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Title: New perspective on glycoside hydrolase binding to lignin from pretreated corn stover

Abstract

Background: Non-specific binding of cellulases to lignin has been implicated as a major factor in the loss of cellulase activity during biomass conversion to sugars. It is believed that this binding may strongly impact process economics through loss of enzyme activities during hydrolysis and enzyme recycling scenarios. The current model suggests glycoside hydrolase activities are lost though non-specific/non-productive binding of carbohydrate-binding domains to lignin, limiting catalytic site access to the carbohydrate components of the cell wall. Results: In this study, we compared component enzyme affinities of a commercial Trichoderma reesei cellulase formulation, Cellic CTec2, towards extracted corn stover lignin using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and p-nitrophenyl substrate activities to monitor component binding, activity loss, and total protein binding. Protein binding was strongly affected by pH and ionic strength. β-D-glucosidases and xylanases, which do not have carbohydrate-binding modules (CBMs) and are basic proteins, demonstrated the strongest binding at low ionic strength, suggesting that CBMs are not the dominant factor in enzyme adsorption to lignin. Despite strong adsorption to insoluble lignin, β-D-glucosidase and xylanase activities remained high, with process yields decreasing only 4–15 % depending on lignin concentration. Conclusion: We propose that specific enzyme adsorption to lignin from a mixture ofmore » biomass-hydrolyzing enzymes is a competitive affinity where β-D-glucosidases and xylanases can displace CBM interactions with lignin. Process parameters, such as temperature, pH, and salt concentration influence the individual enzymes’ affinity for lignin, and both hydrophobic and electrostatic interactions are responsible for this binding phenomenon. Moreover, our results suggest that concern regarding loss of critical cell wall degrading enzymes to lignin adsorption may be unwarranted when complex enzyme mixtures are used to digest biomass.« less

Authors:
; ; ; ; ; ; ; ; ; ;
Publication Date:
Research Org.:
National Renewable Energy Laboratory (NREL), Golden, CO (United States); Alliance for Sustainable Energy, LLC (United States)
Sponsoring Org.:
USDOE Office of Energy Efficiency and Renewable Energy (EERE), Sustainable Transportation Office. Bioenergy Technologies Office (BETO)
OSTI Identifier:
1618630
Alternate Identifier(s):
OSTI ID: 1236147; OSTI ID: 1239800
Report Number(s):
NREL/JA-2700-64797
Journal ID: ISSN 1754-6834; 214; PII: 397
Grant/Contract Number:  
AC36–08GO28308; AC36-08GO28308
Resource Type:
Published Article
Journal Name:
Biotechnology for Biofuels
Additional Journal Information:
Journal Name: Biotechnology for Biofuels Journal Volume: 8 Journal Issue: 1; Journal ID: ISSN 1754-6834
Publisher:
Springer Science + Business Media
Country of Publication:
Netherlands
Language:
English
Subject:
09 BIOMASS FUELS; lignin; glycosyl hydrolase; enzyme binding; cellulase; biomass; pretreatment; Glycoside hydrolase

Citation Formats

Yarbrough, John M., Mittal, Ashutosh, Mansfield, Elisabeth, Taylor, II, Larry E., Hobdey, Sarah E., Sammond, Deanne W., Bomble, Yannick J., Crowley, Michael F., Decker, Stephen R., Himmel, Michael E., and Vinzant, Todd B. New perspective on glycoside hydrolase binding to lignin from pretreated corn stover. Netherlands: N. p., 2015. Web. doi:10.1186/s13068-015-0397-6.
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Yarbrough, John M., Mittal, Ashutosh, Mansfield, Elisabeth, Taylor, II, Larry E., Hobdey, Sarah E., Sammond, Deanne W., Bomble, Yannick J., Crowley, Michael F., Decker, Stephen R., Himmel, Michael E., & Vinzant, Todd B. New perspective on glycoside hydrolase binding to lignin from pretreated corn stover. Netherlands. https://doi.org/10.1186/s13068-015-0397-6
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Yarbrough, John M., Mittal, Ashutosh, Mansfield, Elisabeth, Taylor, II, Larry E., Hobdey, Sarah E., Sammond, Deanne W., Bomble, Yannick J., Crowley, Michael F., Decker, Stephen R., Himmel, Michael E., and Vinzant, Todd B. Fri . "New perspective on glycoside hydrolase binding to lignin from pretreated corn stover". Netherlands. https://doi.org/10.1186/s13068-015-0397-6.
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@article{osti_1618630,
title = {New perspective on glycoside hydrolase binding to lignin from pretreated corn stover},
author = {Yarbrough, John M. and Mittal, Ashutosh and Mansfield, Elisabeth and Taylor, II, Larry E. and Hobdey, Sarah E. and Sammond, Deanne W. and Bomble, Yannick J. and Crowley, Michael F. and Decker, Stephen R. and Himmel, Michael E. and Vinzant, Todd B.},
abstractNote = {Background: Non-specific binding of cellulases to lignin has been implicated as a major factor in the loss of cellulase activity during biomass conversion to sugars. It is believed that this binding may strongly impact process economics through loss of enzyme activities during hydrolysis and enzyme recycling scenarios. The current model suggests glycoside hydrolase activities are lost though non-specific/non-productive binding of carbohydrate-binding domains to lignin, limiting catalytic site access to the carbohydrate components of the cell wall. Results: In this study, we compared component enzyme affinities of a commercial Trichoderma reesei cellulase formulation, Cellic CTec2, towards extracted corn stover lignin using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and p-nitrophenyl substrate activities to monitor component binding, activity loss, and total protein binding. Protein binding was strongly affected by pH and ionic strength. β-D-glucosidases and xylanases, which do not have carbohydrate-binding modules (CBMs) and are basic proteins, demonstrated the strongest binding at low ionic strength, suggesting that CBMs are not the dominant factor in enzyme adsorption to lignin. Despite strong adsorption to insoluble lignin, β-D-glucosidase and xylanase activities remained high, with process yields decreasing only 4–15 % depending on lignin concentration. Conclusion: We propose that specific enzyme adsorption to lignin from a mixture of biomass-hydrolyzing enzymes is a competitive affinity where β-D-glucosidases and xylanases can displace CBM interactions with lignin. Process parameters, such as temperature, pH, and salt concentration influence the individual enzymes’ affinity for lignin, and both hydrophobic and electrostatic interactions are responsible for this binding phenomenon. Moreover, our results suggest that concern regarding loss of critical cell wall degrading enzymes to lignin adsorption may be unwarranted when complex enzyme mixtures are used to digest biomass.},
doi = {10.1186/s13068-015-0397-6},
journal = {Biotechnology for Biofuels},
number = 1,
volume = 8,
place = {Netherlands},
year = {Fri Dec 18 00:00:00 EST 2015},
month = {Fri Dec 18 00:00:00 EST 2015}
}
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Journal Article:
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Publisher's Version of Record
https://doi.org/10.1186/s13068-015-0397-6
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Cited by: 71 works
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Figures / Tables:

Fig. 1 Fig. 1: Current paradigm of carbohydrate binding modules (CBMs) having the highest affinity toward lignin. Here Cel7A with a CBM adsorbs to lignin and is sequestered away from the cellulose, rendering it ineffective. Other enzymes without CBMs do not adsorb to lignin
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Works referenced in this record:

Product inhibition of cellulases studied with 14C-labeled cellulose substrates
journal, January 2013

Access of cellulase to cellulose and lignin for poplar solids produced by leading pretreatment technologies
journal, May 2009

  • Kumar, Rajeev; Wyman, Charles E.
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The adsorption and enzyme activity profiles of specific Trichoderma reesei cellulase/xylanase components when hydrolyzing steam pretreated corn stover
journal, March 2012

Inhibition of the Trichoderma reesei cellulases by cellobiose is strongly dependent on the nature of the substrate : Product Inhibition in Cellulose Hydrolysis
journal, March 2004

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The impacts of deacetylation prior to dilute acid pretreatment on the bioethanol process
journal, January 2012

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  • Biotechnology for Biofuels, Vol. 5, Issue 1
  • DOI: 10.1186/1754-6834-5-8

BLGA protein solutions at high ionic strength: Vanishing attractive interactions and “frustrated” aggregation
journal, July 2002

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Enzymatic Saccharification of Lignocelluloses Should be Conducted at Elevated pH 5.2–6.2
journal, October 2012

Visualizing lignin coalescence and migration through maize cell walls following thermochemical pretreatment
journal, December 2008

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Structural features affecting biomass enzymatic digestibility
journal, June 2008

Evaluation of novel fungal cellulase preparations for ability to hydrolyze softwood substrates – evidence for the role of accessory enzymes
journal, July 2005

Adsorption of Trichoderma reesei CBH I and EG II and their catalytic domains on steam pretreated softwood and isolated lignin
journal, January 2004

Effect of lignin content on changes occurring in poplar cellulose ultrastructure during dilute acid pretreatment
journal, October 2014

Inhibitory effect of lignin during cellulose bioconversion: The effect of lignin chemistry on non-productive enzyme adsorption
journal, April 2013

Evaluating endoglucanase Cel7B-lignin interaction mechanisms and kinetics using quartz crystal microgravimetry: Interaction Kinetics of Cel7B and Lignin
journal, July 2015

  • Pfeiffer, Katherine A.; Sorek, Hagit; Roche, Christine M.
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The isolation, characterization and effect of lignin isolated from steam pretreated Douglas-fir on the enzymatic hydrolysis of cellulose
journal, March 2011

Role of Functional Groups in Lignin Inhibition of Enzymatic Hydrolysis of Cellulose to Glucose
journal, March 2008

Purification and characterization of a novel cellobiohydrolase (PdCel6A) from Penicillium decumbens JU-A10 for bioethanol production
journal, September 2011

The Billion-Ton Biofuels Vision
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Predicting Enzyme Adsorption to Lignin Films by Calculating Enzyme Surface Hydrophobicity
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Effect of enzyme supplementation at moderate cellulase loadings on initial glucose and xylose release from corn stover solids pretreated by leading technologies
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  • DOI: 10.1002/bit.22068
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    Works referencing / citing this record:

    The effect of alkali-soluble lignin on purified core cellulase and hemicellulase activities during hydrolysis of extractive ammonia-pretreated lignocellulosic biomass
    journal, June 2018

    • Zhou, Linchao; da Costa Sousa, Leonardo; Dale, Bruce E.
    • Royal Society Open Science, Vol. 5, Issue 6
    • DOI: 10.1098/rsos.171529

    Cellulases adsorb reversibly on biomass lignin: DJAJADI et al.
    journal, October 2018

    • Djajadi, Demi T.; Pihlajaniemi, Ville; Rahikainen, Jenni
    • Biotechnology and Bioengineering, Vol. 115, Issue 12
    • DOI: 10.1002/bit.26820

    Effect of cellulolytic enzyme binding on lignin isolated from alkali and acid pretreated switchgrass on enzymatic hydrolysis
    journal, November 2019

    Recovering cellulase and increasing glucose yield during lignocellulosic hydrolysis using lignin-MPEG with a sensitive pH response
    journal, January 2019

    • Cai, Cheng; Bao, Yu; Zhan, Xuejuan
    • Green Chemistry, Vol. 21, Issue 5
    • DOI: 10.1039/c8gc04059a

    Stimulation and inhibition of enzymatic hydrolysis by organosolv lignins as determined by zeta potential and hydrophobicity
    journal, June 2017

    Penicillium citrinum UFV1 β-glucosidases: purification, characterization, and application for biomass saccharification
    journal, August 2018

    • da Costa, Samara G.; Pereira, Olinto Liparini; Teixeira-Ferreira, André
    • Biotechnology for Biofuels, Vol. 11, Issue 1
    • DOI: 10.1186/s13068-018-1226-5
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      Figures / Tables found in this record:

      Fig. 1 (p. 3)figure
      Fig. 2 (p. 3)figure
      Fig. 3 (p. 4)figure
      Fig. 4 (p. 5)figure
      Table 1 (p. 5)table
      Fig. 5 (p. 6)figure
      Table 2 (p. 6)table
      Fig. 6 (p. 7)figure
      Table 3 (p. 7)table
      Fig. 7 (p. 8)figure
      Table 4 (p. 8)table
      Fig. 8 (p. 9)figure
      Fig. 9 (p. 10)figure
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