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The Use of Carbohydrate Binding Modules (CBMs) to Monitor Changes in Fragmentation and Cellulose Fiber Surface Morphology during Cellulase- and Swollenin-induced Deconstruction of Lignocellulosic Substrates*

  • Keith Gourlay
    Affiliations
    From the Forest Products Biotechnology/Bioenergy Group, Department of Wood Science, Faculty of Forestry, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada and
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  • Jinguang Hu
    Affiliations
    From the Forest Products Biotechnology/Bioenergy Group, Department of Wood Science, Faculty of Forestry, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada and
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  • Valdeir Arantes
    Affiliations
    From the Forest Products Biotechnology/Bioenergy Group, Department of Wood Science, Faculty of Forestry, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada and
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  • Merja Penttilä
    Affiliations
    the VTT Technical Research Centre of Finland, Metallimiehenkuja 2 (Espoo), FI-02044 VTT, Finland
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  • Jack N. Saddler
    Correspondence
    To whom correspondence should be addressed: Forest Products Biotechnology/ Bioenergy Group, Dept. of Wood Science, Faculty of Forestry, University of British Columbia, 2424 Main Mall, Vancouver, British Columbia V6T 1Z4, Canada. Tel.: 604-822-9741; E-mail: [email protected]
    Affiliations
    From the Forest Products Biotechnology/Bioenergy Group, Department of Wood Science, Faculty of Forestry, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada and
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  • Author Footnotes
    * This work was supported by the Natural Sciences and Engineering Council of Canada (NSERC) and the Bioconversion Network.
Open AccessPublished:December 19, 2014DOI:https://doi.org/10.1074/jbc.M114.627604
      Although the actions of many of the hydrolytic enzymes involved in cellulose hydrolysis are relatively well understood, the contributions that amorphogenesis-inducing proteins might contribute to cellulose deconstruction are still relatively undefined. Earlier work has shown that disruptive proteins, such as the non-hydrolytic non-oxidative protein Swollenin, can open up and disaggregate the less-ordered regions of lignocellulosic substrates. Within the cellulosic fraction, relatively disordered, amorphous regions known as dislocations are known to occur along the length of the fibers. It was postulated that Swollenin might act synergistically with hydrolytic enzymes to initiate biomass deconstruction within these dislocation regions. Carbohydrate binding modules (CBMs) that preferentially bind to cellulosic substructures were fluorescently labeled. They were imaged, using confocal microscopy, to assess the distribution of crystalline and amorphous cellulose at the fiber surface, as well as to track changes in surface morphology over the course of enzymatic hydrolysis and fiber fragmentation. Swollenin was shown to promote targeted disruption of the cellulosic structure at fiber dislocations.
      Background: Fiber fragmentation is thought to occur at dislocations, which are potential targets for the non-hydrolytic protein, Swollenin.
      Results: Changes in cellulose morphology within dislocations were assessed using fluorescent CBMs; Swollenin appeared to promote fragmentation at these sites.
      Conclusion: Swollenin targets and disrupts cellulose at fiber dislocations.
      Significance: Fragmentation is a key step in cellulose deconstruction and is enhanced by the actions of Swollenin.

      Introduction

      The effective enzymatic hydrolysis of lignocellulosic substrates to fermentable sugars, for subsequent conversion to fuels and chemicals, could help the world transition from a fossil fuel-based economy to a global society that is more reliant upon renewable resources. To try to achieve the high sugar concentrations that can be obtained from sugar cane or corn, high-cellulosic solids reaction conditions are desirable as they reduce water consumption, enable the use of smaller reaction vessels, reduce distillation/separation costs for the desired end product and, consequently, reduce the overall capital and operating costs of the bioconversion process (
      • Huang W.-D.
      • Zhang Y.-H.P.
      Analysis of biofuels production from sugar based on three criteria: thermodynamics, bioenergetics, and product separation.
      ). However, high solids hydrolysis slurries have inherent issues with ease of mixing, pumpability, and mass transfer limitations during enzyme diffusion and adsorption to the substrate (
      • Kristensen J.B.
      • Felby C.
      • Jørgensen H.
      Yield-determining factors in high-solids enzymatic hydrolysis of lignocellulose.
      ). Recent work has demonstrated that rapid fiber fragmentation at the very early stages of hydrolysis leads to a dramatic reduction in slurry viscosity, which alleviates some of the issues associated with high solids hydrolysis (
      • Kristensen J.B.
      • Felby C.
      • Jørgensen H.
      Yield-determining factors in high-solids enzymatic hydrolysis of lignocellulose.
      ). It has been suggested that fiber fragmentation is primarily induced by the endoglucanase family of enzymes (
      • Halliwell G.
      • Riaz M.
      The formation of short fibres from native cellulose by components of Trichoderma koningii cellulase.
      ,
      • Walker L.P.
      • Wilson D.B.
      • Irvin D.C.
      • McQuire C.
      • Price M.
      Fragmentation of cellulose by the major Thermomonospora fusca cellulases, Trichoderma reesei CBHI, and their mixtures.
      ), although other proteins present in the cellulolytic mixture, such as the more recently characterized oxidative enzymes (
      • Harris P.V.
      • Welner D.
      • McFarland K.C.
      • Re E.
      • Navarro Poulsen J.-C.
      • Brown K.
      • Salbo R.
      • Ding H.
      • Vlasenko E.
      • Merino S.
      • Xu F.
      • Cherry J.
      • Larsen S.
      • Lo Leggio L.
      Stimulation of lignocellulosic biomass hydrolysis by proteins of glycoside hydrolase family 61: structure and function of a large, enigmatic family.
      ,
      • Quinlan R.J.
      • Sweeney M.D.
      • Lo Leggio L.
      • Otten H.
      • Poulsen J.-C.N.
      • Johansen K.S.
      • Krogh K.B.R.M.
      • Jørgensen C.I.
      • Tovborg M.
      • Anthonsen A.
      • Tryfona T.
      • Walter C.P.
      • Dupree P.
      • Xu F.
      • Davies G.J.
      • Walton P.H.
      Insights into the oxidative degradation of cellulose by a copper metalloenzyme that exploits biomass components.
      ) and amorphogenesis-inducing proteins (
      • Arantes V.
      • Saddler J.N.
      Access to cellulose limits the efficiency of enzymatic hydrolysis: the role of amorphogenesis.
      ,
      • Gourlay K.
      • Hu J.
      • Arantes V.
      • Andberg M.
      • Saloheimo M.
      • Penttilä M.
      • Saddler J.
      Swollenin aids in the amorphogenesis step during the enzymatic hydrolysis of pretreated biomass.
      ,
      • Saloheimo M.
      • Paloheimo M.
      • Hakola S.
      • Pere J.
      • Swanson B.
      • Nyyssönen E.
      • Bhatia A.
      • Ward M.
      • Penttilä M.
      Swollenin, a Trichoderma reesei protein with sequence similarity to the plant expansins, exhibits disruption activity on cellulosic materials.
      ), have also been shown to contribute to the overall deconstruction process.
      A considerable amount of earlier work has defined “cellulase mixtures” as a protein mixture containing predominantly endo- and exoglucanases and perhaps β-glucosidase. However, it is now largely recognized that, to achieve effective lignocellulose deconstruction of more realistic biomass substrates, the enzyme mixture must also contain accessory enzymes/proteins such as hemicellulases, lytic polysaccharide monooxygenases, and non-hydrolytic/non-oxidative proteins that have been shown to facilitate biomass degradation through the opening up of the lignocellulosic matrix (
      • Harris P.V.
      • Welner D.
      • McFarland K.C.
      • Re E.
      • Navarro Poulsen J.-C.
      • Brown K.
      • Salbo R.
      • Ding H.
      • Vlasenko E.
      • Merino S.
      • Xu F.
      • Cherry J.
      • Larsen S.
      • Lo Leggio L.
      Stimulation of lignocellulosic biomass hydrolysis by proteins of glycoside hydrolase family 61: structure and function of a large, enigmatic family.
      ,
      • Arantes V.
      • Saddler J.N.
      Access to cellulose limits the efficiency of enzymatic hydrolysis: the role of amorphogenesis.
      ,
      • Hu J.
      • Arantes V.
      • Saddler J.N.
      The enhancement of enzymatic hydrolysis of lignocellulosic substrates by the addition of accessory enzymes such as xylanase: is it an additive or synergistic effect?.
      ,
      • Hu J.
      • Arantes V.
      • Pribowo A.
      • Gourlay K.
      • Saddler J.N.
      Substrate factors that influence the synergistic interaction of AA9 and cellulases during the enzymatic hydrolysis of biomass.
      ).
      The non-hydrolytic non-oxidative proteins (also known as “disruptive” or “amorphogenesis-inducing” proteins) are of interest (
      • Arantes V.
      • Saddler J.N.
      Access to cellulose limits the efficiency of enzymatic hydrolysis: the role of amorphogenesis.
      ,
      • Saloheimo M.
      • Paloheimo M.
      • Hakola S.
      • Pere J.
      • Swanson B.
      • Nyyssönen E.
      • Bhatia A.
      • Ward M.
      • Penttilä M.
      Swollenin, a Trichoderma reesei protein with sequence similarity to the plant expansins, exhibits disruption activity on cellulosic materials.
      ,
      • Jäger G.
      • Girfoglio M.
      • Dollo F.
      • Rinaldi R.
      • Bongard H.
      • Commandeur U.
      • Fischer R.
      • Spiess A.C.
      • Büchs J.
      How recombinant swollenin from Kluyveromyces lactis affects cellulosic substrates and accelerates their hydrolysis.
      ) due to their apparent ability to disrupt the structural matrix of biomass, consequently facilitating the subsequent depolymerization of the carbohydrate polymers by hydrolytic and oxidative enzymes (
      • Arantes V.
      • Saddler J.N.
      Access to cellulose limits the efficiency of enzymatic hydrolysis: the role of amorphogenesis.
      ). These disruptive proteins include examples from plants (Expansins) (
      • Cosgrove D.J.
      Loosening of plant cell walls by expansins.
      ), bacteria (Expansin-like proteins (
      • Kerff F.
      • Amoroso A.
      • Herman R.
      • Sauvage E.
      • Petrella S.
      • Filée P.
      • Charlier P.
      • Joris B.
      • Tabuchi A.
      • Nikolaidis N.
      • Cosgrove D.J.
      Crystal structure and activity of Bacillus subtilis YoaJ (EXLX1), a bacterial expansin that promotes root colonization.
      ,
      • Lee H.J.
      • Lee S.
      • Ko H.-J.
      • Kim K.H.
      • Choi I.-G.
      An expansin-like protein from Hahella chejuensis binds cellulose and enhances cellulase activity.
      ) and some carbohydrate binding modules (CBMs)
      The abbreviations used are: CBM
      carbohydrate binding module
      FQA
      fiber quality analyzer
      AMCA-X
      (6-((7-amino-4-methylcoumarin-3-acetyl)amino)hexanoic acid
      DMSO
      dimethyl sulfoxide.
      (
      • Din N.
      • Gilkes N.R.
      • Tekant B.
      • Miller R.C.
      • Warren R.A.J.
      • Kilburn D.G.
      Non-hydrolytic disruption of cellulose fibres by the binding domain of a bacterial cellulase.
      )), and fungi (Swollenin (
      • Gourlay K.
      • Hu J.
      • Arantes V.
      • Andberg M.
      • Saloheimo M.
      • Penttilä M.
      • Saddler J.
      Swollenin aids in the amorphogenesis step during the enzymatic hydrolysis of pretreated biomass.
      ,
      • Saloheimo M.
      • Paloheimo M.
      • Hakola S.
      • Pere J.
      • Swanson B.
      • Nyyssönen E.
      • Bhatia A.
      • Ward M.
      • Penttilä M.
      Swollenin, a Trichoderma reesei protein with sequence similarity to the plant expansins, exhibits disruption activity on cellulosic materials.
      ,
      • Jäger G.
      • Girfoglio M.
      • Dollo F.
      • Rinaldi R.
      • Bongard H.
      • Commandeur U.
      • Fischer R.
      • Spiess A.C.
      • Büchs J.
      How recombinant swollenin from Kluyveromyces lactis affects cellulosic substrates and accelerates their hydrolysis.
      ), Loosenin (
      • Quiroz-Castañeda R.E.
      • Martínez-Anaya C.
      • Cuervo-Soto L.I.
      • Segovia L.
      • Folch-Mallol J.L.
      Loosenin, a novel protein with cellulose-disrupting activity from Bjerkandera adusta.
      ), and CBMs (
      • Lee I.
      • Evans B.R.
      • Woodward J.
      The mechanism of cellulase action on cotton fibers: evidence from atomic force microscopy.
      )). These proteins have also been shown to promote a variety of disruptive effects on cellulosic biomass, including filter paper dispersion, crystallinity reduction, particle size reduction, swelling of cotton fibers, and roughening of cotton fiber surface (
      • Arantes V.
      • Saddler J.N.
      Access to cellulose limits the efficiency of enzymatic hydrolysis: the role of amorphogenesis.
      ).
      Swollenin, in particular, has been shown to effectively swell cotton fibers (
      • Saloheimo M.
      • Paloheimo M.
      • Hakola S.
      • Pere J.
      • Swanson B.
      • Nyyssönen E.
      • Bhatia A.
      • Ward M.
      • Penttilä M.
      Swollenin, a Trichoderma reesei protein with sequence similarity to the plant expansins, exhibits disruption activity on cellulosic materials.
      ), disperse filter paper squares (
      • Jäger G.
      • Girfoglio M.
      • Dollo F.
      • Rinaldi R.
      • Bongard H.
      • Commandeur U.
      • Fischer R.
      • Spiess A.C.
      • Büchs J.
      How recombinant swollenin from Kluyveromyces lactis affects cellulosic substrates and accelerates their hydrolysis.
      ), roughen filter paper fibers (
      • Jäger G.
      • Girfoglio M.
      • Dollo F.
      • Rinaldi R.
      • Bongard H.
      • Commandeur U.
      • Fischer R.
      • Spiess A.C.
      • Büchs J.
      How recombinant swollenin from Kluyveromyces lactis affects cellulosic substrates and accelerates their hydrolysis.
      ), reduce the particle size of a number of model cellulosic substrates (
      • Jäger G.
      • Girfoglio M.
      • Dollo F.
      • Rinaldi R.
      • Bongard H.
      • Commandeur U.
      • Fischer R.
      • Spiess A.C.
      • Büchs J.
      How recombinant swollenin from Kluyveromyces lactis affects cellulosic substrates and accelerates their hydrolysis.
      ), enhance the accessibility of cotton fibers (
      • Gourlay K.
      • Arantes V.
      • Saddler J.N.
      Use of substructure-specific carbohydrate binding modules to track changes in cellulose accessibility and surface morphology during the amorphogenesis step of enzymatic hydrolysis.
      ), and enhance hydrolysis (
      • Gourlay K.
      • Hu J.
      • Arantes V.
      • Andberg M.
      • Saloheimo M.
      • Penttilä M.
      • Saddler J.
      Swollenin aids in the amorphogenesis step during the enzymatic hydrolysis of pretreated biomass.
      ,
      • Kang K.
      • Wang S.
      • Lai G.
      • Liu G.
      • Xing M.
      Characterization of a novel swollenin from Penicillium oxalicum in facilitating enzymatic saccharification of cellulose.
      ). Recent work has shown that Swollenin targets the less-ordered regions of the cellulose, rather than directly disrupting the more crystalline regions of the substrate (
      • Gourlay K.
      • Hu J.
      • Arantes V.
      • Andberg M.
      • Saloheimo M.
      • Penttilä M.
      • Saddler J.
      Swollenin aids in the amorphogenesis step during the enzymatic hydrolysis of pretreated biomass.
      ,
      • Gourlay K.
      • Arantes V.
      • Saddler J.N.
      Use of substructure-specific carbohydrate binding modules to track changes in cellulose accessibility and surface morphology during the amorphogenesis step of enzymatic hydrolysis.
      ), and enhances the solubilization of the hemicellulosic fraction of steam-pretreated corn stover when it synergistically interacted with family 10 and 11 xylanases, promoting a 3-fold increase in xylose released by these hydrolases (
      • Gourlay K.
      • Hu J.
      • Arantes V.
      • Andberg M.
      • Saloheimo M.
      • Penttilä M.
      • Saddler J.
      Swollenin aids in the amorphogenesis step during the enzymatic hydrolysis of pretreated biomass.
      ). It appears that, although Swollenin does to some extent enhance the disruption of well structured components of cellulosic substrates, its primary role is facilitating the disruption of the less-ordered regions of pretreated lignocellulosic substrates (
      • Gourlay K.
      • Hu J.
      • Arantes V.
      • Andberg M.
      • Saloheimo M.
      • Penttilä M.
      • Saddler J.
      Swollenin aids in the amorphogenesis step during the enzymatic hydrolysis of pretreated biomass.
      ,
      • Gourlay K.
      • Arantes V.
      • Saddler J.N.
      Use of substructure-specific carbohydrate binding modules to track changes in cellulose accessibility and surface morphology during the amorphogenesis step of enzymatic hydrolysis.
      ).
      It is possible that Swollenin targets the dislocations within lignocellulosic fibers, as these regions have been shown to be enriched in disordered/amorphous cellulose (
      • Thygesen L.G.
      • Hidayat B.J.
      • Johansen K.S.
      • Felby C.
      Role of supramolecular cellulose structures in enzymatic hydrolysis of plant cell walls.
      ). Fiber dislocations (also called kinks, micro-compressions, irregularities, and slip planes (
      • Nyholm K
      • Ander P.
      • Bardage S.
      • Daniel G.
      Dislocations in pulp fibres — their origin, characteristics and importance — a review.
      )) have been observed in a range of plant species, including softwood, hemp, flax, and wheat (
      • Thygesen L.G.
      • Hidayat B.J.
      • Johansen K.S.
      • Felby C.
      Role of supramolecular cellulose structures in enzymatic hydrolysis of plant cell walls.
      ,
      • Thygesen L.G.
      • Eder M.
      • Burgert I.
      Dislocations in single hemp fibres: investigations into the relationship of structural distortions and tensile properties at the cell wall level.
      ,
      • Baley C.
      Influence of kink bands on the tensile strength of flax fibers.
      ). These dislocations are present in untreated biomass fibers and can also be induced during processing steps (
      • Eder M.
      • Terziev N.
      • Daniel G.
      • Burgert I.
      The effect of (induced) dislocations on the tensile properties of individual Norway spruce fibres.
      ). Typically, the fiber structure within the dislocations contains surface features perpendicular to the direction of the microfibrils. Dislocations have also been shown to contain more amorphous cellulose with less order than the surrounding fiber, likely due to the distortion of the crystalline cellulose microfibrils (
      • Ander P.
      • Hildén L.
      • Daniel G.
      Cleavage of softwood kraft pulp fibres by HCl and cellulases.
      ), although other work has suggested that microfibrils continue through these dislocations (
      • Hidayat B.J.
      • Felby C.
      • Johansen K.S.
      • Thygesen L.G.
      Cellulose is not just cellulose: a review of dislocations as reactive sites in the enzymatic hydrolysis of cellulose microfibrils.
      ). Earlier work has suggested that these dislocations within the fibers make up weak points that are rapidly hydrolyzed by cellulases (
      • Thygesen L.G.
      • Hidayat B.J.
      • Johansen K.S.
      • Felby C.
      Role of supramolecular cellulose structures in enzymatic hydrolysis of plant cell walls.
      ).
      In the work described here, we tried to better elucidate the potential role that Swollenin might play in fiber fragmentation by assessing various macroscopic fiber properties over the course of enzymatic hydrolysis of dissolving pulp and a range of pretreated substrates. Fiber quality analysis (FQA) was used to quantify the fiber length/particle size of cellulosic fibers, although the “settleability” of the pulp (i.e. how densely the pulp fibers compact after being allowed to settle in solution) was used as a secondary indicator of fiber length, as shorter fibers enable denser settling than longer ones.
      To try to quantify possible changes at the microfibril level, a CBM adsorption technique (
      • Gourlay K.
      • Arantes V.
      • Saddler J.N.
      Use of substructure-specific carbohydrate binding modules to track changes in cellulose accessibility and surface morphology during the amorphogenesis step of enzymatic hydrolysis.
      ) was also used. As described earlier by Boraston et al. (
      • Boraston A.B.
      • Bolam D.N.
      • Gilbert H.J.
      • Davies G.J.
      Carbohydrate-binding modules: fine-tuning polysaccharide recognition.
      ), CBMs can be broadly categorized into the three types: Type A, which binds to well ordered (crystalline) substrates via a planar hydrophobic surface composed of tryptophan and tyrosine residues; Type B, which binds via a binding cleft to individual carbohydrate chains; and Type C, which binds via a binding pocket to chain ends and smaller carbohydrate molecules (
      • Boraston A.B.
      • Bolam D.N.
      • Gilbert H.J.
      • Davies G.J.
      Carbohydrate-binding modules: fine-tuning polysaccharide recognition.
      ). The CBM adsorption technique used in the work described here compares the adsorption of a Type A and a Type B CBM with cellulose where the Type A CBM has been shown to preferentially adsorb to the more crystalline regions of the cellulose surface (
      • McLean B.W.
      • Boraston A.B.
      • Brouwer D.
      • Sanaie N.
      • Fyfe C.A.
      • Warren R.A.J.
      • Kilburn D.G.
      • Haynes C.A.
      Carbohydrate-binding modules recognize fine substructures of cellulose.
      ), whereas the Type B CBM preferentially binds to the more amorphous regions (
      • Najmudin S.
      • Guerreiro C.I.P.D.
      • Carvalho A.L.
      • Prates J.A.M.
      • Correia M.A.S.
      • Alves V.D.
      • Ferreira L.M.A.
      • Romão M.J.
      • Gilbert H.J.
      • Bolam D.N.
      • Fontes C.M.G.A.
      Xyloglucan is recognized by carbohydrate-binding modules that interact with β-glucan chains.
      ). Several previous studies have used CBMs with different specificities for crystalline and amorphous regions of the cellulose to try to reveal the differences in surface morphology between the surrounding fiber and the cellulose within fiber dislocations (
      • Thygesen L.G.
      • Hidayat B.J.
      • Johansen K.S.
      • Felby C.
      Role of supramolecular cellulose structures in enzymatic hydrolysis of plant cell walls.
      ,
      • Hidayat B.J.
      • Felby C.
      • Johansen K.S.
      • Thygesen L.G.
      Cellulose is not just cellulose: a review of dislocations as reactive sites in the enzymatic hydrolysis of cellulose microfibrils.
      ,
      • Filonova L.
      • Kallas A.M.
      • Greffe L.
      • Johansson G.
      • Teeri T.T.
      • Daniel G.
      Analysis of the surfaces of wood tissues and pulp fibers using carbohydrate-binding modules specific for crystalline cellulose and mannan.
      ,
      • Kawakubo T.
      • Karita S.
      • Araki Y.
      • Watanabe S.
      • Oyadomari M.
      • Takada R.
      • Tanaka F.
      • Abe K.
      • Watanabe T.
      • Honda Y.
      • Watanabe T.
      Analysis of exposed cellulose surfaces in pretreated wood biomass using carbohydrate-binding module (CBM)-cyan fluorescent protein (CFP).
      ,
      • Ding S.-Y.
      • Xu Q.
      • Ali M.K.
      • Baker J.O.
      • Bayer E.A.
      • Barak Y.
      • Lamed R.
      • Sugiyama J.
      • Rumbles G.
      • Himmel M.E.
      Versatile derivatives of carbohydrate-binding modules for imaging of complex carbohydrates approaching the molecular level of resolution.
      ). However, no clear consensus was reached, with some Type A CBMs binding to dislocations (
      • Thygesen L.G.
      • Hidayat B.J.
      • Johansen K.S.
      • Felby C.
      Role of supramolecular cellulose structures in enzymatic hydrolysis of plant cell walls.
      ,
      • Filonova L.
      • Kallas A.M.
      • Greffe L.
      • Johansson G.
      • Teeri T.T.
      • Daniel G.
      Analysis of the surfaces of wood tissues and pulp fibers using carbohydrate-binding modules specific for crystalline cellulose and mannan.
      ), whereas other Type A CBMs did not localize to these dislocations (
      • Kawakubo T.
      • Karita S.
      • Araki Y.
      • Watanabe S.
      • Oyadomari M.
      • Takada R.
      • Tanaka F.
      • Abe K.
      • Watanabe T.
      • Honda Y.
      • Watanabe T.
      Analysis of exposed cellulose surfaces in pretreated wood biomass using carbohydrate-binding module (CBM)-cyan fluorescent protein (CFP).
      ,
      • Ding S.-Y.
      • Xu Q.
      • Ali M.K.
      • Baker J.O.
      • Bayer E.A.
      • Barak Y.
      • Lamed R.
      • Sugiyama J.
      • Rumbles G.
      • Himmel M.E.
      Versatile derivatives of carbohydrate-binding modules for imaging of complex carbohydrates approaching the molecular level of resolution.
      ). However, all of the Type B CBMs tested to date do appear to localize to fiber dislocations (
      • Filonova L.
      • Kallas A.M.
      • Greffe L.
      • Johansson G.
      • Teeri T.T.
      • Daniel G.
      Analysis of the surfaces of wood tissues and pulp fibers using carbohydrate-binding modules specific for crystalline cellulose and mannan.
      ,
      • Kawakubo T.
      • Karita S.
      • Araki Y.
      • Watanabe S.
      • Oyadomari M.
      • Takada R.
      • Tanaka F.
      • Abe K.
      • Watanabe T.
      • Honda Y.
      • Watanabe T.
      Analysis of exposed cellulose surfaces in pretreated wood biomass using carbohydrate-binding module (CBM)-cyan fluorescent protein (CFP).
      ,
      • Ding S.-Y.
      • Xu Q.
      • Ali M.K.
      • Baker J.O.
      • Bayer E.A.
      • Barak Y.
      • Lamed R.
      • Sugiyama J.
      • Rumbles G.
      • Himmel M.E.
      Versatile derivatives of carbohydrate-binding modules for imaging of complex carbohydrates approaching the molecular level of resolution.
      ).
      As described in more detail below, by monitoring fiber dimensions and assessing the binding profile of the fluorescently tagged CBMs on the fiber surface, we were able to show that Swollenin targeted the amorphous regions within the dislocations of cellulose fibers, promoting fiber fragmentation at these dislocations.

      Acknowledgments

      We thank Novozymes, Bagsværd, Denmark for supplying enzymes and Martina Andberg and Markku Saloheimo of VTT for Swollenin.

      REFERENCES

        • Huang W.-D.
        • Zhang Y.-H.P.
        Analysis of biofuels production from sugar based on three criteria: thermodynamics, bioenergetics, and product separation.
        Energy Environ. Sci. 2011; 4: 784-792
        • Kristensen J.B.
        • Felby C.
        • Jørgensen H.
        Yield-determining factors in high-solids enzymatic hydrolysis of lignocellulose.
        Biotechnol. Biofuels. 2009; 2: 11
        • Halliwell G.
        • Riaz M.
        The formation of short fibres from native cellulose by components of Trichoderma koningii cellulase.
        Biochem J. 1970; 116: 35-42
        • Walker L.P.
        • Wilson D.B.
        • Irvin D.C.
        • McQuire C.
        • Price M.
        Fragmentation of cellulose by the major Thermomonospora fusca cellulases, Trichoderma reesei CBHI, and their mixtures.
        Biotechnol. Bioeng. 1992; 40: 1019-1026
        • Harris P.V.
        • Welner D.
        • McFarland K.C.
        • Re E.
        • Navarro Poulsen J.-C.
        • Brown K.
        • Salbo R.
        • Ding H.
        • Vlasenko E.
        • Merino S.
        • Xu F.
        • Cherry J.
        • Larsen S.
        • Lo Leggio L.
        Stimulation of lignocellulosic biomass hydrolysis by proteins of glycoside hydrolase family 61: structure and function of a large, enigmatic family.
        Biochemistry (Mosc.). 2010; 49: 3305-3316
        • Quinlan R.J.
        • Sweeney M.D.
        • Lo Leggio L.
        • Otten H.
        • Poulsen J.-C.N.
        • Johansen K.S.
        • Krogh K.B.R.M.
        • Jørgensen C.I.
        • Tovborg M.
        • Anthonsen A.
        • Tryfona T.
        • Walter C.P.
        • Dupree P.
        • Xu F.
        • Davies G.J.
        • Walton P.H.
        Insights into the oxidative degradation of cellulose by a copper metalloenzyme that exploits biomass components.
        Proc. Natl. Acad. Sci. 2011; 108: 15079-15084
        • Arantes V.
        • Saddler J.N.
        Access to cellulose limits the efficiency of enzymatic hydrolysis: the role of amorphogenesis.
        Biotechnol. Biofuels. 2010; 3: 4
        • Gourlay K.
        • Hu J.
        • Arantes V.
        • Andberg M.
        • Saloheimo M.
        • Penttilä M.
        • Saddler J.
        Swollenin aids in the amorphogenesis step during the enzymatic hydrolysis of pretreated biomass.
        Bioresour. Technol. 2013; 142: 498-503
        • Saloheimo M.
        • Paloheimo M.
        • Hakola S.
        • Pere J.
        • Swanson B.
        • Nyyssönen E.
        • Bhatia A.
        • Ward M.
        • Penttilä M.
        Swollenin, a Trichoderma reesei protein with sequence similarity to the plant expansins, exhibits disruption activity on cellulosic materials.
        Eur. J. Biochem. 2002; 269: 4202-4211
        • Hu J.
        • Arantes V.
        • Saddler J.N.
        The enhancement of enzymatic hydrolysis of lignocellulosic substrates by the addition of accessory enzymes such as xylanase: is it an additive or synergistic effect?.
        Biotechnol. Biofuels. 2011; 4: 36
        • Hu J.
        • Arantes V.
        • Pribowo A.
        • Gourlay K.
        • Saddler J.N.
        Substrate factors that influence the synergistic interaction of AA9 and cellulases during the enzymatic hydrolysis of biomass.
        Energy Environ. Sci. 2014; 7: 2308-2315
        • Jäger G.
        • Girfoglio M.
        • Dollo F.
        • Rinaldi R.
        • Bongard H.
        • Commandeur U.
        • Fischer R.
        • Spiess A.C.
        • Büchs J.
        How recombinant swollenin from Kluyveromyces lactis affects cellulosic substrates and accelerates their hydrolysis.
        Biotechnol. Biofuels. 2011; 4: 33
        • Cosgrove D.J.
        Loosening of plant cell walls by expansins.
        Nature. 2000; 407: 321-326
        • Kerff F.
        • Amoroso A.
        • Herman R.
        • Sauvage E.
        • Petrella S.
        • Filée P.
        • Charlier P.
        • Joris B.
        • Tabuchi A.
        • Nikolaidis N.
        • Cosgrove D.J.
        Crystal structure and activity of Bacillus subtilis YoaJ (EXLX1), a bacterial expansin that promotes root colonization.
        Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 16876-16881
        • Lee H.J.
        • Lee S.
        • Ko H.-J.
        • Kim K.H.
        • Choi I.-G.
        An expansin-like protein from Hahella chejuensis binds cellulose and enhances cellulase activity.
        Mol. Cells. 2010; 29: 379-385
        • Din N.
        • Gilkes N.R.
        • Tekant B.
        • Miller R.C.
        • Warren R.A.J.
        • Kilburn D.G.
        Non-hydrolytic disruption of cellulose fibres by the binding domain of a bacterial cellulase.
        Nat. Biotechnol. 1991; 9: 1096-1099
        • Quiroz-Castañeda R.E.
        • Martínez-Anaya C.
        • Cuervo-Soto L.I.
        • Segovia L.
        • Folch-Mallol J.L.
        Loosenin, a novel protein with cellulose-disrupting activity from Bjerkandera adusta.
        Microb. Cell Fact. 2011; 10: 8
        • Lee I.
        • Evans B.R.
        • Woodward J.
        The mechanism of cellulase action on cotton fibers: evidence from atomic force microscopy.
        Ultramicroscopy. 2000; 82: 213-221
        • Gourlay K.
        • Arantes V.
        • Saddler J.N.
        Use of substructure-specific carbohydrate binding modules to track changes in cellulose accessibility and surface morphology during the amorphogenesis step of enzymatic hydrolysis.
        Biotechnol. Biofuels. 2012; 5: 51
        • Kang K.
        • Wang S.
        • Lai G.
        • Liu G.
        • Xing M.
        Characterization of a novel swollenin from Penicillium oxalicum in facilitating enzymatic saccharification of cellulose.
        BMC Biotechnol. 2013; 13: 42
        • Thygesen L.G.
        • Hidayat B.J.
        • Johansen K.S.
        • Felby C.
        Role of supramolecular cellulose structures in enzymatic hydrolysis of plant cell walls.
        J. Ind. Microbiol. Biotechnol. 2011; 38: 975-983
        • Nyholm K
        • Ander P.
        • Bardage S.
        • Daniel G.
        Dislocations in pulp fibres — their origin, characteristics and importance — a review.
        Nord. Pulp Pap. Res. J. 2001; 16: 376-384
        • Thygesen L.G.
        • Eder M.
        • Burgert I.
        Dislocations in single hemp fibres: investigations into the relationship of structural distortions and tensile properties at the cell wall level.
        J. Mater. Sci. 2007; 42: 558-564
        • Baley C.
        Influence of kink bands on the tensile strength of flax fibers.
        J. Mater. Sci. 2004; 39: 331-334
        • Eder M.
        • Terziev N.
        • Daniel G.
        • Burgert I.
        The effect of (induced) dislocations on the tensile properties of individual Norway spruce fibres.
        Holzforschung. 2007; 62: 77-81
        • Ander P.
        • Hildén L.
        • Daniel G.
        Cleavage of softwood kraft pulp fibres by HCl and cellulases.
        BioResources. 2008; 3: 477-490
        • Hidayat B.J.
        • Felby C.
        • Johansen K.S.
        • Thygesen L.G.
        Cellulose is not just cellulose: a review of dislocations as reactive sites in the enzymatic hydrolysis of cellulose microfibrils.
        Cellulose. 2012; 19: 1481-1493
        • Boraston A.B.
        • Bolam D.N.
        • Gilbert H.J.
        • Davies G.J.
        Carbohydrate-binding modules: fine-tuning polysaccharide recognition.
        Biochem. J. 2004; 382: 769-781
        • McLean B.W.
        • Boraston A.B.
        • Brouwer D.
        • Sanaie N.
        • Fyfe C.A.
        • Warren R.A.J.
        • Kilburn D.G.
        • Haynes C.A.
        Carbohydrate-binding modules recognize fine substructures of cellulose.
        J. Biol. Chem. 2002; 277: 50245-50254
        • Najmudin S.
        • Guerreiro C.I.P.D.
        • Carvalho A.L.
        • Prates J.A.M.
        • Correia M.A.S.
        • Alves V.D.
        • Ferreira L.M.A.
        • Romão M.J.
        • Gilbert H.J.
        • Bolam D.N.
        • Fontes C.M.G.A.
        Xyloglucan is recognized by carbohydrate-binding modules that interact with β-glucan chains.
        J. Biol. Chem. 2006; 281: 8815-8828
        • Filonova L.
        • Kallas A.M.
        • Greffe L.
        • Johansson G.
        • Teeri T.T.
        • Daniel G.
        Analysis of the surfaces of wood tissues and pulp fibers using carbohydrate-binding modules specific for crystalline cellulose and mannan.
        Biomacromolecules. 2007; 8: 91-97
        • Kawakubo T.
        • Karita S.
        • Araki Y.
        • Watanabe S.
        • Oyadomari M.
        • Takada R.
        • Tanaka F.
        • Abe K.
        • Watanabe T.
        • Honda Y.
        • Watanabe T.
        Analysis of exposed cellulose surfaces in pretreated wood biomass using carbohydrate-binding module (CBM)-cyan fluorescent protein (CFP).
        Biotechnol. Bioeng. 2010; 105: 499-508
        • Ding S.-Y.
        • Xu Q.
        • Ali M.K.
        • Baker J.O.
        • Bayer E.A.
        • Barak Y.
        • Lamed R.
        • Sugiyama J.
        • Rumbles G.
        • Himmel M.E.
        Versatile derivatives of carbohydrate-binding modules for imaging of complex carbohydrates approaching the molecular level of resolution.
        BioTechniques. 2006; 41 (438–440, passim): 435-436
        • Karlsson J.
        • Saloheimo M.
        • Siika-Aho M.
        • Tenkanen M.
        • Penttilä M.
        • Tjerneld F.
        Homologous expression and characterization of Cel61A (EG IV) of Trichoderma reesei.
        Eur. J. Biochem. 2001; 268: 6498-6507
        • Ong E.
        • Gilkes N.R.
        • Miller Jr., R.C.
        • Warren R.A.
        • Kilburn D.G.
        The cellulose-binding domain (CBDCex) of an exoglucanase from Cellulomonas fimi: production in Escherichia coli and characterization of the polypeptide.
        Biotechnol. Bioeng. 1993; 42: 401-409
        • Tu M.
        • Chandra R.P.
        • Saddler J.N.
        Recycling cellulases during the hydrolysis of steam exploded and ethanol pretreated lodgepole pine.
        Biotechnol. Prog. 2007; 23: 1130-1137
        • Arantes V.
        • Saddler J.N.
        Cellulose accessibility limits the effectiveness of minimum cellulase loading on the efficient hydrolysis of pretreated lignocellulosic substrates.
        Biotechnol. Biofuels. 2011; 4: 3
        • Berlin A.
        • Maximenko V.
        • Bura R.
        • Kang K.-Y.
        • Gilkes N.
        • Saddler J.
        A rapid microassay to evaluate enzymatic hydrolysis of lignocellulosic substrates.
        Biotechnol. Bioeng. 2006; 93: 880-886
        • Berezin I.V.
        • Rabinovich M.L.
        • Sinitsyn A.P.
        [Applicability of quantitative kinetic spectrophotometric method for glucose determination].
        Biokhimiia. 1977; 42: 1631-1636
        • Starcher B.
        A ninhydrin-based assay to quantitate the total protein content of tissue samples.
        Anal. Biochem. 2001; 292: 125-129
        • Nishiyama Y.
        • Langan P.
        • Chanzy H.
        Crystal structure and hydrogen-bonding system in cellulose Iβ from synchrotron X-ray and neutron fiber diffraction.
        J. Am. Chem. Soc. 2002; 124: 9074-9082
        • Clarke K.
        • Li X.
        • Li K.
        The mechanism of fiber cutting during enzymatic hydrolysis of wood biomass.
        Biomass Bioenergy. 2011; 35: 3943-3950
        • Tozzi E.J.
        • McCarthy M.J.
        • Lavenson D.M.
        • Cardona M.
        • Powell L.R.
        • Karuna N.
        • McCarthy M.J.
        • Jeoh T.
        Effect of fiber structure on yield stress during enzymatic conversion of cellulose.
        AIChE J. 2014; 60: 1582-1590
        • Del Rio L.F.
        • Chandra R.P.
        • Saddler J.N.
        Fibre size does not appear to influence the ease of enzymatic hydrolysis of organosolv-pretreated softwoods.
        Bioresour. Technol. 2012; 107: 235-242
        • Gao S.
        • You C.
        • Renneckar S.
        • Bao J.
        • Zhang Y.-H.P.
        New insights into enzymatic hydrolysis of heterogeneous cellulose by using carbohydrate-binding module 3 containing GFP and carbohydrate-binding module 17 containing CFP.
        Biotechnol. Biofuels. 2014; 7: 24
        • Arantes V.
        • Gourlay K.
        • Saddler J.N.
        The enzymatic hydrolysis of pretreated pulp fibers predominantly involves “peeling/erosion” modes of action.
        Biotechnol. Biofuels. 2014; 7: 87
        • Du R.
        • Huang R.
        • Su R.
        • Zhang M.
        • Wang M.
        • Yang J.
        • Qi W.
        • He Z.
        Enzymatic hydrolysis of lignocellulose: SEC-MALLS analysis and reaction mechanism.
        RSC Adv. 2013; 3: 1871
        • Fox J.M.
        • Jess P.
        • Jambusaria R.B.
        • Moo G.M.
        • Liphardt J.
        • Clark D.S.
        • Blanch H.W.
        A single-molecule analysis reveals morphological targets for cellulase synergy.
        Nat. Chem. Biol. 2013; 9: 356-361
        • Hildén L.
        • Daniel G.
        • Johansson G.
        Use of a fluorescence labelled, carbohydrate-binding module from Phanerochaete chrysosporium Cel7D for studying wood cell wall ultrastructure.
        Biotechnol. Lett. 2003; 25: 553-558