Quantifying Cellulose Accessibility During Enzyme-Mediated Deconstruction Using 2 Fluorescence-Tagged Carbohydrate-Binding Modules

Peter Ciesielski, Vera Novy, Kevin Aissa, Fredrik Nielsen, Suzana Straus, Christopher Hunt, Jack Saddler

Research output: Contribution to journalArticlepeer-review

36 Scopus Citations


Two fluorescence-tagged carbohydrate-binding modules (CBMs), which specifically bind to crystalline (CBM2a-RRedX) and paracrystalline (CBM17-FITC) cellulose, were used to differentiate the supramolecular cellulose structures in bleached softwood Kraft fibers during enzyme-mediated hydrolysis. Differences in CBM adsorption were elucidated using confocal laser scanning microscopy (CLSM), and the structural changes occurring during enzyme-mediated deconstruction were quantified via the relative fluorescence intensities of the respective probes. It was apparent that a high degree of order (i.e., crystalline cellulose) occurred at the cellulose fiber surface, which was interspersed by zones of lower structural organization and increased cellulose accessibility. Quantitative image analysis, supported by 13C NMR, scanning electron microscopy (SEM) imaging, and fiber length distribution analysis, showed that enzymatic degradation predominates at these zones during the initial phase of the reaction, resulting in rapid fiber fragmentation and an increase in cellulose surface crystallinity. By applying this method to elucidate the differences in the enzyme-mediated deconstruction mechanisms, this work further demonstrated that drying decreased the accessibility of enzymes to these disorganized zones, resulting in a delayed onset of degradation and fragmentation. The use of fluorescence-tagged CBMs with specific recognition sites provided a quantitative way to elucidate supramolecular substructures of cellulose and their impact on enzyme accessibility. By designing a quantitative method to analyze the cellulose ultrastructure and accessibility, this study gives insights into the degradation mechanism of cellulosic substrates.

Original languageAmerican English
Pages (from-to)22545-22551
Number of pages7
JournalProceedings of the National Academy of Sciences of the United States of America
Issue number45
StatePublished - 5 Nov 2019

Bibliographical note

Publisher Copyright:
© 2019 National Academy of Sciences. All rights reserved.

NREL Publication Number

  • NREL/JA-2700-75305


  • Analysis
  • Carbohydrate-binding modules
  • Cellulose structure
  • Confocal laser scanning microscopy
  • Enzyme accessibility
  • Quantitative image
  • Supramolecular


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