Search Menu
DOE PAGES title logo U.S. Department of Energy
Office of Scientific and Technical Information
Search Scholarly Publications

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 by13C 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 designingmore » a quantitative method to analyze the cellulose ultrastructure and accessibility, this study gives insights into the degradation mechanism of cellulosic substrates.« less

Authors:
 [1]; ORCiD logo [1];  [1];  [1];  [2];  [2]; ORCiD logo [1]
+ Show Author Affiliations
  1. Univ. of British Columbia, Vancouver, BC (Canada)
  2. National Renewable Energy Lab. (NREL), Golden, CO (United States)
Publication Date:
Research Org.:
National Renewable Energy Laboratory (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
Journal ID: ISSN 0027-8424
Grant/Contract Number:  
AC36-08GO28308
Resource Type:
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; Journal ID: ISSN 0027-8424
Publisher:
National Academy of Sciences
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, Aïssa, Kevin, Nielsen, Fredrik, Straus, Suzana K., Ciesielski, Peter, 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.
Copy to clipboard
Novy, Vera, Aïssa, Kevin, Nielsen, Fredrik, Straus, Suzana K., Ciesielski, Peter, Hunt, Christopher G., & Saddler, Jack. Quantifying cellulose accessibility during enzyme-mediated deconstruction using 2 fluorescence-tagged carbohydrate-binding modules. United States. https://doi.org/10.1073/pnas.1912354116
Copy to clipboard
Novy, Vera, Aïssa, Kevin, Nielsen, Fredrik, Straus, Suzana K., Ciesielski, Peter, Hunt, Christopher G., and Saddler, Jack. Mon . "Quantifying cellulose accessibility during enzyme-mediated deconstruction using 2 fluorescence-tagged carbohydrate-binding modules". United States. https://doi.org/10.1073/pnas.1912354116. https://www.osti.gov/servlets/purl/1573198.
Copy to clipboard
@article{osti_1573198,
title = {Quantifying cellulose accessibility during enzyme-mediated deconstruction using 2 fluorescence-tagged carbohydrate-binding modules},
author = {Novy, Vera and Aïssa, Kevin and Nielsen, Fredrik and Straus, Suzana K. and Ciesielski, Peter 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 by13C 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 = {Mon Oct 21 00:00:00 EDT 2019},
month = {Mon Oct 21 00:00:00 EDT 2019}
}
Copy to clipboard
Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record
https://doi.org/10.1073/pnas.1912354116
Copyright Statement
Other availability
Search WorldCat Search WorldCat to find libraries that may hold this journal
Citation Metrics:
Cited by: 31 works
Citation information provided by
Web of Science
Save / Share:
Export Metadata
Save to My Library
You must Sign In or Create an Account in order to save documents to your library.

Works referenced in this record:

Substrate and Enzyme Characteristics that Limit Cellulose Hydrolysis
journal, October 1999

  • Mansfield, S. D.; Mooney, C.; Saddler, J. N.
  • Biotechnology Progress, Vol. 15, Issue 5
  • DOI: 10.1021/bp9900864

The binding specificity and affinity determinants of family 1 and family 3 cellulose binding modules
journal, January 2003

  • Lehtio, J.; Sugiyama, J.; Gustavsson, M.
  • Proceedings of the National Academy of Sciences, Vol. 100, Issue 2
  • DOI: 10.1073/pnas.212651999

Automatic measurement of sister chromatid exchange frequency.
journal, July 1977

  • Zack, G. W.; Rogers, W. E.; Latt, S. A.
  • Journal of Histochemistry & Cytochemistry, Vol. 25, Issue 7
  • DOI: 10.1177/25.7.70454

Molecular-Level Origins of Biomass Recalcitrance: Decrystallization Free Energies for Four Common Cellulose Polymorphs
journal, April 2011

  • Beckham, Gregg T.; Matthews, James F.; Peters, Baron
  • The Journal of Physical Chemistry B, Vol. 115, Issue 14
  • DOI: 10.1021/jp1106394

Mechanism of the enzymatic hydrolysis of cellulose: Effects of major structural features of cellulose on enzymatic hydrolysis
journal, January 1980

  • Fan, L. T.; Lee, Yong-Hyun; Beardmore, David H.
  • Biotechnology and Bioengineering, Vol. 22, Issue 1
  • DOI: 10.1002/bit.260220113

Dislocations in Norway spruce fibres and their effect on properties of pulp and paper
journal, February 2005

  • Terziev, Nasko; Daniel, Geoffrey; Marklund, Ann
  • Holzforschung, Vol. 59, Issue 2
  • DOI: 10.1515/HF.2005.025

CellProfiler™: free, versatile software for automated biological image analysis
journal, January 2007

  • Lamprecht, Michael R.; Sabatini, David M.; Carpenter, Anne E.
  • BioTechniques, Vol. 42, Issue 1
  • DOI: 10.2144/000112257

Use of Carbohydrate Binding Modules To Elucidate the Relationship between Fibrillation, Hydrolyzability, and Accessibility of Cellulosic Substrates
journal, November 2018

Cellulose crystallinity - a key predictor of the enzymatic hydrolysis rate: Cellulose crystallinity
journal, February 2010

Dislocations in pulp fibres – their origin, characteristics and importance – a review
journal, December 2001

A single-molecule analysis reveals morphological targets for cellulase synergy
journal, April 2013

  • Fox, Jerome M.; Jess, Phillip; Jambusaria, Rakesh B.
  • Nature Chemical Biology, Vol. 9, Issue 6
  • DOI: 10.1038/nchembio.1227

Measurement of cellulase activities
journal, January 1987

Versatile derivatives of carbohydrate-binding modules for imaging of complex carbohydrates approaching the molecular level of resolution
journal, October 2006

  • Ding, Shi-You; Xu, Qi; Ali, Mursheda K.
  • BioTechniques, Vol. 41, Issue 4
  • DOI: 10.2144/000112244

Techno-economic evaluation of bioethanol production from three different lignocellulosic materials
journal, May 2008

Calculating sugar yields in high solids hydrolysis of biomass
journal, February 2011

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

Elucidation of cellulose accessibility, hydrolysability and reactivity as the major limitations in the enzymatic hydrolysis of cellulose
journal, March 2012

Assessing the susceptibility of hemp fibre to the formation of dislocations during processing
journal, July 2016

The effect of (induced) dislocations on the tensile properties of individual Norway spruce fibres
journal, January 2008

  • Eder, Michaela; Terziev, Nasko; Daniel, Geoffrey
  • Holzforschung, Vol. 62, Issue 1
  • DOI: 10.1515/HF.2008.011

Visualisation of dislocations in hemp fibres: A comparison between scanning electron microscopy (SEM) and polarized light microscopy (PLM)
journal, September 2006

  • Thygesen, Lisbeth G.; Bilde-Sørensen, Jørgen B.; Hoffmeyer, Preben
  • Industrial Crops and Products, Vol. 24, Issue 2
  • DOI: 10.1016/j.indcrop.2006.03.009

Role of supramolecular cellulose structures in enzymatic hydrolysis of plant cell walls
journal, September 2010

  • Thygesen, Lisbeth Garbrecht; Hidayat, Budi Juliman; Johansen, Katja Salomon
  • Journal of Industrial Microbiology & Biotechnology, Vol. 38, Issue 8
  • DOI: 10.1007/s10295-010-0870-y

Dislocations in single hemp fibres—investigations into the relationship of structural distortions and tensile properties at the cell wall level
journal, December 2006

  • Thygesen, Lisbeth G.; Eder, Michaela; Burgert, Ingo
  • Journal of Materials Science, Vol. 42, Issue 2
  • DOI: 10.1007/s10853-006-1113-5

The molecular structure within dislocations in Cannabis sativa fibres studied by polarised Raman microspectroscopy
journal, June 2013

Do Enzymatic Hydrolyzability and Simons' Stain Reflect the Changes in the Accessibility of Lignocellulosic Substrates to Cellulase Enzymes?
journal, December 2001

  • Esteghlalian, A. R.; Bilodeau, M.; Mansfield, S. D.
  • Biotechnology Progress, Vol. 17, Issue 6
  • DOI: 10.1021/bp0101177

Integrating enzyme fermentation in lignocellulosic ethanol production: life-cycle assessment and techno-economic analysis
journal, February 2017

  • Olofsson, Johanna; Barta, Zsolt; Börjesson, Pål
  • Biotechnology for Biofuels, Vol. 10, Issue 1
  • DOI: 10.1186/s13068-017-0733-0

Characterization of industrial and laboratory pulp fibres using HCl, Cellulase and FiberMaster analysis
journal, January 2005

Cell-wall recovery after irreversible deformation of wood
journal, November 2003

  • Keckes, Jozef; Burgert, Ingo; Frühmann, Klaus
  • Nature Materials, Vol. 2, Issue 12
  • DOI: 10.1038/nmat1019

Ab Initio Structure Determination and Functional Characterization Of CBM36
journal, July 2004

  • Jamal-Talabani, Sheelan; Boraston, Alisdair B.; Turkenburg, Johan P.
  • Structure, Vol. 12, Issue 7
  • DOI: 10.1016/j.str.2004.04.022

Investigation of the dislocation of natural fibres by Fourier-transform infrared spectroscopy
journal, March 2011

Analysis of the Surfaces of Wood Tissues and Pulp Fibers Using Carbohydrate-Binding Modules Specific for Crystalline Cellulose and Mannan
journal, January 2007

  • Filonova, Lada; Kallas, Åsa M.; Greffe, Lionel
  • Biomacromolecules, Vol. 8, Issue 1
  • DOI: 10.1021/bm060632z

Analysis of exposed cellulose surfaces in pretreated wood biomass using carbohydrate-binding module (CBM)–cyan fluorescent protein (CFP)
journal, February 2010

  • Kawakubo, Takeshi; Karita, Shuichi; Araki, Yuko
  • Biotechnology and Bioengineering, Vol. 105, Issue 3
  • DOI: 10.1002/bit.22550

Ramped-Amplitude Cross Polarization in Magic-Angle-Spinning NMR
journal, October 1994

  • Metz, G.; Wu, X. L.; Smith, S. O.
  • Journal of Magnetic Resonance, Series A, Vol. 110, Issue 2
  • DOI: 10.1006/jmra.1994.1208

Recent advances in understanding the role of cellulose accessibility in enzymatic hydrolysis of lignocellulosic substrates
journal, June 2014

Cellulose crystallinity index: measurement techniques and their impact on interpreting cellulase performance
journal, January 2010

  • Park, Sunkyu; Baker, John O.; Himmel, Michael E.
  • Biotechnology for Biofuels, Vol. 3, Issue 1
  • DOI: 10.1186/1754-6834-3-10

Recognition of cello-oligosaccharides by a family 17 carbohydrate-binding module: an X-ray crystallographic, thermodynamic and mutagenic study 1 1Edited by R. Huber
journal, December 2001

  • Notenboom, Valerie; Boraston, Alisdair B.; Chiu, Patrick
  • Journal of Molecular Biology, Vol. 314, Issue 4
  • DOI: 10.1006/jmbi.2001.5153

Substrate-Related Factors Affecting Enzymatic Saccharification of Lignocelluloses: Our Recent Understanding
journal, November 2012

Metadata matters: access to image data in the real world
journal, May 2010

  • Linkert, Melissa; Rueden, Curtis T.; Allan, Chris
  • The Journal of Cell Biology, Vol. 189, Issue 5
  • DOI: 10.1083/jcb.201004104

Fiber Quality Analysis: OpTest Fiber Quality Analyzer versus L&W Fiber Tester
journal, November 2011

  • Li, Bin; Bandekar, Rohan; Zha, Quanqing
  • Industrial & Engineering Chemistry Research, Vol. 50, Issue 22
  • DOI: 10.1021/ie201631q

Straightforward, effective calibration of SPINAL-64 decoupling results in the enhancement of sensitivity and resolution of biomolecular solid-state NMR
journal, April 2011

  • Comellas, Gemma; Lopez, Jakob J.; Nieuwkoop, Andrew J.
  • Journal of Magnetic Resonance, Vol. 209, Issue 2
  • DOI: 10.1016/j.jmr.2010.12.011

A Mini-review on the Applications of Cellulose-Binding Domains in Lignocellulosic Material Utilizations
journal, May 2015

Quantification of dislocations in hemp fibers using acid hydrolysis and fiber segment length distributions
journal, December 2007

A Threshold Selection Method from Gray-Level Histograms
journal, January 1979

Nanomechanics of cellulose deformation reveal molecular defects that facilitate natural deconstruction
journal, April 2019

  • Ciesielski, Peter N.; Wagner, Ryan; Bharadwaj, Vivek S.
  • Proceedings of the National Academy of Sciences
  • DOI: 10.1073/pnas.1900161116

Cellulose is not just cellulose: a review of dislocations as reactive sites in the enzymatic hydrolysis of cellulose microfibrils
journal, August 2012

Proteomics of protein trafficking by in vivo tissue-specific labeling
journal, April 2021

Consolidated bioprocessing of lignocellulose for production of glucaric acid by an artificial microbial consortium
journal, April 2021

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

Analysis of exposed cellulose surfaces in pretreated wood biomass using carbohydrate-binding module (CBM)–cyan fluorescent protein (CFP)
journal, February 2010

  • Kawakubo, Takeshi; Karita, Shuichi; Araki, Yuko
  • Biotechnology and Bioengineering, Vol. 105, Issue 3
  • DOI: 10.1002/bit.22550

Calculating sugar yields in high solids hydrolysis of biomass
journal, February 2011

Elucidation of cellulose accessibility, hydrolysability and reactivity as the major limitations in the enzymatic hydrolysis of cellulose
journal, March 2012

Physical characterization of enzymatically modified kraft pulp fibers
journal, September 1997

Analysis of the Surfaces of Wood Tissues and Pulp Fibers Using Carbohydrate-Binding Modules Specific for Crystalline Cellulose and Mannan
journal, January 2007

  • Filonova, Lada; Kallas, Åsa M.; Greffe, Lionel
  • Biomacromolecules, Vol. 8, Issue 1
  • DOI: 10.1021/bm060632z

Molecular-Level Origins of Biomass Recalcitrance: Decrystallization Free Energies for Four Common Cellulose Polymorphs
journal, April 2011

  • Beckham, Gregg T.; Matthews, James F.; Peters, Baron
  • The Journal of Physical Chemistry B, Vol. 115, Issue 14
  • DOI: 10.1021/jp1106394

Carbohydrate-binding modules: fine-tuning polysaccharide recognition
journal, September 2004

  • Boraston, Alisdair B.; Bolam, David N.; Gilbert, Harry J.
  • Biochemical Journal, Vol. 382, Issue 3
  • DOI: 10.1042/bj20040892

Nanomechanics of cellulose deformation reveal molecular defects that facilitate natural deconstruction
journal, April 2019

  • Ciesielski, Peter N.; Wagner, Ryan; Bharadwaj, Vivek S.
  • Proceedings of the National Academy of Sciences
  • DOI: 10.1073/pnas.1900161116

The binding specificity and affinity determinants of family 1 and family 3 cellulose binding modules
journal, January 2003

  • Lehtio, J.; Sugiyama, J.; Gustavsson, M.
  • Proceedings of the National Academy of Sciences, Vol. 100, Issue 2
  • DOI: 10.1073/pnas.212651999

Carbohydrate-binding Modules Recognize Fine Substructures of Cellulose
journal, August 2002

  • McLean, Bradley W.; Boraston, Alisdair B.; Brouwer, Darren
  • Journal of Biological Chemistry, Vol. 277, Issue 52
  • DOI: 10.1074/jbc.m204433200

Automatic measurement of sister chromatid exchange frequency.
journal, July 1977

  • Zack, G. W.; Rogers, W. E.; Latt, S. A.
  • Journal of Histochemistry & Cytochemistry, Vol. 25, Issue 7
  • DOI: 10.1177/25.7.70454

Cellulose crystallinity index: measurement techniques and their impact on interpreting cellulase performance
journal, January 2010

  • Park, Sunkyu; Baker, John O.; Himmel, Michael E.
  • Biotechnology for Biofuels, Vol. 3, Issue 1
  • DOI: 10.1186/1754-6834-3-10

Integrating enzyme fermentation in lignocellulosic ethanol production: life-cycle assessment and techno-economic analysis
journal, February 2017

  • Olofsson, Johanna; Barta, Zsolt; Börjesson, Pål
  • Biotechnology for Biofuels, Vol. 10, Issue 1
  • DOI: 10.1186/s13068-017-0733-0

Versatile derivatives of carbohydrate-binding modules for imaging of complex carbohydrates approaching the molecular level of resolution
journal, October 2006

  • Ding, Shi-You; Xu, Qi; Ali, Mursheda K.
  • BioTechniques, Vol. 41, Issue 4
  • DOI: 10.2144/000112244

CellProfiler™: free, versatile software for automated biological image analysis
journal, January 2007

  • Lamprecht, Michael R.; Sabatini, David M.; Carpenter, Anne E.
  • BioTechniques, Vol. 42, Issue 1
  • DOI: 10.2144/000112257
    Sort by:
    [ × clear filter / sort ]

    Similar Records in DOE PAGES and OSTI.GOV collections: