William E.; Miscall, Joel; Ramirez, Kelsey J.; Rorrer, Nicholas A.; Zahn, Michael; Pickford, Andrew R.; McGeehan, John E.; Beckham, Gregg T.ChemSusChem (2022), 15 (1), e202101932CODEN: CHEMIZ; ISSN:1864-5631. (Wiley-VCH Verlag GmbH & Co. KGaA)There is keen interest to develop new technologies to recycle the plastic poly(ethylene terephthalate) (PET). To this end, the use of PET-hydrolyzing enzymes has shown promise for PET deconstruction to its monomers, terephthalate (TPA) and ethylene glycol (EG). Here, the Ideonella sakaiensis PETase wild-type enzyme was compared to a previously reported improved variant (W159H/S238F). The thermostability of each enzyme was compared and a 1.45 S resoln. structure of the mutant was described, highlighting changes in the substrate binding cleft compared to the wild-type enzyme. Subsequently, the performance of the wild-type and variant enzyme was compared as a function of temp., substrate morphol., and reaction mixt. compn. These studies showed that reaction temp. had the strongest influence on performance between the two enzymes. It was also shown that both enzymes achieved higher levels of PET conversion for substrates with moderate crystallinity relative to amorphous substrates. Finally, the impact of product accumulation on reaction progress was assessed for the hydrolysis of both PET and bis(2-hydroxyethyl) terephthalate (BHET). Each enzyme displayed different inhibition profiles to mono(2-hydroxyethyl) terephthalate (MHET) and TPA, while both were sensitive to inhibition by EG. Overall, this study highlights the importance of reaction conditions, substrate selection, and product accumulation for catalytic performance of PET-hydrolyzing enzymes, which have implications for enzyme screening in the development of enzyme-based polyester recycling.
- 51Lu, H.; Diaz, D. J.; Czarnecki, N. J.; Zhu, C.; Kim, W.; Shroff, R.; Acosta, D. J.; Alexander, B.; Cole, H.; Zhang, Y. J. Deep learning redesign of PETase for practical PET degrading applications. bioRxiv 2021, DOI: 10.1101/2021.10.10.463845There is no corresponding record for this reference.
- 52de Castro, A. M.; Carniel, A.; Nicomedes Junior, J.; da Conceição Gomes, A.; Valoni, É. Screening of commercial enzymes for poly(ethylene terephthalate) (PET) hydrolysis and synergy studies on different substrate sources. J. Ind. Microbiol. Biotechnol. 2017, 44, 835– 844, DOI: 10.1007/s10295-017-1942-z52https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1cvptlGkug%253D%253D&md5=894461cc3c0f4dcfaae0d80dc91a3b0aScreening of commercial enzymes for poly(ethylene terephthalate) (PET) hydrolysis and synergy studies on different substrate sourcesde Castro Aline Machado; Nicomedes Junior Jose; da Conceicao Gomes Absai; Valoni Erika; Carniel AdrianoJournal of industrial microbiology & biotechnology (2017), 44 (6), 835-844 ISSN:.Poly(ethylene terephthalate) (PET) is one of the most consumed plastics in the world. The development of efficient technologies for its depolymerization for monomers reuse is highly encouraged, since current recycling rates are still very low. In this study, 16 commercial lipases and cutinases were evaluated for their abilities to catalyze the hydrolysis of two PET samples. Humicola insolens cutinase showed the best performance and was then used in reactions on other PET sources, solely or in combination with the efficient mono(hydroxyethyl terephthalate)-converting lipase from Candida antarctica. Synergy degrees of the final titers of up to 2.2 (i.e., more than double of the concentration when both enzymes were used, as compared to their use alone) were found, with increased terephthalic acid formation rates, reaching a maximum of 59,989 μmol/L (9.36 g/L). These findings open up new possibilities for the conversion of post-consumer PET packages into their minimal monomers, which can be used as drop in at existing industrial facilities.
- 53Gamerith, C.; Zartl, B.; Pellis, A.; Guillamot, F.; Marty, A.; Acero, E. H.; Guebitz, G. M. Enzymatic recovery of polyester building blocks from polymer blends. Process Biochem. 2017, 59, 58– 64, DOI: 10.1016/j.procbio.2017.01.00453https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtVChu7s%253D&md5=443c19eaea24bd703646878c11313174Enzymatic recovery of polyester building blocks from polymer blendsGamerith, Caroline; Zartl, Barbara; Pellis, Alessandro; Guillamot, Frederique; Marty, Alain; Acero, Enrique Herrero; Guebitz, Georg M.Process Biochemistry (Oxford, United Kingdom) (2017), 59 (Part_A), 58-64CODEN: PBCHE5; ISSN:1359-5113. (Elsevier Ltd.)In this study we investigated the ability of a cutinase from Thermobifida cellulosilytica (Thc_Cut1) to hydrolyze poly(ethylene terephthalate) (PET) moieties in different polymer blends. The compn. of various materials including com. available bottles and packaging was detd. using Fourier Transform IR spectroscopy (FT-IR) and Differential Scanning Calorimetry (DSC). When incubated with PET blended with polyethylene (PE) or polyamide (PA) from packaging and bottles without prior sepn., Thc_Cut1 selectively hydrolyzed the PET moieties releasing terephthalic acid (TPA) and mono(2-hydroxyethyl) terephthalate (MHET). Polymer blends were hydrolyzed in an up to 9 times higher extent compared to higher cryst. pure PET. The influence of various parameters like temp., particle size, crystallinity and product inhibition on hydrolysis of PET moieties by Thc_Cut1 was investigated. The amt. of products released was up to 10 times higher when the incubation temp. was increased from 40 °C to 60 °C. The smaller the particle size the higher the hydrolysis rates were. Interestingly, semi-cryst. (24%) PET from bottles was hydrolyzed faster than powder from amorphous PET films (12%). An inhibitory effect of bis(2-hydroxyethyl) terephthalate (BHET) on hydrolysis of PET by Thc_Cut1 was obsd.
- 54Castro, A. M. d.; Carniel, A.; Stahelin, D.; Chinelatto Junior, L. S.; Honorato, H. d. A.; de Menezes, S. M. C. High-fold improvement of assorted post-consumer poly(ethylene terephthalate) (PET) packages hydrolysis using Humicola insolens cutinase as a single biocatalyst. Process Biochem. 2019, 81, 85– 91, DOI: 10.1016/j.procbio.2019.03.00654https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXkvVegurs%253D&md5=fe9b8a35851a08c00e68134feb16483cHigh-fold improvement of assorted post-consumer poly(ethylene terephthalate) (PET) packages hydrolysis using Humicola insolens cutinase as a single biocatalystCastro, Aline Machado de; Carniel, Adriano; Stahelin, Diego; Chinelatto Junior, Luiz Silvino; Honorato, Hercilio de Angeli; de Menezes, Sonia Maria CabralProcess Biochemistry (Oxford, United Kingdom) (2019), 81 (), 85-91CODEN: PBCHE5; ISSN:1359-5113. (Elsevier Ltd.)The dissemination of technologies for poly(ethylene terephthalate) (PET) recycling is of paramount importance in the context of the plastics circular economy. One of the most promising alternatives is to use enzymes as catalysts for PET depolymn. to its monomers, but this route still needs improvement, esp. regarding titer and productivity. In the present work, a sequential approach comprised of fractional factorial and central composite rotatable designs, the path of steepest ascent and one-way evaluation of variable effect, was performed to address these limitations, during assorted post-consumer PET (PC-PET) hydrolysis catalyzed by Humicola insolens cutinase. The highest terephthalic acid concn. and productivity during PC-PET hydrolysis were 100.9 mM (16.8 g/L) and 14.4 mM/day, corresponding to overall improvements of 10-fold and 20-fold, resp. These data are among the best results described so far for enzyme-catalyzed hydrolysis of used PET packages. Also, the use of a single enzyme system, instead of multiple biocatalysts to achieve final conversion of PET to its monomers, lowers the process complexity and costs.
- 55Kari, J.; Andersen, M.; Borch, K.; Westh, P. An Inverse Michaelis–Menten Approach for Interfacial Enzyme Kinetics. ACS Catal. 2017, 7, 4904– 4914, DOI: 10.1021/acscatal.7b0083855https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtVSjtrjK&md5=414b5391cab840baf87cfdbef2f4a2cbAn inverse Michaelis-Menten approach for interfacial enzyme kineticsKari, Jeppe; Andersen, Morten; Borch, Kim; Westh, PeterACS Catalysis (2017), 7 (7), 4904-4914CODEN: ACCACS; ISSN:2155-5435. (American Chemical Society)Interfacial enzyme reactions are ubiquitous both in vivo and in tech. applications, but anal. of their kinetics remains controversial. In particular, it is unclear whether conventional Michaelis-Menten theory, which requires a large excess of substrate, can be applied. Here, an extensive exptl. study of the enzymic hydrolysis of insol. cellulose by cellobiohydrolase Cel7A and cellulase/endoglucanase Cel12A indeed showed that the conventional approach had a limited applicability. Instead, we argue that, unlike bulk reactions, interfacial enzyme catalysis may reach a steady-state condition in the opposite exptl. limit, where the concn. of enzyme far exceeded the molar concn. of accessible surface sites. Under this condition, an "inverse Michaelis-Menten equation", where the roles of enzyme and substrate had been swapped, proved to be readily applicable. We suggest that this inverted approach provides a general tool for kinetic analyses of interfacial enzyme reactions and that its analogy to established theory provides a bridge to the accumulated understanding of steady-state enzyme kinetics. Finally, we show that the ratio of parameters from conventional and inverted Michaelis-Menten anal. reveals the d. of enzyme attack sites on the substrate surface as probed by one specific enzyme. This d., which is an analog to a molar substrate concn. for interfacial reactions, was shown to vary strongly even among related enzymes. This difference reflected how the enzyme discriminates between local differences in surface structure on the substrate.
- 56Andersen, M.; Kari, J.; Borch, K.; Westh, P. Michaelis-Menten equation for degradation of insoluble substrate. Math. Biosci. 2018, 296, 93– 97, DOI: 10.1016/j.mbs.2017.11.01156https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXntVCitQ%253D%253D&md5=863c7ae829e6ced4495ee36206ac2500Michaelis-Menten equation for degradation of insoluble substrateAndersen, Morten; Kari, Jeppe; Borch, Kim; Westh, PeterMathematical Biosciences (2018), 296 (), 93-97CODEN: MABIAR; ISSN:0025-5564. (Elsevier Inc.)Kinetic studies of homogeneous enzyme reactions where both the substrate and enzyme are sol. have been well described by the Michaelis-Menten (MM) equation for more than a century. However, many reactions are taking place at the interface of a solid substrate and enzyme in soln. Such heterogeneous reactions are abundant both in vivo and in industrial application of enzymes but it is not clear whether traditional enzyme kinetic theory developed for homogeneous catalysis can be applied. Since the molar concn. of surface accessible sites (attack-sites) often is unknown for a solid substrate it is difficult to assess whether the requirement of the MM equation is met. In this paper we study a simple kinetic model, where removal of attack sites expose new ones which preserve the total accessible substrate, and denote this approach the substrate conserving model. The kinetic equations are solved in closed form, both steady states and progress curves, for any admissible values of initial conditions and rate consts. The model is shown to merge with the MM equation and the reverse MM equation when these are valid. The relation between available molar concn. of attack sites and mass load of substrate is analyzed and this introduces an extra parameter to the equations. Various exptl. setups to practically and reliably est. all parameters are discussed.
- 57Bååth, J. A.; Borch, K.; Jensen, K.; Brask, J.; Westh, P. Comparative Biochemistry of Four Polyester (PET) Hydrolases. ChemBioChem 2021, 22, 1627– 1637, DOI: 10.1002/cbic.20200079357https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXjsFKit7o%253D&md5=f005b3f1062a71972d3445ab85a36fcbComparative Biochemistry of Four Polyester (PET) Hydrolases**Baath, Jenny Arnling; Borch, Kim; Jensen, Kenneth; Brask, Jesper; Westh, PeterChemBioChem (2021), 22 (9), 1627-1637CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)The potential of bioprocessing in a circular plastic economy has strongly stimulated research into the enzymic degrdn. of different synthetic polymers. Particular interest has been devoted to the commonly used polyester, poly(ethylene terephthalate) (PET), and a no. of PET hydrolases have been described. However, a kinetic framework for comparisons of PET hydrolases (or other plastic-degrading enzymes) acting on the insol. substrate has not been established. Herein, we propose such a framework, which we have tested against kinetic measurements for four PET hydrolases. The anal. provided values of kcat and KM, as well as an apparent specificity const. in the conventional units of M-1s-1. These parameters, together with exptl. values for the no. of enzyme attack sites on the PET surface, enabled comparative analyses. A variant of the PET hydrolase from Ideonella sakaiensis was the most efficient enzyme at ambient conditions; it relied on a high kcat rather than a low KM. Moreover, both sol. and insol. PET fragments were consistently hydrolyzed much faster than intact PET. This suggests that interactions between polymer strands slow down PET degrdn., whereas the chem. steps of catalysis and the low accessibility assocd. with solid substrate were less important for the overall rate. Finally, the investigated enzymes showed a remarkable substrate affinity, and reached half the satn. rate on PET when the concn. of attack sites in the suspension was only about 50 nM. We propose that this is linked to nonspecific adsorption, which promotes the nearness of enzyme and attack sites.
- 58Werner, A. Z.; Clare, R.; Mand, T. D.; Pardo, I.; Ramirez, K. J.; Haugen, S. J.; Bratti, F.; Dexter, G. N.; Elmore, J. R.; Huenemann, J. D. Tandem chemical deconstruction and biological upcycling of poly(ethylene terephthalate) to beta-ketoadipic acid by Pseudomonas putida KT2440. Metab. Eng. 2021, 67, 250– 261, DOI: 10.1016/j.ymben.2021.07.00558https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhsFOktrrL&md5=b97b521b29dad963f5d5bd148889814aTandem chemical deconstruction and biological upcycling of poly(ethylene terephthalate) to β-ketoadipic acid by Pseudomonas putida KT2440Werner, Allison Z.; Clare, Rita; Mand, Thomas D.; Pardo, Isabel; Ramirez, Kelsey J.; Haugen, Stefan J.; Bratti, Felicia; Dexter, Gara N.; Elmore, Joshua R.; Huenemann, Jay D.; Peabody, George L. V.; Johnson, Christopher W.; Rorrer, Nicholas A.; Salvachua, Davinia; Guss, Adam M.; Beckham, Gregg T.Metabolic Engineering (2021), 67 (), 250-261CODEN: MEENFM; ISSN:1096-7176. (Elsevier B.V.)Poly(ethylene terephthalate) (PET) is the most abundantly consumed synthetic polyester and accordingly a major source of plastic waste. The development of chemocatalytic approaches for PET depolymn. to monomers offers new options for open-loop upcycling of PET, which can leverage biol. transformations to higher-value products. To that end, here we perform four sequential metabolic engineering efforts in Pseudomonas putida KT2440 to enable the conversion of PET glycolysis products via: (i) ethylene glycol utilization by constitutive expression of native genes, (ii) terephthalate (TPA) catabolism by expression of tphA2IIA3IIBIIA1II from Comamonas and tpaK from Rhodococcus jostii, (iii) bis(2-hydroxyethyl) terephthalate (BHET) hydrolysis to TPA by expression of PETase and MHETase from Ideonella sakaiensis, and (iv) BHET conversion to a performance-advantaged bioproduct, β-ketoadipic acid (βKA) by deletion of pcaIJ. Using this strain, we demonstrate prodn. of 15.1 g/L βKA from BHET at 76% molar yield in bioreactors and conversion of catalytically depolymd. PET to βKA. Overall, this work highlights the potential of tandem catalytic deconstruction and biol. conversion as a means to upcycle waste PET.
- 59Blundell, D. J.; MacKerron, D. H.; Fuller, W.; Mahendrasingam, A.; Martin, C.; Oldman, R. J.; Rule, R. J.; Riekel, C. Characterization of strain-induced crystallization of poly(ethylene terephthalate) at fast draw rates using synchrotron radiation. Polymer 1996, 37, 3303– 3311, DOI: 10.1016/0032-3861(96)88476-X59https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28Xks1Ghtb8%253D&md5=6b6455387aa6cbc8ca8c0bc5d9589530Characterization of strain-induced crystallization of poly(ethylene terephthalate) at fast draw rates using synchrotron radiationBlundell, D. J.; MacKerron, D. H.; Fuller, W.; Mahendrasingam, A.; Martin, C.; Oldman, R. J.; Rule, R. J.; Riekel, C.Polymer (1996), 37 (15), 3303-3311CODEN: POLMAG; ISSN:0032-3861. (Elsevier)Structural changes during fast drawing of poly(ethylene terephthalate) were studied by wide-angle X-ray scattering using synchrotron radiation. Drawing was studied at 80, 90, 100 or 110° to a final draw ratio of ∼4:1 at a draw rate of ∼ 10s-1. Simultaneous video recording of the sample enabled variation in the X-ray pattern to be correlated with local extension. Essentially all oriented crystn. occurred after final extension. Primary crystn. fits a first-order transformation with little change in the rate of crystn. obsd. over the 30° range of temp. These results show that it can be misleading to rely on crystallinity information obtained when samples from interrupted draw expts. are quenched.
- 60Blundell, D. J.; Mahendrasingam, A.; Martin, C.; Fuller, W.; MacKerron, D. H.; Harvie, J. L.; Oldman, R. J.; Riekel, C. Orientation prior to crystallisation during drawing of poly(ethylene terephthalate). Polymer 2000, 41, 7793– 7802, DOI: 10.1016/S0032-3861(00)00128-260https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXltF2rtbo%253D&md5=d978de444c9c294ec68f246f934f37b9Orientation prior to crystallisation during drawing of poly(ethylene terephthalate)Blundell, D. J.; Mahendrasingam, A.; Martin, C.; Fuller, W.; MacKerron, D. H.; Harvie, J. L.; Oldman, R. J.; Riekel, C.Polymer (2000), 41 (21), 7793-7802CODEN: POLMAG; ISSN:0032-3861. (Elsevier Science Ltd.)Wide angle X-ray scattering data have been recorded during the drawing of poly(ethylene terephthalate) (PET) using a wide range of draw rates (0.05-12 s-1), temps. (90-120°C) and draw ratios. The data were analyzed to follow the development of mol. orientation and the onset of crystn. The mol. orientation prior to crystn. has been characterized in terms of the orientation order parameter <P2(cos θ)>. The rate of increase of <P2(cos θ)> with draw ratio decreases with both increasing temp. and decreasing draw rate. A superposition of all the data to a common ref. temp. of 90°C was obtained using a WLF shift factor to provide a master curve showing the dependence of the development of <P2(cos θ)> on draw rate. A comparison of the known chain relaxation motions of PET with the obsd. relation between draw rate and the onset of crystn. provides an explanation of a previous discrepancy in the literature concerning the point of onset of crystn. For draw rates faster than the rate of the chain retraction motion, the onset of crystn. is delayed until the end of the deformation process. For draw rates slower than the chain retraction motion, there is evidence of the onset of crystn. occurring before the end of the deformation process.
- 61Mahendrasingam, A.; Martin, C.; Fuller, W.; Blundell, D. J.; Oldman, R. J.; MacKerron, D. H.; Harvie, J. L.; Riekel, C. Observation of a transient structure prior to strain-induced crystallization in poly(ethylene terephthalate). Polymer 2000, 41, 1217– 1221, DOI: 10.1016/S0032-3861(99)00461-961https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXns12jsL8%253D&md5=6292d5ec77c4441f956f70ff0d6d779cObservation of a transient structure prior to strain-induced crystallization in poly(ethylene terephthalate)Mahendrasingam, A.; Martin, C.; Fuller, W.; Blundell, D. J.; Oldman, R. J.; MacKerron, D. H.; Harvie, J. L.; Riekel, C.Polymer (1999), 41 (3), 1217-1221CODEN: POLMAG; ISSN:0032-3861. (Elsevier Science Ltd.)Using time-resolved X-ray diffraction at the European Synchrotron Radiation Facility we have obsd. a highly oriented weak transient diffraction peak which persists for about 0.2 s prior to strain-induced crystn. during the uniaxial drawing of poly(ethylene terephthalate) (PET) under industrial processing conditions. This structure may be identified with the mesophase structure proposed by a no. of workers to occur during drawing of PET, poly(ethylene naphthalate) (PEN) and random copolymers of PET and PEN. In our studies, the transient structure was not obsd. at draw temps. greater than 90°C nor when the draw conditions resulted in a degree of polymer orientation below a crit. level. The possibility that this transient structure is a precursor of strain-induced crystn. is suggested by our observation of a correlation between the decay of the diffraction assocd. with it and an increase in the intensity of diffraction peaks assocd. with the development of crystn.
- 62Forestier, E.; Combeaud, C.; Guigo, N.; Sbirrazzuoli, N.; Billon, N. Understanding of strain-induced crystallization developments scenarios for polyesters: Comparison of poly(ethylene furanoate), PEF, and poly(ethylene terephthalate), PET. Polymer 2020, 203, 122755, DOI: 10.1016/j.polymer.2020.12275562https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhsVWhs7rF&md5=40ec244b7664224feefd92d55e540a36Understanding of strain-induced crystallization developments scenarios for polyesters: Comparison of poly(ethylene furanoate), PEF, and poly(ethylene terephthalate), PETForestier, Emilie; Combeaud, Christelle; Guigo, Nathanael; Sbirrazzuoli, Nicolas; Billon, NoellePolymer (2020), 203 (), 122755CODEN: POLMAG; ISSN:0032-3861. (Elsevier Ltd.)Specific conditions of strain, stretching, strain rate and temp. are known to be necessary for the strain induced crystn. phenomenon (SIC) to occur. It leads to the formation of a crystal in different amorphous polymers, stretched above their glassy transition. This phenomenon was intensively documented in case of poly(ethylene terephthalate), PET. More recently, some studies focused on SIC development in biobased poly(ethylene furandicarboxylate), PEF. Comparison of these crystn. abilities and crystn. kinetics upon stretching in the two materials allows to describe main differences between them, and to enlighten the role of chain architecture on SIC. To achieve that point, different mech. tensile tests were conducted using well controlled loading paths to explore the different steps of the microstructural changes induced by the stretching and their correlation with mech. behavior. Several macroscopic equivalence in the effects of SIC were found, such as increase in modulus, appearance of organized phase, increase I n α-relaxation temp. despite some differences in chain architecture. Combining both loading-unloading tests and quenching protocols, it was found that inducing more or less strong interactions between constitutive units, and more or less stable cryst. phases, leads to differences in apparent strain induced crystn. kinetics:• PET stretching can induce, prior to main strain hardening step, the formation of re-enforcing intermediate phases (or imperfect crystal) being stable upon unloading and able to be improved upon relaxation or thermal treatments;• PEF stretching exhibits a more "simple" two-steps path with no intermediate phases stable upon unloading. This can be related with the weaker stability of PEF crystal compared to PET (PEF quiescent crystn. temp. and melting temp. are very close to each other), and to the more complex cryst. lattice in PEF (two units are needed instead of one due to furanic cycle). In addn., for PET, Young modulus increases more gradually during strain hardening than for PEF. The final microstructure after stretching is therefore more dependent on thermomech. treatments (annealing or relaxation steps) in PET in comparison to PEF.
- 63Bashir, Z.; Al-Aloush, I.; Al-Raqibah, I.; Ibrahim, M. Evaluation of three methods for the measurement of crystallinity of PET resins, preforms, and bottles. Polym. Eng. Sci. 2000, 40, 2442– 2455, DOI: 10.1002/pen.1137663https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXoslahsbw%253D&md5=a865125bc74ef411008e9c755788e386Evaluation of three methods for the measurement of crystallinity of PET resins, preforms, and bottlesBashir, Z.; Al-Aloush, I.; Al-Raqibah, I.; Ibrahim, M.Polymer Engineering and Science (2000), 40 (11), 2442-2455CODEN: PYESAZ; ISSN:0032-3888. (Society of Plastics Engineers)The control of crystn. is important at all processing stages of the PET bottle industry, from the manuf. of bottle resins to the fabrication of preforms and bottles. The authors evaluate critically 3 methods of crystallinity measurement. They have used d., Differential Scanning Calorimetry (DSC), and Modulated Differential Scanning Calorimetry (MDSC) to study the crystallinity of PET chips, preforms, and bottles. The accuracy, precision, and general validity of each technique and the problems of interpretation are discussed.
- 64Scandola, M.; Focarete, M. L.; Frisoni, G. Simple Kinetic Model for the Heterogeneous Enzymatic Hydrolysis of Natural Poly(3-hydroxybutyrate). Macromolecules 1998, 31, 3846– 3851, DOI: 10.1021/ma980137y64https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXjtlygu7w%253D&md5=a9333839795596c7ca94cc2a2ebe675fSimple Kinetic Model for the Heterogeneous Enzymic Hydrolysis of Natural Poly(3-hydroxybutyrate)Scandola, Mariastella; Focarete, Maria Letizia; Frisoni, GiovannaMacromolecules (1998), 31 (12), 3846-3851CODEN: MAMOBX; ISSN:0024-9297. (American Chemical Society)The kinetics of the enzymic degrdn. of bacterial poly(3-hydroxybutyrate) (PHB) is studied using PHB-depolymerase A from Pseudomonas lemoignei (Tris-HCl buffer, pH 8, 37°). Biodegrdn. expts. are performed on PHB in the form of both compression-molded films and a powder suspension. From WAXS and DSC measurements the two substrates show the same cryst. fraction. The rate of hydrolysis of PHB films is detd. by gravimetrical and also through spectrophotometric quantification of the hydrolysis products at λ = 210 nm. For the suspension of PHB particles, a turbidimetric detn. of the biodegrdn. rate is applied. A simple two-step kinetic model is proposed, which predicts that the hydrolysis rate per unit substrate surface area reaches a plateau at high enzyme concns. The model satisfactorily describes the enzymic degrdn. results of PHB film and PHB powder suspension, provided that the remarkable changes of the exposed area caused by enzymic attack to the latter substrate are taken into account. Anal. of the enzymic degrdn. results yields analogous hydrolysis rate consts. for film (1.48 μg cm-2 min-1) and powder suspension (1.42 μg cm-2 min-1).
- 65Thomsen, T. B.; Hunt, C. J.; Meyer, A. S. Influence of substrate crystallinity and glass transition temperature on enzymatic degradation of polyethylene terephthalate (PET). New Biotechnol. 2022, 69, 28– 35, DOI: 10.1016/j.nbt.2022.02.00665https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XntFCgu78%253D&md5=abdf6a78ca51b882a02a6fb99b03922fInfluence of substrate crystallinity and glass transition temperature on enzymatic degradation of polyethylene terephthalate (PET)Thomsen, Thore Bach; Hunt, Cameron J.; Meyer, Anne S.New Biotechnology (2022), 69 (), 28-35CODEN: NBEIBR; ISSN:1871-6784. (Elsevier B.V.)This work examines the significance of the degree of crystallinity (XC) of polyethylene terephthalate (PET) and the PET glass transition temp. (Tg) on enzymic degrdn. of PET at elevated temps. using two engineered, thermostable PET degrading enzymes: LCCICCG, a variant of the leaf-branch compost cutinase, and DuraPETase, evolved from the Ideonella sakaiensis PETase. The XC was systematically varied by thermal annealing of PET disks (O 6 mm, thickness 1 mm). The XC affected the enzymic product release rate that essentially ceased at XC 22-27% for the LCCICCG and at XC ∼17% for the DuraPETase. SEM revealed that enzymic treatment produced cavities on the PET surface when the XC was > 10% but resulted in a smooth surface on amorphous PET (XC ∼10%). The Tg of amorphous PET disks decreased from 75 °C to 60 °C during 24 h pre-soaking in water at 65 °C, while the XC remained unchanged. Enzymic reaction on pre-soaked disks at 68 °C, i.e. above the Tg, did not affect the enzymic product release rate catalyzed by LCCICCG. These findings improve the understanding of enzymic PET degrdn. and have implications for development of efficient enzymic PET upcycling processes.
- 66Barth, M.; Honak, A.; Oeser, T.; Wei, R.; Belisário-Ferrari, M. R.; Then, J.; Schmidt, J.; Zimmermann, W. A dual enzyme system composed of a polyester hydrolase and a carboxylesterase enhances the biocatalytic degradation of polyethylene terephthalate films. Biotechnol. J. 2016, 11, 1082– 1087, DOI: 10.1002/biot.20160000866https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtVWkt7rL&md5=37b71dbedbbcdc97e07df882075b6ffdA dual enzyme system composed of a polyester hydrolase and a carboxylesterase enhances the biocatalytic degradation of polyethylene terephthalate filmsBarth, Markus; Honak, Annett; Oeser, Thorsten; Wei, Ren; Belisario-Ferrari, Matheus R.; Then, Johannes; Schmidt, Juliane; Zimmermann, WolfgangBiotechnology Journal (2016), 11 (8), 1082-1087CODEN: BJIOAM; ISSN:1860-6768. (Wiley-VCH Verlag GmbH & Co. KGaA)TfCut2 from Thermobifida fusca KW3 and the metagenome-derived LC-cutinase are bacterial polyester hydrolases capable of efficiently degrading polyethylene terephthalate (PET) films. Since the enzymic PET hydrolysis is inhibited by the degrdn. intermediate mono-(2-hydroxyethyl) terephthalate (MHET), a dual enzyme system consisting of a polyester hydrolase and the immobilized carboxylesterase TfCa from Thermobifida fusca KW3 was employed for the hydrolysis of PET films at 60°C. HPLC anal. of the reaction products obtained after 24 h of hydrolysis showed an increased amt. of sol. products with a lower proportion of MHET in the presence of the immobilized TfCa. The results indicated a continuous hydrolysis of the inhibitory MHET by the immobilized TfCa and demonstrated its advantage as a second biocatalyst in combination with a polyester hydrolase for an efficient degrdn. oft PET films. The dual enzyme system with LC-cutinase produced a 2.4-fold higher amt. of degrdn. products compared to TfCut2 after a reaction time of 24 h confirming the superior activity of his polyester hydrolase against PET films.
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