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A High‐Throughput Investigation of the Binding Specificity of Carbohydrate‐Binding Modules for Synthetic and Natural Polymers

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Rennison, Andrew Philip, Fernandez‐Macgregor, Jaime, Melot, Julie, Durbesson, Fabien, Tandrup, Tobias, Westh, Peter, Vincentelli, Renaud, Møller, Marie Sofie·2025·ChemSusChem
DOI 10.1002/cssc.202500468PMC PMC12330340

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Abstract

Carbohydrate‐binding modules (CBMs) are noncatalytic domains that enhance enzyme binding to substrates. Type A CBMs show potential for engineering plastic‐degrading enzymes due to their affinity for synthetic polymers. This study presents a high‐throughput screening pipeline for characterizing the affinity and specificity of type A CBMs towards the synthetic polymers polyethylene terephthalate (PET), polystyrene (PS), and polyethylene (PE), and the polysaccharides cellulose, chitin, and starch. ≈800 CBMs from the families CBM2, CBM3, CBM10, and CBM64 are expressed as green fluorescent protein (GFP)‐fusion proteins and tested for binding using a modified holdup assay, which produced up to 10 000 data points per day. The screening identifies ≈150 binders for PET and PE, around 250 for PS, and demonstrates family‐specific binding patterns for avicel, chitin, and starch. To demonstrate practical utility, four CBMs with high PET affinity are fused to the PET hydrolase LCC ICCG , enhancing activity on PET powder by around 5‐fold. These CBM‐enzyme fusions mitigate competitive binding to plastic impurities, improving performance in mixed plastic assays. This work significantly expands the repertoire of CBMs binding to synthetic polymers, advances our understanding of CBM‐substrate interactions, and provides knowledge for engineering enzymes to tackle plastic pollution, particularly where mixed plastics pose significant challenges.

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Figure 1 Setup for the high throughput (HTP) holdup assay. a) The process flow of HTP expression, purification, and binding analysis by the holdup assay. b) The setup of the 384‐well filter plates used in the assays. The purified EGFP‐CBM is added from the 96 well plate, to the 384 well plate in the pattern shown. The bottom right well of each group of four has no substrate added and is used as the control well. c) The conceptual framework of the holdup assay. Substrate is added to the top of the filter plate, followed by the protein, which binds to the substrate at varying levels. Unbound protein is then removed by centrifugation and collected in a 384 black plate; it is detected by the fluorescent signal from the EGFP module. BI values are calculated as a fraction of the bound total, compared to the control wells.
Figure 1 Setup for the high throughput (HTP) holdup assay. a) The process flow of HTP expression, purification, and binding analysis by the holdup assay. b) The setup of the 384‐well filter plates used in the assays. The purified EGFP‐CBM is added from the 96 well plate, to the 384 well plate in the pattern shown. The bottom right well of each group of four has no substrate added and is used as the control well. c) The conceptual framework of the holdup assay. Substrate is added to the top of the filter plate, followed by the protein, which binds to the substrate at varying levels. Unbound protein is then removed by centrifugation and collected in a 384 black plate; it is detected by the fluorescent signal from the EGFP module. BI values are calculated as a fraction of the bound total, compared to the control wells.CC BY-NC · PubMed Central
Figure 2 Histograms showing the binding intensities of EGFP‐CBM constructs to each of the six substrates analyzed. Binders are identified as having a higher BI than the average of lone EGFP binding over the course of several plates, as represented by the dotted line on each histogram. The number of binders from each family and total binders on each particular substrate are seen in the insert on each plot, along with the proportion of confirmed binders from the total tested in that family. Tables of the full dataset of the proteins binding to each of the substrates can be found in Dataset S1 and S2, Supporting Information.
Figure 2 Histograms showing the binding intensities of EGFP‐CBM constructs to each of the six substrates analyzed. Binders are identified as having a higher BI than the average of lone EGFP binding over the course of several plates, as represented by the dotted line on each histogram. The number of binders from each family and total binders on each particular substrate are seen in the insert on each plot, along with the proportion of confirmed binders from the total tested in that family. Tables of the full dataset of the proteins binding to each of the substrates can be found in Dataset S1 and S2, Supporting Information.CC BY-NC · PubMed Central
Figure 3 Phylogenetic trees including proteins with at least one confirmed binder in the holdup assays, decorated with the BI of the three polysaccharide substrates, avicel (red), chitin (blue), and starch (green). a) CBM2, b) CBM3, c) CBM10, and d) CBM64. Shading refers to groupings of proteins that bind specifically to the substrate represented by that color, with the yellow shading representing CBMs that do not bind of any of the substrates. Phylogenetic trees of the same families, decorated with the BI values of the three plastic substrates can be seen in Figure S2, Supporting Information.
Figure 3 Phylogenetic trees including proteins with at least one confirmed binder in the holdup assays, decorated with the BI of the three polysaccharide substrates, avicel (red), chitin (blue), and starch (green). a) CBM2, b) CBM3, c) CBM10, and d) CBM64. Shading refers to groupings of proteins that bind specifically to the substrate represented by that color, with the yellow shading representing CBMs that do not bind of any of the substrates. Phylogenetic trees of the same families, decorated with the BI values of the three plastic substrates can be seen in Figure S2, Supporting Information.CC BY-NC · PubMed Central
Figure 4 Activity and selectivity of fusion enzymes made with LCCICCG and CBMs obtained from the holdup assay. a) Activity of LCCICCG fusion enzymes on PET; 300 nM enzyme was incubated in 50 g L−1 of crystalline PET powder for 1 h at 50 °C. The concentration of degradation products was measured by UV absorbance at 240 nm and quantified using a standard curve of bis(2‐hydroxyethl)‐terephthalate (BHET), which serves as a representative degradation product. Each point represents the mean of triplicate experiments. b) Activity of LCCICCG fusion enzymes on PET in mixtures containing increasing amounts of PS “impurity”; 300 nM enzyme was incubated in mixtures of PET and PS for 1 h at 50 °C. The activity of the enzymes was calculated per unit mass of the PET substrate and then normalized to the activity in the experiments without impurity. Each point represents the mean of triplicate experiments.
Figure 4 Activity and selectivity of fusion enzymes made with LCCICCG and CBMs obtained from the holdup assay. a) Activity of LCCICCG fusion enzymes on PET; 300 nM enzyme was incubated in 50 g L−1 of crystalline PET powder for 1 h at 50 °C. The concentration of degradation products was measured by UV absorbance at 240 nm and quantified using a standard curve of bis(2‐hydroxyethl)‐terephthalate (BHET), which serves as a representative degradation product. Each point represents the mean of triplicate experiments. b) Activity of LCCICCG fusion enzymes on PET in mixtures containing increasing amounts of PS “impurity”; 300 nM enzyme was incubated in mixtures of PET and PS for 1 h at 50 °C. The activity of the enzymes was calculated per unit mass of the PET substrate and then normalized to the activity in the experiments without impurity. Each point represents the mean of triplicate experiments.CC BY-NC · PubMed Central

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Metadata

Authors
Rennison, A. P., Fernandez‐Macgregor, J., Melot, J., Durbesson, F., Tandrup, T., Westh, P., Vincentelli, R., & Møller, M. S.
Affiliations
  • Technical University of Denmark (DK)
  • Laboratory Architecture et Fonction des Macromolecules Biologiques Aix‐Marseille University Luminy Campus 13288 Marseille France
  • Aix-Marseille Université (FR)
Corresponding author
Møller, Marie Sofie
ORCID
Venue
ChemSusChem
Publisher
Wiley
Published
2025-07-14
Volume
18
Issue
16
Pages
e202500468-e202500468
ISSN
  • 1864-5631 (ISSN-L)
  • 1864-564X
DOI
10.1002/cssc.202500468
PMC
PMC12330340
OpenAlex
W4411454387
Cited by
7
References
1
Citations by year
2026: 5 · 2025: 2
FWCI
2.09
Type
journal article
Language
en
Open access
Licence
Locations
  • ChemSusChem (publishedVersion, cc-by-nc)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by-nc)
  • PubMed (publishedVersion)
  • HAL (submittedVersion, cc-by-nc)
Funders
  • Agence Nationale de la Recherche (ANR‐16‐CONV‐0001)
  • French Infrastructure for Integrated Structural Biology (ANR‐10‐INSB‐05‐01)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Enzyme Catalysis and Immobilization
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biotechnology › Enzyme Production and Characterization
  • Physical Sciences › Engineering › Biomedical Engineering › Biofuel production and bioconversion
Keywords
carbohydrate-binding module, synthetic polymers, polyethylene terephthalate, polystyrene, polyethylene, cellulose, chitin, starch, PET hydrolase, plastic-degrading enzymes, mixed plastic waste, plastic pollution
MeSH terms
Polyethylene Terephthalates, Polymers, Protein Binding, Substrate Specificity, High-Throughput Screening Assays

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CoChat. (2026). Citation check: A High‐Throughput Investigation of the Binding Specificity of Carbohydrate‐Binding Modules for Synthetic and Natural Polymers. https://app.cochat.ai/papers/10.1002/cssc.202500468
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Rennison, A. P., Fernandez‐Macgregor, J., Melot, J., Durbesson, F., Tandrup, T., Westh, P., Vincentelli, R., & Møller, M. S. (2025). A High‐Throughput Investigation of the Binding Specificity of Carbohydrate‐Binding Modules for Synthetic and Natural Polymers. ChemSusChem. https://doi.org/10.1002/cssc.202500468