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Use of substructure-specific carbohydrate binding modules to track changes in cellulose accessibility… Gourlay, Keith; Arantes, Valdeir; Saddler, Jack N Jul 24, 2012

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METHODOLOGY Open AccessUse of substructure-specific carbohydrate bindingmodules to track changes in cellulose accessibilityand surface morphology during theamorphogenesis step of enzymatic hydrolysisKeith Gourlay, Valdeir Arantes and Jack N Saddler*AbstractBackground: Cellulose amorphogenesis, described as the non-hydrolytic “opening up” or disruption of a cellulosicsubstrate, is becoming increasingly recognized as one of the key steps in the enzymatic deconstruction of cellulosicbiomass when used as a feedstock for fuels and chemicals production. Although this process is thought to play amajor role in facilitating hydrolysis, the lack of quantitative techniques capable of accurately describing themolecular-level changes occurring in the substrate during amorphogenesis has hindered our understanding of thisprocess.Results: In this work, techniques for measuring changes in cellulose accessibility are reviewed and a newquantitative assay method is described. Carbohydrate binding modules (CBMs) with specific affinities for crystalline(CBM2a) or amorphous (CBM44) cellulose were used to track specific changes in the surface morphology of cottonfibres during amorphogenesis. The extents of phosphoric acid-induced and Swollenin-induced changes to celluloseaccessibility were successfully quantified using this technique.Conclusions: The adsorption of substructure-specific CBMs can be used to accurately quantify the extent ofchanges to cellulose accessibility induced by non-hydrolytic disruptive proteins. The technique provided a quick,accurate and quantitative measure of the accessibility of cellulosic substrates. Expanding the range of CBMs usedfor adsorption studies to include those specific for such compounds as xylan or mannan should also allow for theaccurate quantitative tracking of the accessibility of these and other polymers within the lignocellulosic biomassmatrix.Keywords: Amorphogenesis, Cellulose Accessibility, Swollenin, Cellulose Disruption, Carbohydrate Binding Modules,Enzymatic Hydrolysis, BiofuelsBackgroundOver the past 50 years a considerable amount of re-search has been dedicated to determining the roles ofthe hydrolytic proteins involved in the solubilisation anddepolymerisation of the carbohydrates within the ligno-cellulosic biomass matrix [1-3]. In its native state, cel-lulose chains typically exist in tightly packed bundlesencased within a complex sheath of hemicellulosesand lignin [4-6]. In order for cellulases to hydrolyzethe glycosidic linkages within these chains, they mustfirst be able to diffuse into this dense, heterogeneousmatrix and access the cellulose [7].It is becoming increasingly apparent that enzymaticdeconstruction of cellulose occurs through two distinctsteps. First, an initial disruption of the substrate, the so-called “cellulose amorphogenesis” phase, is thought tobe mediated at least in part by non-hydrolytic disruptiveproteins [8]. This step is required to enhance the acces-sibility of the cellulose to the cellulase enzyme mixture[8] while, in the subsequent step, the cellulase enzymesdiffuse into and hydrolyze the cellulose. Although thebasic functions and mechanisms of the major hydrolytic* Correspondence: jack.saddler@ubc.caForest Products Biotechnology/Bioenergy Group, Department of WoodScience, Faculty of Forestry, University of British Columbia, 2424 Main Mall,Vancouver, BC V6T 1Z4, Canada© 2012 Gourlay et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the CreativeCommons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly cited.Gourlay et al. Biotechnology for Biofuels 2012, 5:51http://www.biotechnologyforbiofuels.com/content/5/1/51enzymes have been studied extensively, little is knownabout the role of the non-hydrolytic proteins that havebeen suggested to be involved in disrupting the substrateprior to hydrolysis [8]. By developing a better under-standing of the role that non-hydrolytic proteins play inthe disruption of lignocellulosic materials, it should bepossible to design more efficient enzyme preparations,thereby bringing us one step closer to achieving an ef-fective, enzyme/sugar-based biorefinery [5,9-11].Protein-induced amorphogenesisSeveral cellulolytic organisms have been shown to pro-duce non-hydrolytic proteins capable of disrupting cellu-losic and lignocellulosic substrates (Reviewed in [8]).While the exact mechanisms by which these proteins dis-rupt the substrate have yet to be fully resolved, qualitativeand semi-quantitative observations have suggested thatthis disruption can be manifested as a delamination, fibril-lation, swelling, loosening, roughening, pitting, weakening,or decrystallization of cellulosic and lignocellulosic sub-strates. The term “amorphogenesis” has been suggested asa way of describing any combination of these phenomenainduced by non-hydrolytic proteins [8].In previous work we [12,13] and other workers [14] havesuggested that accessibility challenges at the macroscopic(fibre), microscopic (fibril) and nanoscopic (microfibril)level restrict effective enzymatic hydrolysis. Interestingly,non-hydrolytic disruptive proteins have been shown to dis-rupt the substrate at each of these three organizationallevels. Thus it is likely that these proteins play a key role inenhancing the effectiveness of enzymatic hydrolysis. For ex-ample, amorphogenesis induced by non-hydrolytic disrup-tive proteins has been observed at, 1) the macroscopiclevel through the dispersion of adjacent fibres [15-17], 2) the microscopic level through the loosening/rough-ening/swelling of plant cell walls [16-27], and 3) at thenanoscopic level through the pitting of microfibrils andcellulose decrystallization (Figure 1) [17,23,26,28-30].Recently, it has been suggested [8] that non-hydrolyticdisruptive proteins can be categorized into two distinctgroups. Those with an as yet unknown catalytic mechan-ism such as Swollenin [16,17], Loosenin [27], Expansins[21,31] and several Family 1 and Family 2 CBMs[18,19,23,30] and those thought to act through a directoxidative catalytic mechanism, such as GH61 [32-36]and Family 33 CBMs [37,38] (Table 1). These twogroups of proteins are thought to act in distinctly differ-ent ways on the substrate. For example, several of theproteins with uncharacterized catalytic function, such asSwollenin, Expansins and Loosenin, are thought to pro-mote amorphogenesis through disruption of the hydro-gen bonding network of the substrate (i.e. without directcleavage of the carbohydrate chains) [16,39]. In contrast,the oxidative proteins that act on the substrate do sothrough a direct catalytic oxidative mechanism, whereradical species are generated in close proximity to thecellulose surface, resulting in the direct oxidative cleav-age of the cellulose chains [33-38].A common theme between these two groups of pro-teins appears to be the release of soluble oligomers fromthe substrate. Specifically, Beta-Expansins have beenshown to solubilize both hemicellulose and pectin fromnative maize silk cell walls [31], while the oxidative pro-teins GH61 and CBM33 promote the release of solublecello-oligosaccharides from model cellulosic substrates[33,37,38]. Further evidence supporting the role of non-hydrolytic proteins in cellulose amorphogenesis comesfrom reports that disruptive proteins with unknowncatalytic mechanisms appear to enhance the enzymatichydrolysis of native and pretreated substrates [17,20,23-25,27,29,40-43]. In addition to these proteins, hydrolysisenhancing activity has also been observed for the oxida-tive disruptive proteins. In particular, GH61 has beenshown to significantly reduce the total protein requiredto achieve 70-80% hydrolysis yields of a pretreated cornstover substrate [32]. These results suggest that non-hydrolytic proteins can act within a similar time scale tothat of enzymatic hydrolysis and that they are capable ofpromoting further amorphogenesis within a substratethat has already been disrupted by a physicochemicalpretreatment.These earlier observations encouraged various re-search groups to try to develop a better understandingof the role that non-hydrolytic proteins might play inpromoting the amorphogenesis of lignocellulosic sub-strates, with the expectation that further elucidation oftheir action might help in the development of more effi-cient commercial enzyme preparations. However, one ofthe key limitations in better characterizing the non-hydrolytic proteins involved in biomass deconstructionhas been the lack of simple quantitative techniques formeasuring changes in cellulose accessibility. Varioustechniques have been used, with mixed success, to try toquantify changes in cellulose accessibility and their rela-tive merits are discussed below. We also describe a noveltechnique, using the adsorption of substructure-specificCBMs over short time scales (<30 minutes) to quantifycellulose accessibility [44] and to accurately quantify thedegree of amorphogenesis induced by non-hydrolyticdisruptive proteins.Techniques for measuring amorphogenesisAlthough a range of quantitative techniques can be usedto measure the second (hydrolytic) step of cellulose de-construction [45-47], few if any current techniques areable to provide an accurate quantitative measure of howthe enzyme mixture might increase cellulose accessibilityor what has been termed the amorphogenesis step ofGourlay et al. Biotechnology for Biofuels 2012, 5:51 Page 2 of 14http://www.biotechnologyforbiofuels.com/content/5/1/51enzyme mediated cellulose hydrolysis. This is in partdue to the nature of the end product of each step. Afterenzymatic hydrolysis, the end products (soluble sugars)are readily quantified by high performance liquid chro-matography or by using colorimetric techniques such asthe dinitrosalicylic acid [45] or glucose oxidase assaysFigure 1 Simplified schematic representation of amorphogenesis occurring at three levels of biomass organization. Native plant materialat the nanoscopic (A1), microscopic (B1) and macroscopic (C1) levels of organization. After amorphogenesis induced by non-hydrolytic disruptiveproteins, nanoscopic pitting (A2), microscopic swelling/roughening (B2), and macroscopicfibre dispersion (C2) is observed. These effects occurwithout significant release of sugars from the substrate.Table 1 Non-hydrolytic disruptive proteins and their effects on biomassProteins with unknown catalytic mechanism Putative function ReferencesFamily 1 and 2 CBMs Fibre pitting/roughening, small particle release [18,19,23,30]Swollenin, Loosenin Fibre swelling, microfibril dispersion, dispersion of cellulose aggregates [16,17,27]Expansins Loosening of plant cell walls, solubilization of oligomeric sugars [21,31,39]Expansin-like proteins Loosening of filter paper, dispersion of cellulose aggregates [25,78]Fibril Forming Protein Fibril release from filter paper [15]Proteins with putative oxidative catalytic mechanism Putative function ReferencesGH61 Oxidative cleavage of crystalline cellulose [33-36]CBM33 Oxidative cleavage of crystalline cellulose [37,38]The non-hydrolytic proteins capable of promoting amorphogenesis of cellulosic and/or lignocellulosic biomass identified to date. Amorphogenesis induced bythese proteins has been shown to manifest as a range of specific disruptive effects.Gourlay et al. Biotechnology for Biofuels 2012, 5:51 Page 3 of 14http://www.biotechnologyforbiofuels.com/content/5/1/51[46,47]. In contrast, the end products of the amorpho-genesis step are challenging to describe, let alone quan-tify [17].A major challenge in trying to quantify amorphogen-esis is that the method or technique has to be versatileenough to accurately quantify a range of effects occur-ring at different levels of biomass organization, varyingin scale by several orders of magnitude (from the micro-fibril, with typical diameters of 3–5 nm and lengths of100 s to 1000 s of nm, up to the whole fibre, with dia-meters of 5–50 μm and lengths of 1–4 mm) [5,48,49].This point was recently highlighted by Jäger et al. (2011),who noted that no single technique has the capacity tosimultaneously quantify the effects occurring at multiplelevels of cell wall organization [17]. As a result, previousattempts to measure these effects have typically madeuse of a suite of complementary qualitative and semi-quantitative techniques [15-20,22-30].The most widely used methods employed to try toconfirm disruptive protein mediated amorphogenesis ofbiomass typically involve the application of qualitativemicroscopic techniques. Light microscopy has been usedto try to assess the macroscopic dispersion of Valoniacell walls and microscopic swelling of cotton fibresinduced by the fungal disruptive protein Swollenin [16].Scanning electron microscopy (SEM) has also been usedto show the microscopic roughening of cotton fibres bySwollenin [17] and by the CBMs from the bacteriaCellulomonas fimi and Clostridium cellulovorans andthe fungus Trichoderma reesei [19,22,26]. Additionally,atomic force microscopy (AFM) has been used to shownanoscopic pitting of cotton microfibrils induced byCBM1 from Trichoderma pseudokoningii S-38 [28].However, while these techniques have provided usefulqualitative information on the effects of disruptive pro-teins on model cellulosic substrates, attempts to quantifythese effects have so far been limited to either monitor-ing changes in crystallinity [17,23,26,29,30], measuringthe release of small particles [19,20,23] or by indirectlyquantifying amorphogenesis by measuring changes inthe ease of hydrolyzability of the substrate induced bythese proteins [17,24,27,42,43]. The various methodspreviously used to try and quantify amorphogenesis arediscussed below.CrystallinityThere are conflicting opinions on how influential cellulosecrystallinity is on limiting enzymatic hydrolysis and the ef-fect that amorphogenesis-inducing proteins might haveon enhancing cellulose hydrolysis. Earlier work usingFourier-transform infrared spectroscopy to assess the in-fluence of CBM1 from T. pseudokoningii S-38 on cottonfibre deconstruction claimed that the addition of CBM1helped reduce substrate crystallinity [30], while the highlysimilar CBM1 from T. reesei when added to WhatmanCF11 cellulose fibres did not appear to result in any de-crease in substrate crystallinity when measured using X-ray diffraction [22]. In contrast, the addition of bacterialderived CBM3a from C. cellulovorans reduced the crystal-linity of cotton fibres when assessed by both Fourier-transform infrared spectroscopy and X-ray diffraction [26]while a recombinant Swollenin, Swo2 from T. pseudoko-ningii S-38 apparently caused an increase in the crystallin-ity of Avicel PH-101 [50]. Conversely, the application of arecombinant Swollenin from T. reesei resulted in a de-crease in the crystallinity of filter paper, alpha-celluloseand Avicel when measured by powder X-ray diffraction[17].Although these non-uniform observations might suggestthat different combinations of disruptive protein and sub-strate result in different changes in crystallinity, it is morelikely that these varied results are due to issues with themethods used to measure crystallinity. These issues includethe interpretation of results from the different methods formeasuring crystallinity and the applicability of extrapolatingcrystallinity measurements to suggest the degree of amor-phogenesis. In earlier work [8] it was suggested that amor-phogenesis primarily resulted from substrate changes suchas cellulose delamination or fibrillation, where relativelylarge, intact fragments, still containing crystalline regions,are released from the bulk of the substrate. Pinto et al.(2004) have also suggested that non-hydrolytic disruptiveproteins could increase the accessibility of cellulosic sub-strates without affecting the crystallinity. These workersreported no decrease in the crystallinity of cotton fibresafter treatment with a non-hydrolytic disruptive protein,while observing a roughening of the cotton fibres as visua-lized by SEM [22]. A possible parallel mechanism is thatthe increase in cellulose accessibility could result from theswelling or loosening of the interactions between microfi-brils, resulting in the overall weakening of the cell wallwhile leaving the crystalline cores of the microfibrils rela-tively untouched. Thus it is possible that any changes in thecrystallinity of the substrate would only occur as a second-ary effect of the more general process of amorphogenesis.Particle release and size reductionEarlier work claiming that certain CBMs could induceamorphogenesis was carried out using CBM2a from C.fimi where their addition to cotton fibres resulted in therelease of small particles without a concomitant release ofreducing sugars [19,20]. However, although this techniquecould semi-quantitatively describe the release of particlesfrom the substrate, it provided no characterization of theresidual substrate.A related approach to measuring fragmentation wasemployed by two independent groups to study theeffects of the Swollenin proteins AfSwo1 and TasSwo1Gourlay et al. Biotechnology for Biofuels 2012, 5:51 Page 4 of 14http://www.biotechnologyforbiofuels.com/content/5/1/51from Aspergillus fumigatus and Trichoderma asperellum,respectively, on Avicel PH-101 [24,43]. These research-ers used light microscopy to try to quantify size reduc-tion in Avicel particles after incubation with Swollenins,with Chen et al. (2010) using image-analysis software todemonstrate an almost 2-fold reduction in the size ofthe Avicel particles [24].Hydrolysis enhancementSeveral independent research groups have demonstratedenhanced enzymatic hydrolysis of cellulosic and lignocellu-losic substrates after treatment with non-hydrolytic disrup-tive proteins [17,20,23-25,27,29,32,40-43]. Although thiswork collectively suggests that these disruptive proteins areindeed capable of enhancing the hydrolyzability of modeland native cellulosic substrates, the “degree of hydrolysisenhancement” method is an indirect approach to try toquantify cellulose amorphogenesis. For example, it is pos-sible that some of the enhancement of hydrolysis observedafter addition of disruptive proteins could be due to theseproteins binding to and blocking lignin, thereby preventingnon-productive adsorption of cellulases to the lignin[51,52], rather than through a direct “disruptive” effect.Additionally, the somewhat contradictory results observedwhen similar Family 2 CBMs from Cel6A and Xyn10Afrom C. fimi were used to test for hydrolysis enhancementplaces further doubt on the suitability of using enhance-ment of substrate hydrolyzability as a tool for accuratelyquantifying amorphogenesis [20,53].More recent attempts at quantifying cellulose disruptioninduced by non-hydrolytic proteins have exploited thesynergism observed between these proteins and endoglu-canases specific for amorphous regions of cellulose[27,43]. This method uses quantification of sugar releasedby endoglucanases from a disrupted cellulosic substrate asan indirect measure of the degree of amorphogenesis ofthe substrate induced by the non-hydrolytic disruptiveproteins. Although this technique has been successfullyapplied to “semi-quantitatively” measure the disruptiveeffects of Swollenin on Avicel [43] and Loosenin on cottonfibres [27], one drawback of this technique is the specifi-city of the endoglucanases for amorphous cellulose[27,43]. As the endoglucanases employed are specific foramorphous cellulose this approach will only work well ifthe amorphogenesis step results in a simple decrystaliza-tion of cellulose. However, it will not measure the otherpossible influences of enhanced cellulose accessibility suchas the splitting, delaminating or loosening of the cellulose,which could occur without any significant changes in thecrystallinity of the cellulose.Other potential methods for quantifying amorphogenesisWhile each of the putative indications of amorphogenesis,such as delamination, fibrillation, swelling, loosening,roughening, pitting, weakening or decrystallization of thesubstrate can all be thought of as distinct processes, if theyplay a role in amorphogenesis we should be able to in-crease access of the enzymes to the cellulose without asignificant increase in the release of reducing sugars. Sev-eral groups have tried to develop methods of accuratelyquantifying changes in the accessibility of lignocellulosicsubstrates, with many of these techniques modified fromtraditional pulp and paper procedures [54]. Althoughthese techniques have previously only been used toassess the overall accessibility of the substrate, in-cluding accessibility to the lignin and hemicellulosesas well as the cellulose, some of these techniquesalso have potential for quantifying the amorphogen-esis step.Techniques with potential for quantifying cellulose ac-cessibility and the amorphogenesis step include measuringthe water retention value, mean fibre size, nitrogen ad-sorption capacity and mercury porosimetry of the cellu-losic substrate. Or, performing techniques such as soluteexclusion, differential scanning calorimetry, time-domainnuclear magnetic resonance, Simons’ Staining and proteinadsorption (Reviewed in [54]). One of the benefits of thesemodified pulp and paper techniques is that they can moni-tor changes in the cellulose accessibility at the macro-scopic, microscopic and nanoscopic levels. For example,fibre size measurements give an indication of macroscopicchanges in accessibility while techniques such as mercuryporosimetry and solute exclusion can be used to quantifychanges in pore size distribution at the microscopic level[54]. The ability to monitor changes in the substrate at themacroscopic, microscopic and nanoscopic levels wouldlikely be of great value when quantifying cellulose amor-phogenesis, as this phenomenon could occur at each ofthese levels.Two techniques with potential for quantifying changes incellulose accessibility include differential scanning calorim-etry [55,56] and time-domain nuclear magnetic resonance[57], which were developed to assess pore volume and dis-tribution within cellulosic and lignocellulosic materials.These techniques can be used to differentiate between pri-mary bound water (water directly bound to the substratesurface with severe restrictions on conformational changes),secondary bound water (water in close proximity to thesubstrate where hydrogen bonding networks propagatingfrom polar groups at the substrate surface and capillaryforces place some degree of conformational constraints onthe water molecules) and the free/bulk water (water distalto the substrate, which is not conformationally constrained)[57]. Thus a quantitative measurement of the total water-accessible surface area of the substrate can be obtainedwhile providing an insight into the overall pore size distri-bution. Although the differential scanning calorimetry andtime-domain nuclear magnetic resonance methods haveGourlay et al. Biotechnology for Biofuels 2012, 5:51 Page 5 of 14http://www.biotechnologyforbiofuels.com/content/5/1/51not yet been used to quantify overall cellulose accessibility,it is likely that these techniques could be adapted to providesome quantitative information on the extent and mechan-ism of disruption induced by non-hydrolytic disruptiveproteins.An alternative technique with potential for measuringcellulose accessibility involves the use of protein adsorp-tion to try to quantify the amount of cellulose in thesubstrate that is accessible to enzymes [7,12,44,58]. Thistechnique makes use of the cellulases themselves as ac-cessibility probes, where either a mixture of cellulases ormonocomponent cellulases are incubated with the sub-strate followed by quantifying the amount of protein thatis adsorbed to the substrate. While this technique mightprovide a good indication of the amount of accessiblesurface area of the substrate, there are two key problemsto be overcome when using this approach. First, unlessthe adsorption study is carried out at low temperatures(which will not be representative of hydrolysis reactionconditions!), the cellulases will hydrolyze the substrate,thereby changing the substrate accessibility during thecourse of the assay. Secondly, cellulases are known toadsorb unproductively to lignin, which restricts the ac-curate quantification of cellulose accessibility [51,59].Other recent attempts at using cellulases to quantify ac-cessibility have involved the production of a fluorescently-tagged non-hydrolytic CBM to enhance the accuracy andsensitivity of the protein adsorption technique [44]. Thistechnique makes use of BSA blocking to overcome pro-blems with lignin-binding [60]. However, one potentialdrawback is that different CBMs recognize different sub-structures within the substrate [61,62]. Thus, it is possiblethat a probe making use of a single CBM might primarilybe quantifying the accessibility of a specific cellulosic sub-structure, rather than the overall accessibility of the cellu-lose. An alternative strategy might be to use cellulaseinhibitors that limit or prevent substrate hydrolysis duringprotein adsorption experiments at more typical substratehydrolysis temperatures (i.e. 50°C). However, while inhibi-tors such as hexachloropalladate have been shown to in-hibit Cel7a from T. reesei, it has only a limited effect onmost of the other enzymes present in commercial cellulasepreparations [63,64]. Thus the inhibition approach wouldonly work when using monocomponent cellulases for theadsorption studies.Another procedure for measuring accessibility is theSimons’ stain method [65]. This technique involves quanti-fying the adsorption of an anionic direct dye which has ahigher affinity for cellulose than to lignin and hemicellu-lose [65,66]. The amount of dye bound to the cellulosicsubstrate gives a good indication of the total amount ofcellulose accessible to cellulase within the substrate [7].Overall, these methods have proven to be useful in quan-tifying some of the changes that occur in the substrateduring enzymatic hydrolysis. However, there are severaldrawbacks to using these techniques to measure changes incellulose accessibility. For example, the water retentionvalue is known to be insensitive to small changes in fibrecharacteristics and would not be able to detect changes inthe cellulosic component during or after amorphogenesis.Another major drawback of many of these techniques isthat they measure the overall accessibility of the substrate,including the amount of accessible lignin and hemicellu-lose, not just the cellulose. This restricts techniques such asnitrogen adsorption, solute exclusion, differential scanningcalorimetry and time-domain nuclear magnetic resonancefrom being used to assess changes in the specific amount ofaccessible cellulose within the substrate during or after theamorphogenesis process. Finally, some of these techniquesare labour intensive, such as the solute exclusion technique,or require the use of toxic heavy metals, as in determiningmercury porosimetry.To date, no single technique has provided an accuratequantitative measure of the changes occurring within thecellulosic substrate during amorphogenesis. In the workdescribed below we describe a novel method where cellu-lose substructure-specific CBMs have been successfullyused to quantify amorphogenesis by determining changesin the accessibility and surface morphology of cellulosebefore and after treatment with amorphogenesis-inducingagents.Results and discussionAs protein-mediated amorphogenesis is still an evolvingconcept, we initially assessed the sensitivity and reproduci-bility of a CBM-mediated method for quantifying changesin cellulose accessibility by using concentrated phosphoricacid to disrupt cotton fibres to varying degrees of disassoci-ation [67]. The use of harsh acid treatments was intendedto provide an exaggerated range of disrupted substrates,and was not intended to be representative of milder, bio-logical treatments. The disruptive effect of the acid was ini-tially qualitatively assessed using SEM (Figure 2) followedby a quantitative assessment where the adsorption of eachof the substructure-specific CBMs [61] was determined(Figure 3).The CBM2a and CBM44 were used to specifically de-tect the crystalline and amorphous regions of celluloserespectively. Previous work has shown that CBM2a is aType A CBM which binds to cellulose through a flatbinding face incorporating a planar arrangement ofhydrophobic aromatic residues [68,69]. This CBM hasbeen shown by several researchers to be specific forcrystalline cellulose [61,70,71]. Competitive bindingexperiments on PASC have been used to demonstratethat CBM2a has little binding site overlap with a Type BCBM (CBM4-1) known to exclusively recognize theamorphous regions of cellulose while showing no affinityGourlay et al. Biotechnology for Biofuels 2012, 5:51 Page 6 of 14http://www.biotechnologyforbiofuels.com/content/5/1/51for crystalline regions [61,71]. As CBM2a is thought tointeract with 2–3 chains in the ordered crystal lattice[70], in the work reported here, we have defined a crys-talline region as one with 2–3 adjacent crystalline cellu-lose chains. In contrast, the amorphous-cellulosebinding CBM used in this work, CBM44, is a Type BCBM with a binding site comprised of a narrow groovelined with hydrophobic aromatic residues [72]. Thisgroove confers binding specificity to free polysaccharidechains, such as those present in amorphous cellulose,but does not enable binding to the tightly packed chainsfound within crystalline cellulose [72].To try to progressively disrupt cotton fibres in order toassess the ability of CBM adsorption to quantify celluloseaccessibility a range of phosphoric acid concentrationswas used. As the concentration of phosphoric acid wasincreased, an increase in the degree of disruption of thesample, including the splitting, delaminating and roughen-ing of the fibres was apparent (Figure 2). An increase indisruption generally correlated with an increase inbinding of both the amorphous-binding and crystalline-binding CBMs up to a concentration of 77% acid treat-ment (Figure 3). This increase in the combined binding ofboth CBMs provided a good indication that the overall(crystalline and amorphous) cellulose accessibility hadincreased. Surprisingly, the use of increasingly harsh acidtreatments resulted in only a relatively small increase inthe amount of CBM2a bound to the substrate when com-pared to the increase in CBM44 binding. It had beenanticipated that the increasing disruption of the fibresFigure 2 SEM images of cotton fibres disrupted by phosphoric acid treatments. SEM micrographs of cotton fibres after treatment with arange of o-phosphoric acid concentrations. After control treatment (nanopure water, 0% (w/w) acid), cotton fibres appear smooth, with fewsurface features. As the acid concentration was increased to near the point of cellulose dissolution (~73%-78%), manifestations ofamorphogenesis begin to appear at the surface of the cotton fibres. At 74% phosphoric acid, initial signs of splitting, roughening, fibrillation andpeeling/delamination of the fibres appear. As the acid concentration is increased from 74% to 76%, these effects become more pronounced. At77%, the fibre structure has been almost completely destroyed, with large portions of the outer layer of the fibre appearing to peel off, revealinga rough, fibrillated underlying structure. After treatment with 78% phosphoric acid no fibre structure remains. All cellulose present appears tohave been dissolved and reprecipitated into amorphous cellulosic ‘mats’. All images were taken at x1200 magnification and each image depicts arepresentative fibre for the indicated acid concentration.Gourlay et al. Biotechnology for Biofuels 2012, 5:51 Page 7 of 14http://www.biotechnologyforbiofuels.com/content/5/1/51would result in the amorphous-binding CBM44 beingbound more than the crystalline-binding CBM2a. How-ever, as CBM2a recognizes only two to three adjacentchains as being ‘crystalline’, the observed relative increasein CBM2a binding over CBM44 binding as the acid con-centration increased was likely due to the increased solv-ent exposure of small microcrystalline substructureswithin the acid-disrupted cotton fibres.As the acid concentration was further increased from77% to 78%, the SEM micrographs of the cotton showedthat any residual fibre structure had been lost and thatthe substrate now had the form of amorphous cellulosic‘mats’ (Figure 2). These SEM observations complemen-ted the CBM adsorption results, where increasing theacid concentration from 77% to 78% resulted in a largeincrease in the amount of adsorbed CBM44, without sig-nificantly altering the adsorption of CBM2a (Figure 3). Itwas apparent that the specificity of CBM adsorption wasdistinct enough that changes in the surface morphologyof the cellulosic substrates could be readilydifferentiated.After determining that CBM adsorption could be usedto quantify acid-induced changes in cellulose accessibil-ity, we attempted to correlate the degree of substratedisruption (quantified by CBM adsorption) with enzym-atic hydrolyzability. Each of the phosphoric-acid dis-rupted cotton fibre samples was hydrolyzed using acommercial cellulase mixture (30 filter paper units/g cel-lulose of Celluclast 1.5 L, supplemented with 15 cello-biase units/g cellulose of Beta-glucosidase (Novozym188, Novozymes A/S, Bagsværd, Denmark)). The initialhydrolysis rate (defined here as the hydrolysis rate overthe first 30 minutes of the reaction) was plotted againstthe adsorption of each individual CBM, as well as thesum of their adsorptions (Figure 4). The adsorption ofeach individual CBM, and particularly their summedadsorptions, was found to correlate well with theenhanced enzymatic hydrolyzability of the cotton fibres.The steeper slope of the curve for CBM44 when com-pared to CBM2a seemed to indicate that, at least for theinitial stages of hydrolysis, enzymatic hydrolysis rateswere influenced more by the amount of accessibleamorphous cellulose than they were by the amount ofaccessible crystalline cellulose.Interestingly, although the hydrolyzability and CBM44adsorption increased with every incremental increase inphosphoric acid concentration, this was not the case forCBM2a. As the acid concentration was increased from77% to 78%, the adsorption of CBM2a to the substratewas not significantly affected, even as the hydrolyzabilitycontinued to increase. This suggested that any attemptsto correlate changes in substrate accessibility to hydro-lyzability using a specific mono-component cellulasemay be problematic, as some cellulases contain CBMsspecific for crystalline cellulose and might thereforeunderestimate the accessibility of the highly amorphousregions of the substrate.This seemed to indicate that substructure specific CBMscould be used to quantify acid-induced changes in cellu-lose accessibility and that increases in accessibility (asdetermined by CBM adsorption) can provide a good pre-dictor of initial rates of enzymatic hydrolysis.Quantification of Swollenin-induced increases in celluloseaccessibilityAs Swollenin had previously been shown to disrupt mercer-ized cotton fibres [16], we next tried to quantify anychanges in cellulose accessibility and surface morphology ofFigure 3 Adsorption of CBMs to variably-disrupted cotton fibres. Adsorption of crystalline cellulose-specific CBM2a and amorphouscellulose-specific CBM44 to cotton fibres treated with a range of o-phosphoric acid concentrations. Experiments were run in triplicate and errorbars represent one standard deviation from the mean.Gourlay et al. Biotechnology for Biofuels 2012, 5:51 Page 8 of 14http://www.biotechnologyforbiofuels.com/content/5/1/51mercerized cotton fibres treated with Swollenin by lookingat the degree of adsorption of substructure-specific CBMsto the treated fibres. Although mercerization is known tocause a significant reduction in the crystallinity of cellulosicsubstrates, mercerized cellulose has been shown to retainsome adsorptive capacity for crystalline binding CBMs [73].After incubation with Swollenin, binding of both the crys-talline and amorphous-specific CBMs to the mercerizedcotton fibres increased (Figure 5).The increase in binding was more pronounced for-CBM2a than for CBM44 after Swollenin treatment, indicat-ing that the increase in accessibility was not simply due toSwollenin-mediated decrystallization of the cellulose at themicrofibril surface. This suggested that Swollenin might actby promoting the delamination or fibrillation of the sub-strate, or by promoting the “splitting” of microfibrils,thereby exposing new crystalline regions of cellulose to theCBMs.Subsequent SEM micrographs of Swollenin-treated mer-cerized cotton fibres indicated that Swollenin treatmentsresulted in a smoothing of the roughened patches producedduring mercerization (Figure 6). This smoothing effect wasin contrast to the buffer- and BSA-treated mercerized cot-ton fibres, which retained their roughened surface. AfterSwollenin treatment, the roughened patches at the surfaceof the fibres appeared to have been sloughed off, revealingthe smooth, well ordered surface of the underlying cottonfibre. The observed increase in turbidity in the supernatantafter Swollenin treatment (data not shown) was also indica-tive of the release of small particles into solution. It is pos-sible that the roughened patches at the surface of themercerized cotton fibres will contain a higher proportion ofamorphous cellulose than the underlying fibre, as theseprotruding rough regions were more exposed to the NaOHused for mercerization. This treatment has been shown topromote the conversion of crystalline cellulose I intoamorphous cellulose and crystalline cellulose II [74]. It ispossible that the release of these roughened particles fromthe surface of the fibre resulted in an increase in both theamount of exposed amorphous cellulose (primarily on thereleased particles) and the amount of exposed crystallinecellulose (primarily on the newly exposed surface of theunderlying cotton fibre). However, it should be noted thatthe small roughened particles that are released from thesurface of the cotton fibres appear to be approximately100 nm in the shorter direction, and up to 1000 nm in thelonger direction (estimated from Figure 6). Since the cellu-losic cores of cotton microfibrils have diameters of only 3–5 nm and lengths of 100 s to 1000 s of nm [5,48,49], it ispossible that the small, roughened particles released fromthe surface of the cotton fibres still contained significantamounts of crystalline cellulose.Although it was not evident by which specific mechanismthe Swollenin resulted in this “smoothing” effect, it is pos-sible that Swollenin acts in a similar manner to the Expan-sin family of proteins, which have been shown to weakenplant cell walls through disruption of the hydrogen bondingnetwork between plant cell wall polymers [39]. If Swollenindisrupts hydrogen bonding in a similar mode to Expansinsthis might also explain how Swollenin appears to both dis-rupt the cell wall structure of Whatman filter paper No. 1fibres and result in the swelling of cotton fibres [16,17].R² = 0.9828R² = 0.9728 R² = 0.997901002003004005006007008000 10 20 30 40 50 60 70Initial Hydrolysis Rate ug/mL glucose/hourmg CBM Bound/mg CottonCBM2aCBM44Sum CBMsFigure 4 Initial hydrolysis rate vs adsorption of CBMs. Initial hydrolysis rate (calculated after 30 minutes of hydrolysis) of acid-disruptedcotton fibres increases with increasing CBM adsorption. Each data point represents a cotton fibre sample treated with a different concentration ofo-phosphoric acid and hydrolyzed with the same enzyme loading. Experiments were run in triplicate and error bars represent one standarddeviation from the mean.Gourlay et al. Biotechnology for Biofuels 2012, 5:51 Page 9 of 14http://www.biotechnologyforbiofuels.com/content/5/1/51These results indicated that substructure-specific CBMscould not only be used to track changes in cellulose acces-sibility after harsh acid treatments, but could also be usedto track changes in surface morphology after the milder,Swollenin-induced, amorphogenesis. This technique hasseveral advantages over current alternatives as it provideda direct, quantitative method able to consolidate changesin multiple substrate characteristics. Specifically, changesin the amounts of accessible amorphous cellulose, access-ible crystalline cellulose and the total (amorphous andcrystalline) accessible cellulose can all be quantified. It wasalso apparent that this method could help better indicatethe mode of action of non-hydrolytic, disruptive/amorpho-genesis-inducing proteins and has potential to yield novelinsights into the mechanisms of glycosyl hydrolases andthe other accessory enzymes involved in lignocellulose de-construction. Additionally, this technique has the potentialto facilitate comparisons of the disruptive capabilities ofvarious non-hydrolytic proteins which might promote anincrease in cellulose accessibility to the more traditional,hydrolytic components of the cellulase enzyme mixture.It is also possible that CBMs with specificities for certainhemicelluloses, such as xylan or mannan, might be able tobe used to track changes in the accessibility of these poly-mers during pretreatment, amorphogenesis and hydrolysisof softwoods, hardwoods and agricultural residues. In pre-vious work, Filonova et al., (2007) demonstrated the useof fluorescently-tagged mannan-specific CBM’s to quantifythe accessibility of mannan in wood tissues and pulp, afterapplying a protein-based lignin-blocking technique to pre-vent non-specific adsorption of the CBMs to lignin [75].The use of CBM-specific antibodies, or conjugation ofCBMs to distinct fluorophores, have been used to providedirect visualization of the locations of the different47.6%22.1%38.0%0510152025Sum CBMsCBM44CBM2amg CBM Bound/g CottonControlSwolleninFigure 5 Adsorption of CBMs to Swollenin-treated cotton fibres. Swollenin-induced changes in the accessibility and surface morphology ofmercerized cotton fibres quantified using CBM adsorption. The % values represent the % increase in adsorption of the CBMs after Swollenintreatment. A BSA negative protein control was found to have no significant effect on the extent of binding of either CBM. At least threereplicates were performed for each sample. Error bars represent one standard deviation from the mean.Figure 6 Effect of Swollenin on mercerized cotton fibres imaged by SEM. The surface of the control fibre (Left) appears roughened due tothe mercerization treatment. The rough features on the surface of the mercerized cotton fibres appear to have been sloughed off by the actionof Swollenin (Right). Images are of representative fibres for the indicated treatment, and are at 10000x magnification.Gourlay et al. Biotechnology for Biofuels 2012, 5:51 Page 10 of 14http://www.biotechnologyforbiofuels.com/content/5/1/51polymers or substructures at the substrate surface [75,76].Thus, by utilizing a suite of different CBMs with specifici-ties for a range of structural features of the substrate, itmight be possible to track changes in the morphology ofthe substrate during pretreatment and hydrolysis whilebetter quantifying the role that enzyme access to the cellu-lose plays in limiting the rate and extent of enzymatichydrolysis.ConclusionsPrevious attempts to try and quantify the “cellulose swel-ling/delamination” or the “amorphogenesis step” of cellu-lose hydrolysis have tended to make use of a suite ofcomplementary qualitative and semi-quantitative techni-ques. While these techniques have provided some useful in-formation regarding the effects of amorphogenesis-inducing proteins on (ligno) cellulosic substrates, they havetypically provided little insight into the mode of action ofthese proteins. A novel technique, using non-hydrolyticsubstructure-specific CBMs capable of quantitatively meas-uring changes in cellulose accessibility and surface morph-ology was successfully used to track changes in thecellulose during Swollenin-induced amorphogenesis. Thisnovel method provided useful insights into how proteinssuch as Swollenin might increase cellulose accessibility bynon-hydrolytic mechanisms such as the swelling or delam-ination of the cellulose substructures.MethodsProteinsCBM44 from Clostridium thermocellum was purchasedfrom NZYTech (Lisbon, Portugal, CR0049). CBM2a fromCellulomonas fimi was provided by Dr. Douglas Kilburnfrom the University of British Columbia, Canada, after re-combinant expression and purification from E. coli. Re-combinant Swollenin (Swo1) was generously provided byVTT Technical Research Centre, Finland. Briefly, theSwollenin was expressed in Trichoderma reesei as ahistidine-tagged protein and purified in two chromato-graphic steps.Phosphoric acid-induced amorphogenesisPhosphoric acid-disrupted cotton fibres were prepared fol-lowing a protocol similar to that described by Zhang et al.(2006) [67]. Briefly, ice-cold o-phosphoric acid (Fisher Sci-entific, Canada, A242) solutions were produced at variousconcentrations and 14.5 mL was added to 50 mL centrifugetubes containing 0.2 g cotton fibres (Sigma-Aldrich, St.Louis MO, USA, C6663) pre-wetted with 0.5 mL nanopurewater to give final o-phosphoric acid concentrations of 0–78% w/w. Samples were incubated for one hour on ice withoccasional mixing. Ice-cold nanopure water (35 mL) wasslowly added to each sample, followed by centrifugation at10,000 g for 15 minutes. The fibres were resuspended in50 mL nanopure water and washed a further 4 times with50 mL nanopure water, followed by one wash with 50 mL20 mM Na2CO3 (Fisher Scientific, Pittsburg PA, USA,S263) and 2 subsequent washes in 50 mL nanopure water.The cotton fibres were then lyophilized overnight.Swollenin-induced amorphogenesisPrior to Swollenin treatment, 200 mg cotton fibres(Sigma-Aldrich, St. Louis MO, USA, C6663) were mercer-ized in 50 mL 25% (w/w) ice-cold NaOH for 15 minutes.The mercerized fibres were washed thoroughly with nano-pure water then lyophilized overnight. Dried cotton fibres(50 mg) were weighed into 2 mL screwcap tubes. Swolle-nin or BSA (10 μg/mg cotton in 50 mM sodium acetatebuffer, pH 5), or buffer alone was added and the sampleswere incubated overnight at 50°C in a FinepcrCombiSV12 hybridization incubator at 30 rpm. Protein wasremoved from the samples by extensive washing withnanopure water. Samples were lyophilized overnight priorto CBM adsorption and microscopy studies.Turbidity measurementsSupernatants from the Swollenin- or control-treatedmercerized cotton fibres were transferred to 1 mL plasticcuvettes, and the optical density at 600 nm was read ona Varian Cary 50 Bio Spectrophotometer. Sodium acetatebuffer (50 mM, pH 5) was used as a blank. Samples wererun in triplicate.Scanning electron microscopyLyophilized cotton fibres were mounted on aluminumSEM stubs using double sided tape and sputter-coatedwith 10 nm Au/Pd (80:20 mix) then imaged on a HitachiS-2600 VP-SEM (Tokyo, Japan).CBM adsorptionCBM2a and CBM44 were made up to 500 μg/mL in50 mM sodium acetate buffer, pH 5, and added to 5 mg ofphosphoric acid-treated or Swollenin-treated cotton fibresto a final CBM concentration of 50 μg/mg cotton. Sampleswere incubated for 30 minutes at 20°C in FinepcrCombiSV12 hybridization incubator at 30 rpm then centrifuged at16,000 g for 10 minutes in a benchtop centrifuge. Theamount of CBM bound to the cotton was calculated bymeasuring the absorbance of the supernatant at 280 nmand determining the concentration of the residual CBM inthe supernatant using the calculated molar extinction coef-ficients of 27,625 M-1 and 27,365 M-1 for CBM2a andCBM44, respectively [77]. The amount of CBM bound tothe cotton was calculated by subtracting the amount of theresidual CBM in the supernatant from the original amountof CBM added to the sample.Gourlay et al. Biotechnology for Biofuels 2012, 5:51 Page 11 of 14http://www.biotechnologyforbiofuels.com/content/5/1/51Enzymatic HydrolysisCotton samples were hydrolyzed using 5 mg substrate in1 mL 50 mM sodium acetate buffer, pH 5, at 50°C for 30minutes, with an enzyme loading of 30 filter paper unitsCelluclast (Novozyme, USA) per gram cotton and sup-plemental β-glucosidase (Novozymes 188, Novozymes,Bagsværd, Denmark) at 1:2 cellobiase units/filter paperunit. After hydrolysis, the enzymes were heat inactivatedat 100°C for 10 minutes, samples were centrifuged at16,000 g in a benchtop centrifuge for 10 minutes, andthe glucose concentration of the supernatant was deter-mined using the glucose oxidase assay [46,47].AbbreviationsCBM: Carbohydrate binding module; SEM: Scanning electron microscopy;BSA: Bovine serum albumin.Competing interestsThe authors declare that they have no competing interests.AcknowledgementsThe Natural Sciences and Engineering Research Council of Canada (NSERC),Natural Resources Canada (NRCan) and Genome BC are gratefullyacknowledged for the support of this work. We particularly want to thank Dr.Doug Kilburn and Emily Kwan at UBC for generously donating CBM2a, aswell as Dr. Merja Penttilä and Martina Andberg at VTT Technical ResearchCentre of Finland for their generous donation of purified Swollenin.Authors' contributionsKG designed and carried out the experiments, analyzed results and draftedthe manuscript. 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