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

Abstract

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. Bymore » designing a quantitative method to analyze the cellulose ultrastructure and accessibility, this study gives insights into the degradation mechanism of cellulosic substrates.« less

Authors:
 [1];  [1];  [1];  [1]; ORCiD logo [2];  [3];  [1]
  1. University of British Columbia
  2. National Renewable Energy Laboratory (NREL), Golden, CO (United States)
  3. U.S. Department of Agriculture
Publication Date:
Research Org.:
National Renewable Energy Lab. (NREL), Golden, CO (United States)
Sponsoring Org.:
USDOE Office of Energy Efficiency and Renewable Energy (EERE)
OSTI Identifier:
1573198
Report Number(s):
NREL/JA-2700-75305
Grant/Contract Number:  
AC36-08GO28308
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Proceedings of the National Academy of Sciences of the United States of America
Additional Journal Information:
Journal Volume: 116; Journal Issue: 45
Country of Publication:
United States
Language:
English
Subject:
09 BIOMASS FUELS; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; carbohydrate-binding modules; enzyme accessibility; supramolecular cellulose structure; confocal laser scanning microscopy; quantitative image analysis; FCICPL

Citation Formats

Novy, Vera, Aissa, Kevin, Nielsen, Fredrik, Straus, Suzana K., Ciesielski, Peter N, Hunt, Christopher G., and Saddler, Jack. Quantifying Cellulose Accessibility During Enzyme-Mediated Deconstruction Using 2 Fluorescence-Tagged Carbohydrate-Binding Modules. United States: N. p., 2019. Web. doi:10.1073/pnas.1912354116.
Novy, Vera, Aissa, Kevin, Nielsen, Fredrik, Straus, Suzana K., Ciesielski, Peter N, Hunt, Christopher G., & Saddler, Jack. Quantifying Cellulose Accessibility During Enzyme-Mediated Deconstruction Using 2 Fluorescence-Tagged Carbohydrate-Binding Modules. United States. doi:10.1073/pnas.1912354116.
Novy, Vera, Aissa, Kevin, Nielsen, Fredrik, Straus, Suzana K., Ciesielski, Peter N, Hunt, Christopher G., and Saddler, Jack. Mon . "Quantifying Cellulose Accessibility During Enzyme-Mediated Deconstruction Using 2 Fluorescence-Tagged Carbohydrate-Binding Modules". United States. doi:10.1073/pnas.1912354116.
@article{osti_1573198,
title = {Quantifying Cellulose Accessibility During Enzyme-Mediated Deconstruction Using 2 Fluorescence-Tagged Carbohydrate-Binding Modules},
author = {Novy, Vera and Aissa, Kevin and Nielsen, Fredrik and Straus, Suzana K. and Ciesielski, Peter N and Hunt, Christopher G. and Saddler, Jack},
abstractNote = {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.},
doi = {10.1073/pnas.1912354116},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
number = 45,
volume = 116,
place = {United States},
year = {2019},
month = {10}
}

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Works referenced in this record:

Toward an aggregated understanding of enzymatic hydrolysis of cellulose: Noncomplexed cellulase systems
journal, November 2004

  • Zhang, Yi-Heng Percival; Lynd, Lee R.
  • Biotechnology and Bioengineering, Vol. 88, Issue 7, p. 797-824
  • DOI: 10.1002/bit.20282