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Access to cellulose limits the efficiency of enzymatic hydrolysis: the role of amorphogenesis Arantes, Valdeir; Saddler, Jack N Feb 23, 2010

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REVIEW Open AccessAccess to cellulose limits the efficiency ofenzymatic hydrolysis: the role of amorphogenesisValdeir Arantes, Jack N Saddler*AbstractThe efficient enzymatic saccharification of cellulose at low cellulase (protein) loadings continues to be a challengefor commercialization of a process for bioconversion of lignocellulose to ethanol. Currently, effective pretreatmentfollowed by high enzyme loading is needed to overcome several substrate and enzyme factors that limit rapid andcomplete hydrolysis of the cellulosic fraction of biomass substrates. One of the major barriers faced by cellulaseenzymes is their limited access to much of the cellulose that is buried within the highly ordered and tightlypacked fibrillar architecture of the cellulose microfibrils. Rather than a sequential ‘shaving’ or ‘planing’ of thecellulose fibrils from the outside, it has been suggested that these inaccessible regions are disrupted or loosenedby non-hydrolytic proteins, thereby increasing the cellulose surface area and making it more accessible to thecellulase enzyme complex. This initial stage in enzymatic saccharification of cellulose has been termedamorphogenesis. In this review, we describe the various amorphogenesis-inducing agents that have beensuggested, and their possible role in enhancing the enzymatic hydrolysis of cellulose.ReviewContinuing interest in the utilization of renewable bio-mass resources for the production of alternative fuelshas brought increasing attention on the technical bottle-necks that still need to be resolved and how the variabil-ity of different lignocellulosic materials might influencethe efficiency of enzymatic hydrolysis.Over the past 40 to 50 years, many excellent researchgroups have been assessing the ability of carbohydrate-degrading enzymes to depolymerize the cellulosic com-ponent of lignocellulosic substrates into soluble, fermen-table sugars. However the efficient, rapid and completeenzymatic hydrolysis of lignocellulosic materials usinglow protein loadings has proven to be one of the majortechnical and economical bottlenecks in the overall bio-conversion process of lignocellulose to biofuels [1-4].Several factors related to the substrates (such as lig-nin/hemicellulose association, degree of cellulose crystal-linity and polymerization, extent of surface area) andenzymes (such as end-product inhibition, need forsynergism, irreversible enzyme adsorption) have beensuggested to account for the recalcitrance of cellulose toenzymatic hydrolysis [5]. However, there is still consid-erable disagreement in the literature regarding the rela-tive importance of each of these factors, and ourunderstanding of how enzymes completely hydrolyzecellulose is still far from complete.Enzymatic saccharification of cellulose is generallydescribed as a heterogeneous reaction system in whichcellulases in an aqueous environment react with the inso-luble, macroscopic and structured cellulose, containinghighly ordered and less ordered regions. Unsatisfactorily,the majority of the research directed at understandingthe mechanisms of cellulose biodegradation has given lit-tle attention to the existence and the influence that thefibrillar architecture of the cellulose fibril network willhave on the enzyme reactivity and consequential courseof heterogeneous cellulase reactions.In order for cellulases to efficiently hydrolyze cellulosicsubstrates, they must first be able to access the cellulosechains that are tightly packed in the form of insolublemicrofibrils encased in hemicellulose and lignin [5].Previous work has shown that the ability of cellulaseenzymes such as Trichoderma reesei cellobiohydrolase(CBH)I to access the cellulose chains within the microfi-brils embedded in fiber walls is significantly limited,probably due to the enzyme’s ability to access only thesurface layers of the microfibrils [5]. Although cellulose* Correspondence: jack.saddler@ubc.caForestry Products Biotechnology/Bioenergy Group, Department of WoodScience, Faculty of Forestry, University of British Columbia, 2424 Main Mall,Vancouver BC, V6T 1Z4, CanadaArantes and Saddler Biotechnology for Biofuels 2010, 3:4http://www.biotechnologyforbiofuels.com/content/3/1/4© 2010 Arantes and Saddler; 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.could be slowly eroded by surface shaving or planing, ithas been proposed that, to achieve efficient enzymaticsaccharification, cellulose chains in the highly orderedand tightly packed regions of microfibrils should ratherbe delaminated, disrupted or loosened, thereby increasingthe surface area and making the individual cellulosemolecules more accessible and available for interactionswith cellulose-degrading enzymes. Fiber swelling andfragmentation of cellulose aggregations into short fibershave been observed during enzymatic hydrolysis of cellu-lose before any detectable amount of reducing sugars isreleased [6-9]. This initial stage in enzymatic saccharifica-tion of cellulose has been termed amorphogenesis [6].The original mechanistic model for enzymatic degra-dation of cellulose postulated by the pioneering work ofMandel and Reese introduced the C1-Cx model [10,11].They hypothesized that an unknown component of thecellulase system (C1, the so called ‘swelling factor’)opens up the cellulose matrix, allowing this now moreaccessible substrate to be depolymerized by the trulyhydrolytic enzymes (Cx) [10,11]. Although many hydro-lytic enzymes that could account for the suggested Cxaction have been identified and characterized, so far theidentification and characterization of the C1 factorremains elusive.CBH 1, along with a number of other proteins (expan-sins, expansin-like proteins, swollenin), contains a poly-saccharide binding surface. These proteins have beensuggested to be able to non-hydrolytically loosen or dis-rupt the packaging of the cellulose fibril network. Thecellulose-disrupting activity of such proteins has recentlybeen shown to interact synergistically with cellulaseenzymes when they are used to hydrolyze insoluble cel-lulose, apparently by increasing the accessibility of thecellulose to the enzymes [12,13]. In this review paper,we provide an overview of these amorphogenesis-indu-cing agents and their interactions with cellulose. Inaddition, their potential for possible application in theenzymatic saccharification of cellulose-containing mate-rials for biofuel production is also discussed. The struc-tural arrangement of the cellulose chains in the fibrillararchitecture, and their accessibility and reactivity arealso briefly outlined. The use of enzymes and their com-ponents is expected to radically influence the way wecurrently think cellulose is organized within the plantcell wall.Cellulose: structure, accessibility and reactivityCellulose, an insoluble polymer consisting of b-(1-4)-linked glucose residues [14-16], has been the subject ofintense research for more than a century, and newinsights into a better understanding of its moleculararchitecture continue to emerge [15,16]. It is wellknown that native cellulose molecules (cellulose I) arefound in fibril form, and that its molecular architecturehas a high degree of individuality, depending on itssource (cell wall layer or plant type) [16].Briefly, the visually dominant structural features ofcellulose in higher plants are cellulose microfibrils withdiameter of 2-10 nm, cross-linked by other cell wallcomponents such as xyloglucans [15,16]. Microfibrils areunbranched fibrils composed of approximately 30-36glucan chains aggregated laterally by means of hydrogenbonding and van der Waals forces to produce crystallinestructures [15]. Microcrystalline cellulose has beenshown to be made up of two different crystal phases: Iaand Ib [15,16]. Although considerable progress has beenmade in elucidating the crystal structures of cellulose inmicrofibrils, they are still not well understood [15,16],and a deeper understanding of cellulose structure isrequired if we are to overcome the natural recalcitranceof lignocellulosic substrates. It is likely that these crystalstructures affect the rate of diffusion of reactants andthus play an important role in the accessibility and reac-tivity of cellulose.Previous work by Krässing [14] has shown that ahigher degree of fibrillar aggregation produces a morecompact fiber structure, with fewer, smaller intersticesresulting in a smaller internal accessible surface area.An important feature of the highly ordered regions isthat the cellulose chains are packed so tightly that evensmall molecules such as water cannot penetrate thesehighly organized structural entities [14]. The limitedaccessibility to these regions leads to alteration of theirreactivity to swelling and reactive agents such as cellu-lases. With this type of structure, it is apparent thatonly the cellulose molecules situated on the surface ofthese aggregations would be susceptible to the degradingactions of enzymes.If cellulose hydrolysis only occurs on the surface ofthe cellulose aggregations, the available surface area is apotential determinant of the maximum rate of hydrolysisthat can be achieved. It has been proposed that thetightly packed cellulose regions are a major factor incontributing to the resistance of cellulose to degrada-tion, by limiting the accessibility to cellulases [17,18]. In1985, Coughlan [6] coined the term ‘amorphogenesis’ tosuggest a possible mechanism by which the dispersion,swelling or delamination of cellulosic substrate occurred,resulting in a reduction in the degree of fibrillar aggre-gation and/or crystallinity, and the creation of a largeraccessible surface by increasing the reactive internal sur-face. Consequently, amorphogenesis enhances the reac-tivity of the fibrous cellulosic substrates by increasingthe amount of cellulose directly accessible to theenzymes.It has been suggested (Figure 1) that as cellulases needto adsorb onto the surface of the insoluble celluloseArantes and Saddler Biotechnology for Biofuels 2010, 3:4http://www.biotechnologyforbiofuels.com/content/3/1/4Page 2 of 11before hydrolysis, the inaccessible bulk of the substrateis structurally loosened to increase the molecular disor-der of the tightly packed regions in the fibrous cellulosicnetwork and to expose the cellulose chains buriedwithin the microfibrils while they remain molecularlyalmost unchanged (amorphogenesis) (Figure 1a) [6].Once the cellulose network is accessible to the enzymes,the synergistic action of endo- and exo-glucanases pro-mote the fragmentation of accessible molecules to solu-ble cello-oligosaccharides (cellulosic molecules with adegree of polymerization of < 6 units) (Figure 1b),which are quickly hydrolyzed, mostly to cellobiose (Fig-ure 1c). This component of the proposed mechanismseems likely to occur, as cello-oligosaccharides areFigure 1 Proposed mechanism for cellulose amorphogenesis/depolymerization by cellulases (adapted from [6]). Amorphogenesis (A)takes place at the macromolecular level by non-hydrolytic agents.Arantes and Saddler Biotechnology for Biofuels 2010, 3:4http://www.biotechnologyforbiofuels.com/content/3/1/4Page 3 of 11seldom detected in solution, with cellobiose proving tobe the primary cellulose hydrolysis product in mostnative cellulase systems. In most commercial cellulasesystems, an extraneous source of b-glucosidase is usuallyadded to completely hydrolyze the cellobiose to glucose(Figure 1d), enhancing the overall reaction by minimiz-ing end-product inhibition.Carbohydrate-binding modulesMany carbohydrate-hydrolyzing enzymes, such as cellu-lases and xylanases, are modular proteins with at leasttwo distinct modules: the catalytic module and thecarbohydrate-binding module (CBM) [19]. CBMs arethought to have one or more of the following functions:enzyme concentration on the surface of the substrate/proximity effect (the phase transfer); substrate targeting/selectivity; and disruption of non-hydrolytic crystallinesubstrate. CBMs that are specific for insoluble cellulosecan be grouped into two general categories: those thatinteract with crystalline cellulose (type A CBMs) andthose that interact with non-crystalline cellulose (cello-oligosaccharides in addition to insoluble cellulose) (typeB CBMs, [20] the so-called targeting function). Thesenon-catalytic modules readily adsorb to accessible siteson a cellulose-containing substrate to form a complexheld together by specific, non-covalent, thermodynami-cally favorable bonds [21]. Consequently, the catalyticmodule is aligned with the substrate to establish a high,local concentration of the enzyme on the cellulose sur-face (the so-called proximity function).Various researchers have shown that removal of theCBM component of individual cellulases reduces thehydrolytic activity of the catalytic module on insoluble,crystalline substrates such as microcrystalline cellulose(Avicel), cotton, and filter paper, whereas their activityon soluble or amorphous cellulose remains largely unaf-fected [5,22,23]. In addition, CBMs isolated from bothbacteria and fungi have been suggested to facilitate cel-lulose hydrolysis by physically disrupting the structureof the fibrous cellulosic network and releasing small par-ticles, without showing any detectable hydrolytic activity,which is normally quantified by the release of reducingsugars (the so-called disruptive function) [7,8]. In recentstudies investigating the morphological and structuralchanges of cotton fibers after treatment with purifiedCBM from fungal CBH1, it was found that CBM couldpromote non-hydrolytic disruption of crystalline cellu-lose by weakening and splitting the hydrogen bonds (asobserved by infra-red spectroscopy and X-ray diffrac-tion), thereby freeing cellulose chains [24,25]. Moleculardynamic simulations also provided a nanoscopic view ofthe mechanism, showing that strong and mediumhydrogen bonds decreased dramatically when CBM wasbound to the cellulose surface of cotton fibers [24].Furthermore, CBM treatment of cellulosic fibers (What-man CF11) has also been shown to reduce the interfiberinteraction (disaggregation of agglomerates between thefibers, as observed by scanning electron microscopy)through steric and hydrophobic effects, which wouldincrease the cellulose surface area [26].Earlier workers [8] proposed that CBMs bind to thecellulose fibers and penetrate the fibrillar network atsurface discontinuities, subsequently releasing cellulosefragments that are non-covalently associated with thefiber but bonded to the underlying microfibrils. Theyalso suggested that further penetration by the CBMthen exfoliates the fiber structure, releasing the ends ofcellulose chains, which remain bound to the fiber,resulting in a roughening of the surface. In related work,Lee et al. [27], using atomic force microscopy, observedslightly elongated holes that were left throughout thesurface of cotton fibers after they were treated withhexachloropalladate-inactivated T. reesei CBH I. It wassuggested that these holes were a result of the penetra-tion of the CBM into the cellulose fibers [27]. Incuba-tion of cotton fibers with cellulase from Thermotogamaritima, which lacks CBM, had no effect on the sur-face of cotton fibers.In a similar fashion to the proposed disruptive activityof CBM from CBH1, other CBMs have also been shownto display disruptive activity upon binding onto othernon-soluble polysaccharides such as chitin (a cellulosederivative where the 2-hydroxy group is substituted withan acetamido group) and starch. One such component,CBP21 (~20 kDa), produced by Serratia marcescens,belongs to CBM family 33 and is known to bind to crys-talline b-chitin (which has chitin chains arranged in asimilar fashion to that of cellulose I) and stronglyenhance chitin hydrolysis by chitinases [28,29]. This isthought to be due to increased substrate availabilityafter disruption of the crystalline chitin structure[28,29]. It was suggested that the binding of CBP21 tochitin led to the disruption of the crystalline substratestructure through specific polar interactions that werenot only important for binding, but also for alteration ofthe substrate structure [29]. In related work, Zeltins andSchrempf [28] showed that the chitin-binding proteinCHB1 secreted by Streptomyces olivaceoviridis interactsspecifically with crystalline a-chitin by binding andpenetrating into the structure of the substrate. Anotherexample of a CBM with disruptive activity comes fromthe starch-binding domain (SBD) of Aspergillus nigerglucoamylase I, an exo-acting enzyme that releases glu-cose from the non-reducing ends of the polymer chains[30,31]. This glucoamylase contains an SBD with twobinding sites for starch [30,31]. These sites have beenshown to help with crystalline starch hydrolysis and alsohelp promote disruption of a-glucan interchain bindingArantes and Saddler Biotechnology for Biofuels 2010, 3:4http://www.biotechnologyforbiofuels.com/content/3/1/4Page 4 of 11at the surface of granular starch, thereby enhancingenzymatic degradation of crystalline starches by gluco-amylase I [30,31].Building on the C1-CX model of cellulose hydrolysis,Russian researchers [32-35] proposed a mechanism to tryto explain the dispersion of cellulose [30-33] (Figure 2).They proposed that cellulases are adsorbed to cellulosedefects (disturbances in the crystalline structure of cellu-lose, such as microcracks) (Figure 2a), followed by theirpenetration into the interfibrillar spaces (Figure 2b). Thisconsequently would induce a mechanical action (disper-sion) of the cellulose structure. It was suggested that thepresence of the large enzyme within such a narrow spacecauses an increase in the mechanical pressure exerted onthe cavity walls, swelling the cellulose structure andaccommodating more and more water molecules betweenthe microfibrils (Figure 2c). The water within the defectspenetrates further and further inside the capillary space,breaking the hydrogen bonds between the cellulosechains, resulting in the disassociation of the individualmicrofibrils (Figure 2c). In turn, the adsorbed enzymesprevent the solvated chains and free chain ends from rea-ligning and readhering [32,36].Recent computational simulations have indicated thatthe water solutions in contact with microcrystalline cel-lulose surfaces are highly structured and that thesestructured water layers might inhibit molecular diffusionclose to the cellulose surface [37]. During enzymatichydrolysis, this would limit the approach of cellulasestowards the cellulose surfaces [37]. More recent compu-ter simulation studies with T. reesei CBH I action onmicrocrystalline cellulose Ib showed that the CBMderived from this enzyme showed no tendency to dis-sociate from the cellulose surface [38], although it wasobserved to move about slightly on the surface [39].This finding indicated that the suggested structuredwater layers may not be as problematic as originally sug-gested. Once the enzyme is adsorbed onto the cellulosesurface, a processive hydrolysis mechanism would befaster than a mechanism that requires diffusion awayfrom and subsequent repenetration of the hydrationlayers [37].Figure 2 Schematic representation of amorphogenesis of cellulose fibers mediated by the carbohydrate-binding module (CBM) ofcellobiohydrolase I (CBHI) (adapted from [36]). For clarity, the carbohydrate-binding module is oversized compared with the catalytic domain.Arantes and Saddler Biotechnology for Biofuels 2010, 3:4http://www.biotechnologyforbiofuels.com/content/3/1/4Page 5 of 11Although the functions of CBMs during enzymatichydrolysis of cellulose have not been fully elucidatedand continue to be the subject of research, it seems rea-sonable to believe that the primary role of a CBM is toanchor the catalytic module to cellulose. This anchoringby the CBM is generally accepted to increase the effec-tive concentration of cellulases onto the solid substrate,thereby assisting the enzyme through the phase transferfrom the soluble fraction (enzyme) to the insoluble frac-tion (substrate) [40]. Complementing this, CBM mayalso have a more active role in the depolymerization ofcellulose by influencing the cellulose structure throughthe non-hydrolytic release of single cellulose chainsfrom the highly ordered and tightly packed regions ofmicrofibrils, which might occur by disrupting the inter-microfibrillar associative forces [6,32-36] and by feedingthe newly exposed chains through the tunnel-shapedcatalytic module for hydrolysis.ExpansinsExpansins are plant-derived proteins, which were firstidentified in the early 1990s and are primarily knownfor their unique ‘loosening’ effect on the cellulosic net-work within plant cell walls during growth [41-43].Two families of expansins have currently been charac-terized: a- and b-expansins [44,45]. Although they shareonly about 20% of their amino acid identity, they are ofsimilar size (~27 kDa), contain a number of conservedresidues and characteristic motifs distributed throughoutthe length of the protein, and their predicted secondarystructures share up to 75% identity. However, theyappear to act on different cell wall components [44,45].Expansins usually consist of two domains (D1 andD2) connected by a peptide linker [44,45]. D1 showsstructural and sequence similarity to the catalytic sitefound in family-45 glycosyl hydrolyses (GH45) whosemembers have been characterized as endoglucanases[44-48]. Although D1 has conserved much of theGH45 catalytic site, it lacks hydrolytic activity on cellwall polysaccharides [42,43]. Recently, Yennwar et al.[44] suggested that expansins do not display hydrolyticactivity due to lack of a second aspartate (a key part ofthe catalytic machinery required for glucan hydrolysisby GH45 enzymes) in D1 [44]. D2 was initially specu-lated to be a CBM on the basis of conserved aromaticand polar residues on the surface of the protein[44,46]. However, recent studies on the structure ofexpansins have identified two potential polysaccharidebinding surfaces, one of which corresponds to the bur-ied D2 face contacting D1 [44]. In addition, the find-ings that the linker coupling D1 and D2 is very shortand that the multiple contacts between D1 and D2could enable close coupling of the two domains suggestthat the two domains, when closely packed and alignedtogether, could form a potential polysaccharide-bindingsurface spanning D1 and D2 [44].Most evidence suggests a non-hydrolytic action ofexpansins that enlarges cell wall cavities by bindingpolysaccharides and disrupting non-covalent bondswithin cellulose microfibrils and between other cell wallpolysaccharides attached to the microfibrils [44,45]. Inaddition, Yennawar et al. [44] suggested that expansinsact as a molecular device that uses the strain energystored in a taut cellulose-binding glycan to help dissoci-ate the glycan from the surface of cellulose.Despite the lack of hydrolytic activity in expansinsthemselves, some studies have shown that expansinsenhance the enzymatic hydrolysis of crystalline celluloseby cellulases [49]. It has been proposed that this syner-gistic action is a result of the expansins making the glu-can chains within the microfibrils more accessible to thecellulases [45]. In this model, expansins are believed toact like a zipper opening the crosslinking of cellulosemicrofibrils by ungluing the chains that stick themtogether, which in turn enhances cellulose accessibility,thereby speeding cellulase action [50,51]. For instance,Baker et al. [10], using yellow poplar sawdust pretreatedwith dilute acid, showed that extremely small additionsof expansin along with T. reesei cellulases (ratio ~0.012)was sufficient to induce up to a 13% increase in cellu-lose conversion compared with the sugar yield obtainedwhen cellulase was used alone.Expansin-like proteinsSome proteins produced by bacteria and fungi havebeen shown to have sequence similarity to plant expan-sins [10,11,52-56]. Kerff et al. [52] determined the struc-ture and activities of one of the proteins secreted byBacillus subtilis, a Gram-positive soil bacterium capableof colonizing the surface of plant roots. These authorsconsidered this protein (EXLX1) to be a member of theexpansin superfamily [52], based on its structural simi-larity to plant expansins (including its two-domainstructure, with the precise spatial alignment of the twodomains resulting in an open binding surface spanningboth domains), its ability to bind to cell walls, its plantcell wall extension activity and its lack of hydrolyticactivity against major polysaccharides of the plant cellwall.When EXLX1 was used along with low levels ofT. reesei cellulase enzymes (ratio 10:1) to hydrolyzemicrocrystalline cellulose (Avicel), it enhanced cellulosehydrolysis but not beyond the enhancement observedwith bovine serum albumin (BSA), which was used as anonspecific control [52]. This lower cellulolytic enhancingactivity was attributed to the weak plant cell wall exten-sion activity of EXLX1 (10-fold weaker than that of plantb-expansins) [52]. Under such hydrolysis conditions (highArantes and Saddler Biotechnology for Biofuels 2010, 3:4http://www.biotechnologyforbiofuels.com/content/3/1/4Page 6 of 11EXLX1:cellulase enzymes ratio), it cannot be ruled outthat the higher concentration of EXLX1 in comparison tocellulase enzymes might have resulted in competition forbinding sites between cellulases and EXLX1. BecauseBSA only loosely binds to Avicel [57], competition forbinding sites between cellulases and BSA is not expected.This would explain why the cellulolytic enhancing activityof EXLX1 was lower than that of BSA. In contrast to thecellulolytic enhancing activity observed with EXLX1, theenhancement obtained by the addition of BSA was not aresult of disrupting activity. It has previously been shownthat cellulase enzymes adsorb to the inner wall of thereaction vessel during hydrolysis [58,59]. Thus, whenhigh concentrations of BSA relative to cellulase enzymes(10:1) were used, it is likely that this high protein addi-tion prevented or at least reduced adsorption of cellulasesto the wall of the reaction vessel, resulting in moreenzymes being available to react with the Avicel. Thiswould lead to higher cellulose conversion compared withhydrolysis being carried out in the absence of BSA.In related work, the EXLX1 gene was expressed inEscherichia coli; the purified recombinant protein dis-played cellulose-binding and cellulose-weakening activ-ities towards filter paper, indicating its functionalhomology with plant expansins [11]. Moreover, at muchlower EXLX1:cellulase enzymes ratios than those usedin previous work [52], the recombinant EXLX1 proteinwas found to promote significant cellulolytic enhancingactivity when mixed with a commercial T. reesei cellu-lase mixture during hydrolysis of filter paper. This wasshown when it was compared with the control contain-ing only filter paper and cellulase and with the negativecontrol containing filter paper, BSA and cellulaseenzymes [11]. The ratio of the recombinant EXLX1 pro-tein and cellulase enzymes was found to be a crucialdeterminant of the cellulolytic enhancing activity, withthe highest synergistic activity (5.9-fold) observed at thelowest cellulase loading (0.012 filter paper units (FPU)/gfilter paper) and the highest recombinant protein load-ing (300 μg/g filter paper). However, under this low cel-lulase loading, the cellulose conversion was < 10% of thetheoretical maximum, and at higher cellulase loading(0.6 FPU/g filter paper, giving ~20% cellulose conver-sion), the synergistic activity was insignificant [11].Another example of expansin-like proteins is a proteinisolated from T. reesei, a well-known cellulolytic fungus[53]. This expansin-like protein (named swollenin dueto its ability to swell cotton fibers) contains an amino-terminal fungal-type cellulose-binding module linked tothe plant expansin homologous module [53]. Saloheimoet al. [53] reported the sequence similarity of swolleninto the fibronectin (Fn)III-type repeats of mammaliantitin proteins. These latter proteins have been shown tobe able to unfold and refold easily, allowing the proteinto stretch. This ability might be important for swolleninif its function is to allow slippage of cellulose microfi-brils in plant cell walls, as suggested for expansins.Swollenin has also been shown to disrupt the struc-ture of the cotton fibers, weaken filter paper and pro-mote an apparent dispersion of Valonia cell wallstructure [53]. This ability to disrupt solid substrates isunlikely to be the result of hydrolytic activity, as noreducing sugars were detected [53]. This would seem toindicate that swollenin is inactive against the b-1,4-gly-cosidic bonds in cellulose, suggesting that swollenin mayshare a similar role with expansins in swelling the cellu-losic network within cell walls. Saloheimo et al. [53]have reported that swollenin is an important componentin the enzyme mixture required for degradation of lig-nocellulosic biomass and hence, a potential candidatefor the C1-induced dispersion proposed by Reese et al.[10]. In addition, there is evidence [53] that the swolle-nin gene is regulated in a manner similar to that of theT. reesei cellulase genes, so that low expression levelsoccur in the presence of glucose and high expressionlevels occur in the presence of cellulose [53].The observation that microbial proteins containing anexpansin-like domain, such as swollenin in T. reesei [53]and EXLX1 protein in B. subtilis [52], can enhance rootcolonization, suggest that expansin-type modules havebeen adapted by diverse microbes to facilitate theirinteractions with plants [52]. It seems that several swol-lenin-like activities are displayed by T. reesei, which mayvary in their modes of action but would contributesynergistically to the efficient hydrolysis of the plantpolysaccharides [53]. Similarly to the potential role ofexpansins in enhancing the efficiency of enzymatichydrolysis of cellulose, it has been suggested that swolle-nin would increase the access of cellulases to cellulosechains by promoting dispersion of cellulose aggrega-tions, exposing individual cellulose chains to interactionswith cellulases. Although the cellulolytic enhancingactivity of swollenin has not been assessed, recently achimeric enzyme associating T. reesei swollenin with anAspergillus niger feruloyl esterase was constructed andfound to significantly increase the efficiency of ferulicacid release from lignocellulosic substrate [60].Yellow affinity substanceIt has been shown that some cellulolytic bacteria, espe-cially strains of the thermophilic anaerobic Clostridiumthermocellum, produce an unidentified, yellow, water-insoluble substance when growing on cellulose [61,62].Similarly to CBMs in fungal cellulases, this yellow sub-stance has been shown to have a strong affinity for crys-talline cellulose and to be part of the bacterialcellulolytic system required for efficient enzymaticdegradation of cellulose [61-63]. Production of thisArantes and Saddler Biotechnology for Biofuels 2010, 3:4http://www.biotechnologyforbiofuels.com/content/3/1/4Page 7 of 11‘yellow affinity substance’ has been observed to precedethe production of cellulases and also to be involved inthe hydrolysis of cellulose by facilitating the binding ofthe cellulolytic enzyme complexes to cellulose [61,64].Kopecny et al. [62] showed that endoglucanase and cel-lobiohydrolase activities were increased in the presenceof the yellow affinity substance.Despite some similarities in functions to that ofCBMs, no substantial research has subsequently beenconducted to investigate the exact means by which theyellow substance enhances cellulose saccharification.Other non-hydrolytic proteinsRecently, an unknown non-hydrolytic protein (Zea h), ofapproximately 56 kDa, purified from fresh postharvestcorn stover (the unused plant parts left after harvest),was shown to decrease the hydrogen-bond intensity andcrystallinity index of filter paper [65]. It also increasedthe adsorption of cellulase onto cellulosic substrates,which in turn increased the conversion of cellulose toglucose by a factor of 3.2, and accelerated hydrolysis byincreasing hydrolysis rate of cellulases by a factor of 2[65]. Although the Zea h protein appears to have poten-tial to enhance the cellulolytic activity of cellulaseenzymes, the mechanism involved in this enhancementand the three-dimensional structure of the proteinremain to be resolved.Several fungal proteins with homology to family 61glycosyl hydrolase (GH61) have also been reported toshow cellulolytic enhancing activity on a variety of pre-treated lignocellulosic substrates when combined withT. reesei cellulases [66,67]. For instance, the expressionof Thielavia terrestris GH61 in T. reesei allowed for areduction in protein loading of 1.4-fold to reach 90%conversion of the cellulose in corn stover pretreatedwith steam [67]. Based on the lack of hydrolytic activityof GH61 on pretreated lignocellulosic substrates and ona variety of cellulosic and hemicellulosic model sub-strates, it was suggested that the cellulase-enhancingeffect of such proteins is limited to substrates containingother cell wall-derived materials such as hemicelluloseor lignin [67]. However, no clear correlation wasobserved between the proportion of these non-celluloly-tic components and the degree of enhancementobserved [67]. Although it has not been experimentallyestablished, rather than acting on cellulose microfibrilsthemselves, GH61 proteins could be acting via disrup-tion of non-covalent bonds between cellulose and thenon-cellulolytic materials (as observed with some expan-sins [68]), resulting in increased access of cellulases tothe cellulose microfibrils and enhancing the overall cel-lulolytic activity of the cellulase complex.T. reesei Cel61B, which was previously thought to bean endoglucanase [69], is the only GH61 protein so farto have its three-dimensional structure resolved [70].The structure appears to lack any suitable catalytic cen-tre. However, a possible catalytic role has been specu-lated for the bound cation (nickel or other transitionmetals), given the highly conserved binding site in theGH61 proteins [70]. CBP21, a non-catalytic carbohy-drate binding protein reported to disrupt the insolublecrystalline b-chitin structure and enhance chitin hydro-lysis by chitinases as described earlier, is the proteinwhose structure is most similar to that of Cel61B. It ispossible therefore that Cel61B may also have somedirect or indirect role in the enzymatic degradation ofcellulose [70]. However, the exact mechanism and func-tion of Cel61B and other related GH61 proteins has yetto be fully resolved.Low molecular weight peptides or phenolate-typecompounds produced by ‘brown rot’ wood-decayingfungi (mainly Basidiomycota) are thought to mediate thenon-hydrolytic/nonenzymatic attack of the lignocellulosematrix [71-73]. This attack is thought to increase poresize, consequently enhancing the diffusion of cellulaseswithin the substrate [71-78]. These nonhydrolytic/none-nzymatic reactions mediated by low molecular weightcompounds have been shown to enhance the activity ofcommercial cellulases and brown rot endoglucanasesduring hydrolysis of pure cellulose and various lignocel-lulosic substrates [79,80]. In addition, it has been sug-gested that this initial attack swells the ordered packingof the cellulose chains, exposing new end-groups of thefibrous cellulosic substrate (enhancing accessibility) tothe attack of cellulases, as evidenced by a significantdecrease in the crystallinity of the cotton fibers [81].When the overall modification of milled spruce woodwas examined using pyrolysis-molecular beam massspectrometry coupled with multivariate analyses, it wasapparent that the non-hydrolytic/nonenzymatic-mediated reactions could more readily open the struc-ture of the lignocellulosic matrix, freeing cellulose fibrils[78], which indicated that this non-hydrolytic/nonenzy-matic mechanism could be, in brown rot fungi, a poten-tial candidate for the C1-induced disruption proposed byReese et al. [10].ConclusionConsiderable progress has been made in elucidating thenature, type and mechanism of cellulases when soluble,short chain oligosaccharides are assumed to be the sub-strate. However, when the recalcitrant, largely inaccessi-ble nature of the cellulosic substrate is considered, theexact biochemical mechanisms involved in the delami-nation, dispersion and swelling of cellulose has beenmuch discussed but still remains largely unknown. Ithas been suggested that disruption of the highly orderedand tightly packed regions of the cellulose structureArantes and Saddler Biotechnology for Biofuels 2010, 3:4http://www.biotechnologyforbiofuels.com/content/3/1/4Page 8 of 11facilitates the exposure of inaccessible cellulose chainsburied within these regions, thereby enhancing enzymeaccess to cellulose, which is expected to speed thehydrolytic attack of cellulases. In this context, some pro-teins have been proposed as having an active role in thesolubilization of cellulose by affecting (weakening, swel-ling) the cellulose structure via the non-hydrolyticrelease of the previously enzyme-inaccessible individualcellulose chains. Although the mechanism by whicheach of these proteins attack cellulose has yet to beresolved, the observation that most of these swelling ordelaminating agents contain a (potential) carbohydrate-binding surface may indicate that this binding modulemay play an important role in this non-hydrolytic amor-phogenesis activity. It is apparent that further researchis needed to better understand the possible mechanismsof these proposed amorphogenesis-inducing agents.Moreover, it is also possible that the enzymatic hydroly-sis of cellulose occurs as just an external surface phe-nomenon. However this is unlikely as, althoughrelatively slow, the rate of cellulose hydrolysis indicatesthat there must be some creation of new surfaces withinthe cellulose matrix. However, how this delamination,swelling and dispersion action of the cellulase complexoccurs has yet to be fully determined.AcknowledgementsNatural Sciences and Engineering Research Council of Canada (NSERC),Natural Resources Canada (NRCan), International Energy Agency (IEA),Genome BC and Novozymes are all gratefully acknowledged for the supportof our group’s work. We thank our colleagues within the UBC ForestProducts Biotechnology/Bioenergy Group for stimulating discussions on thetopic of this review.Authors’ contributionsVA and JNS conceptualized, researched and wrote the manuscript. Allauthors read and approved the final manuscript.Competing interestsThe authors declare that they have no competing interests.Received: 13 November 2009 Accepted: 23 February 2010Published: 23 February 2010References1. Walker LP, Wilson DB: Enzymatic hydrolysis of cellulose: An Overview.Biores Technol 1991, 36:3-14.2. 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