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The enhancement of enzymatic hydrolysis of lignocellulosic substrates by the addition of accessory enzymes… Hu, Jinguang; Arantes, Valdeir; Saddler, Jack N Oct 5, 2011

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The enhancement of enzymatic hydrolysis oflignocellulosic substrates by the addition ofaccessory enzymes such as xylanase: is it anadditive or synergistic effect?Hu et al.Hu et al. Biotechnology for Biofuels 2011, 4:36http://www.biotechnologyforbiofuels.com/content/4/1/36 (5 October 2011)RESEARCH Open AccessThe enhancement of enzymatic hydrolysis oflignocellulosic substrates by the addition ofaccessory enzymes such as xylanase: is it anadditive or synergistic effect?Jinguang Hu, Valdeir Arantes and Jack N Saddler*AbstractBackground: We and other workers have shown that accessory enzymes, such as b-glucosidase, xylanase, andcellulase cofactors, such as GH61, can considerably enhance the hydrolysis effectiveness of cellulase cocktails whenadded to pretreated lignocellulosic substrates. It is generally acknowledged that, among the several factors thathamper our current ability to attain efficient lignocellulosic biomass conversion yields at low enzyme loadings, amajor problem lies in our incomplete understanding of the cooperative action of the different enzymes acting onpretreated lignocellulosic substrates.Results: The reported work assessed the interaction between cellulase and xylanase enzymes and their potential toimprove the hydrolysis efficiency of various pretreated lignocellulosic substrates when added at low proteinloadings. When xylanases were added to the minimum amount of cellulase enzymes required to achieve 70%cellulose hydrolysis of steam pretreated corn stover (SPCS), or used to partially replace the equivalent cellulasedose, both approaches resulted in enhanced enzymatic hydrolysis. However, the xylanase supplementationapproach increased the total protein loading required to achieve significant improvements in hydrolysis (anadditive effect), whereas the partial replacement of cellulases with xylanase resulted in similar improvements inhydrolysis without increasing enzyme loading (a synergistic effect). The enhancement resulting from xylanase-aidedsynergism was higher when enzymes were added simultaneously at the beginning of hydrolysis. This co-hydrolysisof the xylan also influenced the gross fiber characteristics (for example, fiber swelling) resulting in increasedaccessibility of the cellulose to the cellulase enzymes. These apparent increases in accessibility enhanced the steampretreated corn stover digestibility, resulting in three times faster cellulose and xylan hydrolysis, a seven-folddecrease in cellulase loading and a significant increase in the hydrolysis performance of the optimized enzymemixture. When a similar xylanase-aided enhancement strategy was assessed on other pretreated lignocellulosicsubstrates, equivalent increases in hydrolysis efficiency were also observed.Conclusions: It was apparent that the ‘blocking effect’ of xylan was one of the major mechanisms that limited theaccessibility of the cellulase enzymes to the cellulose. However, the synergistic interaction of the xylanase andcellulase enzymes was also shown to significantly improve cellulose accessibility through increasing fiber swellingand fiber porosity and also plays a major role in enhancing enzyme accessibility.* Correspondence: jack.saddler@ubc.caForestry Products Biotechnology/Bioenergy Group, Wood ScienceDepartment, University of British Columbia, 2424 Main Mall, Vancouver BC,V6T 1Z4, CanadaHu et al. Biotechnology for Biofuels 2011, 4:36http://www.biotechnologyforbiofuels.com/content/4/1/36© 2011 Hu et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative CommonsAttribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction inany medium, provided the original work is properly cited.BackgroundA crucial step in the bioconversion of lignocellulosicfeedstocks to biofuels is to cost-effectively maximize thesaccharification of the cellulose and hemicellulose com-ponents to fermentable sugars. One of the challenges isthe still too high enzyme costs involved in the saccharifi-cation of the cellulosic component [1,2] and, to a lesserextent, the loss of some of the hemicellulosic sugars dur-ing pretreatment [3]. Thus, in many pretreatment strate-gies such as steam explosion, mild severity conditions areoften used to avoid, or at least minimize, sugar loss dur-ing pretreatment [4]. Under these milder pretreatmentconditions, some of the hemicellulose, mostly xylan inagricultural residues and hardwood, remains associatedwith the cellulosic-rich water insoluble fraction [5]. How-ever, this residual hemicellulose component is known toexert a significant influence on the effectiveness of enzy-matic hydrolysis of its cellulosic component [6-10].The hemicellulose-degrading enzyme activities detectedin most of the commercially available cellulase prepara-tions are too low, or are insufficiently active enough, toachieve significant conversion of the residual hemicellulose[11,12]. Therefore, supplementation of the cellulaseenzymes with the so called ‘accessory enzymes’, such asxylanase-enriched preparations, has been the most com-mon approach to increase the overall fermentable sugaryields from pretreated hardwoods [8,13] and agriculturalresidues [12,14]. Although accessory enzymes offer thepotential to increase substrate digestibility, the highdosages of enzyme supplementation applied in many ofthe past studies can be difficult to justify because of theincreased enzyme costs that are incurred. It is generallyacknowledged that among the several factors that hamperour current ability to attain efficient lignocellulosic bio-mass conversion yields at low enzyme loadings, a majorproblem lies in our incomplete understanding of the coop-erative action of the different enzymes acting on pre-treated lignocellulosic substrates.Hemicellulose, which is generally found in higher con-centrations on the outer surface of cellulose fibers but isalso diffused into the interfibrillar space through fiberpores [15,16], has been proposed to act as a physical bar-rier that limits the accessibility of the cellulase enzymesto the cellulose [7,8,17,18]. In recent work, we and otherworkers have also shown that the limited access of cellu-lase enzymes to the cellulose chains is a key factor whichnecessitates the use of relatively high enzyme dosages toattain effective cellulose hydrolysis [19]. One of the mainbeneficial effects of cellulase supplementation with xyla-nase during biomass saccharification is thought to be theresult of the improved cellulose accessibility as a result ofxylan solubilization [6,12].It has been suggested that xylan forms a sheath oneach cellulose microfibril and that it is also ‘zipped into’the cellulose microfibrils during crystallization, shortlyafter cellulose synthesis [20,21]. Thus it might be antici-pated that the depolymerization of cellulose by cellulaseswithin the fiber would expose the xylan chains naturallytrapped within or between microfibrils to the action ofxylanase. If true, this degradation model might indicatethe potential synergism between xylanase and cellulaseenzymes during lignocellulose hydrolysis. Previous worklooking at xylanase-aided bleaching treatment of Kraftcellulosic pulps showed that the xylanase not onlyhydrolyzed the reprecipitated xylan on the fiber surfacebut also increase pulp fiber porosity, resulting in a sub-stantial increase in the permeability of the cellulosicpulp fibers [18,22,23].The synergistic action among the multiple forms ofhemicellulose-degrading enzymes (for examples, enzymesacting on the xylan backbone and on xylan side chains)and also among the cellulose-degrading enzymes (such asexoglucanases and endoglucanases) has been studiedextensively [9,24-26]. Although synergistic cooperationbetween cellulases and an endoxylanase have beenobserved at low substrate conversion yields [9,27,28], lim-ited work has looked at the interaction between xylanaseand cellulases at conditions relevant to the biofuelsindustry. This is when relatively low enzyme loadings areused to achieve fast and nearly complete hydrolysis of thecellulose. Similarly, the lack of relevant controls in thisprevious body of work has made it difficult to determineif the beneficial effect of the accessory enzyme additionwas a result of a cooperative interaction (synergism) withthe cellulases or merely an additive effect, as theincreased substrate hydrolyzability upon enzyme supple-mentation was typically associated with a correspondingand often substantial increase in protein loading.In the work reported here, the possible additive orsynergistic interaction of xylanase with cellulases wasdetermined during the hydrolysis of steam pretreated cornstover (SPCS) at the minimum enzyme loading requiredto achieve substantial (greater than 70%) cellulose hydroly-sis. Initially, two strategies were evaluated; xylanase sup-plementation of the minimum cellulase dose required foreffective cellulose hydrolysis, and replacement of a portionof the minimum cellulase dose with xylanase while keep-ing the total protein loading constant. The degree ofsynergism (DS) was calculated and compared at variousxylanase:cellulase ratios. To further investigate the xyla-nase-cellulase interaction mechanisms, hydrolysis experi-ments were also carried out by adding the enzymes(cellulases and xylanase) separately, simultaneously andsequentially. The changes in the gross fiber characteristicsof SPCS were also monitored over the course of hydrolysiswith or without xylanase addition. Finally, the beneficialhydrolysis-boosting effect of xylanase was further evalu-ated on a range of lignocellulosic substrates. In all of theHu et al. Biotechnology for Biofuels 2011, 4:36http://www.biotechnologyforbiofuels.com/content/4/1/36Page 2 of 13hydrolysis experiments, controls containing BSA and addi-tional cellulolytic enzymes at equivalent protein loadingsto the xylanase were carried out in an attempt to evaluatewhether xylanolytic activity offers any advantage over sim-ply increasing the protein dose or the overall cellulaseactivity.ResultsThe protein content and specific activities of the enzymepreparationsInitially, the protein concentrations and specific activ-ities of the three commercial glycoside hydrolase pre-parations used in this study were determined andcompared (Table 1). As expected, all preparationsdemonstrated substantial differences in their proteincontent and specific activities towards model substrates.Novozyme 188, which is commonly used as b-glucosi-dase supplementation, displayed the highest proteinconcentration and b-glucosidase activity. The cellulaseenzymes cocktail Celluclast 1.5 L showed the highestactivity towards all of the cellulosic substrates, while theother enzyme preparations had below detectable levelsof filter paper activity and contained very low carboxy-methyl cellulase (CMCase) activity. The Celluclast pre-paration contained low levels of endoxylanase activity,indicating its low hydrolytic capability towards xylan. Incontrast, the Multifect Xylanase showed very highendoxylanase activity relative to the other enzyme pre-parations and very low activities towards the cellulosicsubstrates, indicating its overall low saccharificationactivity towards cellulose.Effect of xylanase supplementation on the enzymatichydrolysis of SPCSIn order to assess the influence of xylanase addition dur-ing the enzymatic hydrolysis of the SPCS substrate, thehydrolysis experiments were carried out over a range ofincreasing dosage of cellulase activity (Celluclast) in theabsence and presence of fixed amounts of xylanase activ-ity (Multifect Xylanase) (Figure 1).In the absence of xylanase, cellulose and xylan hydrolysisincreased with increasing cellulase loading and reached aplateau at a cellulase protein loading of 35 mg/g cellulose(Figure 1). At this enzyme dosage, the cellulose and xylanhydrolysis were about 70% and 80%, respectively. A furtherincrease in cellulase loading beyond 35 mg/g celluloseresulted in only marginally improved hydrolysis yields.Alternatively, when the SPCS substrate was hydrolyzedwith increasing cellulase loading in the presence ofxylanase (60 mg/g cellulose), a significant increase in cellu-lose hydrolysis was observed, with more than 80% of thecellulose hydrolyzed at a cellulase loading of 5 mg/g cellu-lose, compared to only about 45% in the absence of xyla-nase supplementation. As expected, xylan hydrolysis alsoincreased significantly from 56% in the absence of xylanasesupplementation to over 95% with xylanase supplementa-tion. Further increasing the cellulase loading beyond5 mg/g cellulose in the presence of xylanase supplementa-tion did not significantly increase either cellulose or xylanhydrolysis.Interestingly, the addition of xylanase (60 mg/g cellu-lose) alone, in the absence of cellulases, resulted in only60% of the xylan being hydrolyzed and further increasesin xylanase loading did not improve the hydrolysis ofthe xylan (data not shown). However, the addition oflow amounts of cellulases (5 mg/g cellulose) significantlyincreased xylan hydrolysis (Figure 1).It appears that the supplementation of cellulases withxylanase not only substantially increased the rate ofhydrolysis of both the cellulose and xylan, it alsoincreased the extent of hydrolysis. For example, the maxi-mum degree of cellulose and xylan hydrolysis in theabsence of xylanase supplementation were about 75%and 93%, respectively, at a cellulase loading of 95 mg/gcellulose, whereas cellulose and xylan hydrolysis wereabout 80% and 100%, respectively, when xylanase wasadded (60 mg/g cellulose), even at relatively low cellulaseloadings of 5 mg/g cellulose. This increase suggested thatthe residual xylan present in the pretreated SPCS sub-strate played an important role in limiting the ease of cel-lulose hydrolysis.Cellulase replacement with xylanase versus cellulasesupplementation with xylanaseAlthough it was clear that xylanase supplementationimproved both cellulose and xylan hydrolysis at variouslevels of cellulase loading (Figure 1), it appeared that asignificant xylanase-boosting effect was only achievedTable 1 Protein content, filter paper activity and specific activities of the commercial enzyme preparations on modelsubstratesEnzymepreparationProtein content(mg/mL)FPA(FPU/mL)CMCase(U/mL)b-glucosidase(U/mL)CBH 1(U/mL)Xylanase (U/mL)b-xylosidase(U/mL)Celluclast 1.5L 129.2 50.3 474.7 17.0 158.0 438.8 37.8Multifect Xylanase 37.1 n/a 9.0 12.7 2.7 2588.4 22.5Novozym 188 233.4 n/a 15.0 239.0 26.2 32.63 3.9CMCase: carboxymethyl cellulase; CBH1: cellobiohydrolase 1; FPA: filter paper activity; FPU: filter paper unit; n/a: negligible activity detected.Hu et al. Biotechnology for Biofuels 2011, 4:36http://www.biotechnologyforbiofuels.com/content/4/1/36Page 3 of 13when low cellulase loadings were used. To further evalu-ate the interaction between the cellulase and xylanaseenzymes during hydrolysis of SPCS, two sets of hydroly-sis were carried out. In the xylanase supplementationapproach, varying amounts of xylanase (5 to 60 mg/gcellulose) were added to the minimum amount of cellu-lases (35 mg/g cellulose) which had previously beendetermined to be required for 70% of the cellulose to behydrolyzed (Figure 1). In the cellulase replacementapproach, varying amounts of the cellulases werereplaced with xylanase (up to 86% cellulase replacementon a protein basis) while the total amount of enzymeadded, on a protein basis, was kept constant at 35 mg/gcellulose.It was apparent that supplementing the cellulases withvarying amounts of xylanase increased both the celluloseand xylan hydrolysis (Table 2). However, the DS betweencellulases and xylanase was about 1, indicating that theobserved improvement in SPCS hydrolyzability was more aproduct of increased enzyme loading. This could be termedan additive effect, as the saccharification performance ofthe unsupplemented cellulase mixture was 22.8 mg sugars/mg enzymes while the saccharification performance of thexylanase-supplemented cellulase mixture was 21.5 mgsugars/mg enzymes. Alternatively, when relatively smallamounts of the cellulases were replaced with xylanase, aslight increase in the degree of synergism was observed,suggesting synergistic cooperation between the cellulaseand xylanase enzymes (Table 2). Although lower amountsof cellulase enzymes were present under these conditions(71% cellulase and 29% xylanase) as compared to hydrolysisruns with no cellulase replacement (100% cellulase, 35 mg/g), the same levels of SPCS hydrolysis were obtained (74%cellulose and 82% xylan hydrolysis, Table 2). As the per-centage of cellulases replaced by xylanase was increased(up to 86%), the degree of synergism also increased. Underthe conditions tested, the highest degree of synergism wasobserved at a cellulase and xylanase loading of 5 and30 mg/g cellulose, respectively, which resulted in a substan-tial increase in both cellulose and xylan hydrolysis (86%cellulose and 99% xylan hydrolysis, Table 2). It appearsthat with this enzyme ratio, the xylanase and cellulasesworked synergistically to hydrolyze the SPCS, resulting inan enzyme mixture with a relatively high saccharificationperformance (27.3 mg sugars/mg enzymes). Similar SPCShydrolysis yields could be achieved by replacing cellulaseswith xylanase to a total final protein loading of 35 mg/gcellulose, as compared to increasing the enzyme dosage bysupplementing the 35 mg cellulases/g cellulose withincreasing amounts of xylanase (Table 2). As the enzymemixture containing 5 mg of cellulases/g cellulose and30 mg xylanase/g cellulose displayed the highest degree ofFigure 1 Conversion of SPCS at increasing cellulase doses with or without xylanase supplementation (60 mg/g cellulose) after 72 hhydrolysis. Relationship between xylan removal and cellulose conversion after 72 h hydrolysis at various enzyme doses (inset). Full line:hydrolysis in the presence of xylanase; dashed line: hydrolysis in the absence of xylanase. SPCS: steam pretreated corn stover.Hu et al. Biotechnology for Biofuels 2011, 4:36http://www.biotechnologyforbiofuels.com/content/4/1/36Page 4 of 13synergism (1.62) during the hydrolysis of the SPCS sub-strate, this mixture was used to try to better understandthe mechanism behind this observed synergism.Simultaneous, separate, and sequential hydrolysisMost of the previous work in this area has reported anincrease in substrate digestibility upon xylanase supple-mentation, without further explanation of the likelymechanism behind the cooperative interaction betweenboth types of enzymes. To try to further clarify themechanisms behind the observed synergistic cooperationbetween the cellulases and xylanase, various time coursehydrolysis profiles were followed using simultaneous, sepa-rate, and sequential addition of cellulases and xylanase(Figure 2).It was apparent that, regardless of the enzymes additionstrategy used, much of the xylan was solubilized duringthe first three hours of hydrolysis (Figure 2B). The simul-taneous (cellulases plus xylanase) and the separate addi-tion (cellulases) strategies showed similar trends ofincreased xylan removal after 72 hours hydrolysis, result-ing in about 100% and 50% xylan solubilization respec-tively. However, when xylanase was added alone (30 mg/gcellulose), xylan hydrolysis leveled off after 24 hours withonly about 60% of the original xylan solubilized. It is likelythat the readily accessible xylan was hydrolyzed in the firstfew hours of hydrolysis and that in order to access thexylan that is more closely associated with the cellulose and‘buried’ within the fiber structure, the synergistic interac-tion with cellulases is required for more extensive xylansolubilization (Figure 2B). The addition of xylanase aloneleft one third of the original xylan in the SPCS, even ifvery high xylanase loading (100 mg/g cellulose) was used(data not shown). However, when a sequential cellulaseaddition approach was used (Figure 2B), the hydrolysisprofile followed the same trend as when the cellulases andxylanase were added simultaneously at the beginning ofthe hydrolysis. However the final xylan hydrolysis yieldswere lower (Figure 2B).It was apparent that the addition of xylanase alone(30 mg/g cellulose) resulted in limited (5%) cellulosehydrolysis (Figure 3A). However, when cellulases (5 mg/gcellulose) were added to the xylanase pre-hydrolyzedSPCS (sequential hydrolysis, Figure 2A), cellulose hydroly-sis increased sharply from 5% at 24 hours of hydrolysis toabout 53% at 48 hours, which was significantly higher thanthe cellulose hydrolysis achieved when adding only cellu-lases (36% at 48 hours of hydrolysis). This cellulose hydro-lysis yield was also higher than would have been the sumof cellulose hydrolysis obtained with cellulases and xyla-nase when used separately (theoretical conversion). It wasapparent that pre-hydrolyzing SPCS with xylanase resultedin enhanced cellulose hydrolysis when cellulases were sub-sequently added.Table 2 Effect of cellulase supplementation with xylanase and cellulase replacement with xylanase on cellulose andxylan hydrolysis, and on the degree of synergism during hydrolysis of SPCS after 72 hHydrolysis strategy Total protein(mg/g cellulose)Enzyme mixture Xylanase (% total proteinpreparation)DS Cellulosehydrolysis (%)Xylanhydrolysis (%)Xylanasesupplementation40 35 mg C + 5 mgX13 0.99 73.9 80.145 35 mg C + 10mg X22 0.96 74.1 83.645 35 mg C + 10mg BSA0 n/a 72.9 81.395 35 mg C + 60mg X63 1.02 87.1 100Cellulasereplacement35 35 mg C 0 n/a 72.1 81.625 mg C + 10mg X29 0.98 74.1 82.615 mg C + 20mg X57 1.09 74.2 82.610 mg C + 25mg X71 1.31 82.3 98.65 mg C + 30 mgX86 1.62 86.3 99.35 mg C + 30 mgBSA0 n/a 55.8 68.6Separate hydrolysis 5 5 mg C 0 n/a 45.6 56.330 30 mg X 100 n/a 5.2 61.4Separate hydrolysis used as control. BSA: bovine serum albumin; C: cellulases; DS: degree of synergism; SPCS: steam pretreated corn stover; X: xylanase; n/a: notapplicable.Hu et al. Biotechnology for Biofuels 2011, 4:36http://www.biotechnologyforbiofuels.com/content/4/1/36Page 5 of 13A B Figure 2 Time course of SPCS hydrolysis. Separate hydrolysis: (black rhombus) 5 mg cellulases or (black triangle) 30 mg xylanase;Simultaneous hydrolysis: (black circle) 5 mg cellulases and 30 mg xylanase; Sequential hydrolysis: addition of (black square) 5 mg cellulases,(clear circle) 5 mg xylanase and (clear rhombus) 5 mg BSA to pre-hydrolyzed SPCS with 30 mg xylanase for 24 h. Theoretical: (asterisk) sum ofcellulose conversion after hydrolysis with 30 mg xylanase and 5 mg cellulases separately. (A) Cellulose hydrolysis. (B) Xylan hydrolysis. BSA:bovine serum albumin; SPCS: steam pretreated corn stover.Hu et al. Biotechnology for Biofuels 2011, 4:36http://www.biotechnologyforbiofuels.com/content/4/1/36Page 6 of 13BA    CFigure 3 Change in SPCS fiber properties during separate and simultaneous hydrolysis with cellulases and xylanase after 24 h. (A)Fiber length distribution. (B) Average fiber width. (C) Fiber surface area (combination interior/exterior) determined by Simons’ stainingtechnique. Enzyme loading (mg/g cellulose): cellulases (5), xylanase (30) and BSA (30). Control: substrates were incubated at the same conditionwithout the addition of enzymes. BSA: bovine serum albumin; SPCS: steam pretreated corn stover.Hu et al. Biotechnology for Biofuels 2011, 4:36http://www.biotechnologyforbiofuels.com/content/4/1/36Page 7 of 13When both types of enzymes were added simulta-neously (simultaneous hydrolysis), the enzymatic digest-ibility of SPCS was substantially increased (Figure 2A).For example, when the enzymes were added together,after 12 hours hydrolysis about 50% of the cellulose washydrolyzed, whereas in the absence of xylanase, only 20%of the cellulose was hydrolyzed. After 72 hours, approxi-mately 45% of the cellulose was hydrolyzed when usingcellulase enzymes (5 mg/g cellulose) alone, whereas simi-lar hydrolysis yields could be achieved in just 10 hourswhen xylanase was supplied together with cellulases atthe beginning of hydrolysis. It was apparent that thecombined addition of cellulases and xylanase not onlyresulted in higher hydrolysis rates but also substantiallyincreased the extent of SPCS hydrolysis. After 72 hours,cellulose hydrolysis was almost two-fold higher (87%) ascompared to hydrolysis carried out in the absence ofxylanase (45%). To ensure that there were no othermechanisms at play, such as a possible ‘detergent’ effectof added protein, a protein control of BSA was shown tohave almost no effect (Figure 2).Pulp fiber propertiesHemicellulose is known to contribute to fiber strengthand its solubilization is known to influence pulp fiberproperties. One method that has been successfully usedby the pulp and paper sector to evaluate changes at thefiber level is the use of a fiber quality analyzer (FQA). Byusing this equipment we hoped to determine any changesin the fiber dimensions and fiber size distribution of theunhydrolyzed and residual SPCS after simultaneous andseparate hydrolysis with cellulase and xylanase additionover 24 hours (Figure 3A and 3B).When compared to the unhydrolyzed SPCS control,xylanase addition alone did not result in any changes inthe fiber length distribution (Figure 3A). In contrast, cel-lulase treatment resulted in a significant decrease in thepopulation of fibers with lengths less than 0.15 mm andalso a slight increase in the population of fibers withlengths within the range of 0.15 to 0.35 mm (Figure 3A).The latter group of fibers is likely due to the fragmenta-tion of long fibers into shorter fibers, as the average fiberlength after 24 hours decreased from 0.555 mm to about0.270 mm. Earlier work has shown that the smaller fiberswere rapidly hydrolyzed and solubilized [29]. When thexylanase and cellulases were added simultaneously, a dif-ferent pattern was observed, with a significant increase inthe population of fibers in the length range of 0.09 to0.23 mm (Figure 3A). There was also significantly morefiber fragmentation as the average fiber length wasreduced from 0.55 mm to about 0.22 mm. The largeamount of fibers in the length range of 0.09 to 0.23 mmwas also likely due to the higher fragmentation of SPCSfibers, resulting in the higher numbers of shorter fibers.We next measured changes in the fiber width of the resi-dual SPCS after hydrolysis, as this value can provide a gen-eral indication of the degree of fiber swelling (Figure 3B).As was observed with the fiber length values, xylanasetreatment alone promoted only a slight change in the aver-age fiber width, whereas cellulase addition increased thefiber width by about 10%. A significant change in fiberwidth was observed when both the xylanase and cellulaseswere added simultaneously, with the fiber width increasingby about 30% as compared to the untreated fibers, and by20% as compared to the cellulase alone-treated SPCSfibers. This increase in fiber width suggested a significantincrease in fiber swelling as a result of the synergistic coop-eration of the cellulases and xylanase.To see if we could quantify any increases in accessibil-ity of the SPCS fibers to the enzymes, we used theSimons’ stain method, which has previously been shownto provide a good estimation of cellulose accessibility[19]. An increase in the orange dye (DO) to blue dye(DB) ratio after xylanase treatment indicated that moreof the cellulose was accessible, probably due to theremoval of the xylan in the SPCS fibers (Figure 3C).The addition of cellulases alone resulted in a decrease inthe DO:DB ratio primarily because of the decreasingamount of substrate that was available to measure ashydrolysis proceeded.The effect of xylanase supplementation on the enzymatichydrolysis of other pretreated lignocellulosic substratesTo determine if the xylanase-boosting effect observed dur-ing hydrolysis of SPCS could also be observed with otherbiomass substrates, steam pretreated sweet sorghumbagasse (SPSB) and steam pretreated lodgepole pine(SPLP) were hydrolyzed with similar combinations of cel-lulases and xylanase as were carried out with the SPCSsubstrate (Figure 4). The SPSB had a higher xylan contentthan did the SPCS substrate, whereas the xylan content ofthe SPLP was negligible (Table 3). The effectiveness ofhydrolysis of both the SPSB and SPLP substrates wasassessed using a total protein loading of 35 mg/g cellulose(5 mg cellulases plus 30 mg xylanase/g cellulose; 5 mg cel-lulases plus 30 mg BSA/g cellulose). The simultaneousaddition of xylanase and cellulases also significantlyenhanced the cellulose hydrolysis of both the SPSB andSPLP substrates. Interestingly, xylanase supplementationboosted the cellulose hydrolysis of the SPLP substrateeven though the xylan in this substrate was below detect-able levels. This suggested that the mechanism behind thexylanase-boosting effect of cellulose hydrolysis in pre-treated biomass is not solely due to increasing celluloseaccessibility through xylan removal and that it alsoinvolves contributions to changes in fiber morphology aswas shown with the FQA and Simons’ stain valuesobtained during the hydrolysis of the SPCS substrate.Hu et al. Biotechnology for Biofuels 2011, 4:36http://www.biotechnologyforbiofuels.com/content/4/1/36Page 8 of 13DiscussionEffective enzymatic hydrolysis of the cellulose and hemicel-lulose present in pretreated lignocellulosic substrates tofermentable sugars has been shown by many workers torequire a combination of various glycoside hydrolaseswhose combined action is believed to be more efficientthan the sum of the actions of the individual enzymes. Thisis the basis of the so called ‘synergistic enzyme interaction’effect. Many studies have looked at the synergistic interac-tions among the major cellulase components and differentexplanations have been given for their interactions onmodel cellulosic substrate. Hypotheses include the creationof new hydrolysis sites, removal of physical obstacles [25],presence of stereospecific enzyme activities, and the forma-tion of enzyme-enzyme complexes [24,30,31]. Morerecently, we and other workers have shown that someaccessory enzymes, such as b-glucosidase, xylanase and thecellulase enhancing factors such as GH61, can considerablyenhance the hydrolysis effectiveness of cellulase cocktailswhen added to pretreated lignocellulosic substrates [32-34].The major beneficial effect of xylanase supplementa-tion to cellulase enzymes mixtures has frequently beensuggested to be a result of increasing cellulose accessi-bility to the cellulase enzymes due to the removal of thexylan coating on the outer surface of the pretreatedpulp fibers [6,12]. However, despite the potential toenhance hydrolysis yields by xylanase supplementation,one major reservation is the likely substantial increasein total protein loading that would be required, thusincreasing enzyme costs.In the work described here, our initial approach was todefine the minimum amount of cellulase enzymesrequired for effective hydrolysis and to assess the typeand extent of the interaction between xylanase and cellu-lase enzymes in an attempted to improve hydrolysis effi-ciency of pretreated lignocellulose at low proteinFigure 4 Improvement in cellulose hydrolysis yields in the presence of xylanase (30 mg/g cellulose) and cellulases (5 mg/g cellulose)as compared to hydrolysis yields in the presence of only cellulases (5 mg/g cellulose) after 72 h hydrolysis of various substrates:SPCS: steam pretreated corn stover; SPSB: steam pretreated sweet sorghum bagasse; SPLP: steam pretreated lodgepole pine.Table 3 Steam pretreatment conditions and chemical composition of pretreated lignocellulosic substratesSubstrate Pretreatment conditions Composition of pretreated feedstocks AbbreviationAra Gal Glu Xyl Man AILCorn stover 190°C, 5 minutes, 3% SO2 1.0 0.7 56.1 7.0 1.1 27.0 SPCSSweet sorghum bagasse 190°C, 5 minutes, 3% SO2 0.6 0.8 54.3 9.8 1.0 25.8 SPSBLodgepole pine 200°C, 5 minutes, 4% SO2 bdl bdl 46.4 bdl bdl 45.0 SPLPAIL: acid insoluble lignin; Ara: arabinan; bdl: below detectable level; Gal: Galactan; Glu: glucan; Man: mannan; Xyl: XylanHu et al. Biotechnology for Biofuels 2011, 4:36http://www.biotechnologyforbiofuels.com/content/4/1/36Page 9 of 13loadings. It was apparent that the type of interactionbetween xylanase and cellulase enzymes is dependent onthe total enzyme loading and enzymes ratio, as has beenobserved previously [26,35]. A strong synergistic effectwas observed at low cellulase loading and when a highxylanase to cellulase ratio was used. The highest DS(1.62) was observed at a xylanase to cellulase proteinloading of 6:1, which resulted in a considerable reduction(seven-fold) in the total amount of cellulase enzymesrequired for effective cellulose hydrolysis of the SPCSsubstrate. In related work, Bura et al. [8] and Berlin et al.[11] also found that a similar xylanase to cellulase ratioimproved cellulose hydrolysis. However, in these earlierworks it was more of an additive effect rather than a trulysynergistic interaction.There are several possible explanations for the strongsynergistic interaction observed between cellulases andxylanase. As mentioned earlier, xylanase have been sug-gested to remove the xylan coat on the surface of pulpfiber, improving cellulose accessibility to cellulase enzymes[6,8,12]. In a related work, Pauly et al. [20] found that onethird of the xyloglucan present in a dicot plant wasentrapped within the microfibril, and its removal requiredthe action of both cellulase and xyloglucanase enzymes.Xylanases have also been suggested to increase the pro-portion of substrate available for productive cellulaseinteraction due to their unproductive binding to sites onlignin [36,37]. The xylanase-enriched preparation used inthis work belongs to the glycoside hydrolase family 11(GH11), which lacks a carbohydrate binding module [38],and this enzyme component has been shown to preferen-tially bind to lignin [39]. Previous work has shown that lig-nin has very little influence on the xylanase activity ascompared to cellulases and b-glucosidase [40]. Therefore,for the work reported here, an increase in cellulase avail-ability due to xylanase binding to lignin is unlikely to bethe major reason for the observed enhancement of cellu-lase activities in the presence of xylanase.Xylanases have also been shown to result in the solubili-zation of lignin fractions from pretreated lignocellulosicbiomass by breaking down the lignin-carbohydrate com-plex and consequently improving substrate digestibility[18,22]. It is also worth noting that the xylanase-aidedbleaching treatment of cellulosic pulps promoted physicalchanges to the pulp fibers, such as an increase in fiber por-osity and fiber disintegration [18,22,23]. Similar changesduring hydrolysis of lignocellulosic biomass are likely toincrease the available specific surface area of the celluloseto cellulase enzymes and therefore the effectiveness of thecellulases, a process termed amorphogenesis [41].Since it has been predicted that a diverse range of plantbiomass will be needed to satisfy the projected demandsfor advanced biofuels [42], the hydrolysis boosting abilityof xylanase was also evaluated on a range of cellulosicmaterials. It was apparent that xylanase treatment couldsignificantly improve the cellulose hydrolysis of all of thelignocellulosic substrates assessed, even for the steampretreated softwood that contained virtually no xylan.This further supports the proposal that one of the mainbeneficial effects of the synergistic interaction betweenxylanase and cellulases is the substantial change in thegross fiber characteristics (for example, fiber swelling)which were observed. It is likely that this synergismoccurs in a similar fashion to the amorphogenesis effectthat has been suggested in cofactors such as GH61 or cel-lulose binding modules to enhance the effectiveness ofcellulase enzymes [19].In summary, it appears that the observed xylanase-boosting effect during hydrolysis of pretreated lignocel-lulosic biomass is a result of both increased celluloseaccessibility to cellulase enzymes as a result of xylanremoval from pulp fibers, and the synergistic interactionof the xylanase and cellulase enzymes increasing cellu-lose accessibility through increasing fiber swelling andfiber porosity.ConclusionIt was apparent that the overall protein loading requiredto achieve fast, nearly complete hydrolysis of a model cel-lulosic substrate (SPCS) could be significantly reduced bymaking use of the synergistic interaction that occursbetween cellulases and xylanase. It is likely that theadded xylanase enhanced overall hydrolysis by solubiliz-ing xylan, which impedes access to the cellulose. The‘xylanase-boosting’ effect was observed on a range of pre-treated lignocellulosic materials, regardless of their xylancontent. So called accessory enzymes such as xylanasemight offer considerable potential to increase the overallperformance of cellulase enzyme mixtures, while redu-cing the protein loading required to achieve effectivehydrolysis of pretreated lignocellulosic substrates.MethodsLignocellulosic biomass preparation and compositionCorn stover, sweet sorghum bagasse and lodgepole pinewere steam pretreated according to previously describedprocedures [12,43]. The chemical composition of thewater insoluble fraction (cellulose-rich material) afterpretreatment was determined using the modified Klasonlignin method, derived from the TAPPI standardmethod T222 om-88 [3]. The values determined afterthe various pretreatment conditions are described inTable 3.Enzyme preparationsCellulases (Celluclast 1.5L, Novozymes, Franklington, NC),from Trichoderma reesei, b-glucosidase (Novozym 188,Novozymes A/S, Bagsvaerd, Denamark) from AspergillusHu et al. Biotechnology for Biofuels 2011, 4:36http://www.biotechnologyforbiofuels.com/content/4/1/36Page 10 of 13niger, and xylanase (Multifect Xylanase, Genencor US Inc.,Palo Alto, CA) from a genetically modified strain of T. ree-sei were used. The protein content and enzyme specificactivities of the enzyme preparations are summarized inTable 1. The filter paper activity was determined accord-ing to International Union of Pure and Applied Chemistry[44]. Xylanase and CMCase activities were determined asdescribed elsewhere [45]. Cellobiohydrolase 1, b-xylosidaseand b-glucosidase activities were determined usingp-nitrophenyl-b-D-cellobioside, p-nitrophenyl-b-D-xylo-pyranoside, and p-nitrophenyl-b-D-glucopyranoside assubstrates, respectively, as described previously [46]. Pro-tein concentration was measured using the Ninhydrinassay using BSA as the protein standard [47].Enzymatic hydrolysisThe hydrolysis experiments were carried out at 2% (w/v)solids loading in sodium acetate buffer (50 mM, pH 5.0).The reaction mixtures (1 mL) were mechanically shakenin an orbital shaker incubator (Combi-D24 hybridizationincubator, FINEPCR®, Yang-Chung, Seoul, Korea) at 50°Cin accordance with previously described methods [19].Three sets of hydrolysis were carried out. Initially, therequired minimum cellulase loading for efficient cellulosehydrolysis in the absence and presence of xylanase(60 mg/g cellulose) and the effect of xylanase wereassessed by hydrolyzing SPCS at increasing cellulase load-ings (5 to 100 mg/g cellulose). To further assess themechanism of the xylanase-boosting effect during SPCShydrolysis, the hydrolysis of SPCS was carried out usingtwo strategies for enzyme addition. In the first, xylanasesupplementation, varying amounts of xylanase (0 to100 mg/g cellulose) were supplemented to the cellulasemixture (35 mg/g cellulose). In the second, cellulase repla-cement, varying amounts of the total cellulase loading(35 mg/g cellulose) was replaced (up to 86%) with theexact amount of xylanase at a fixed total protein dosage(35 mg/g cellulose). Finally, the SPCS was hydrolyzed withseparate, simultaneous, and sequential additions of cellu-lases (5 mg/g cellulose) and xylanase (30 mg/g cellulose).Sequential hydrolysis was carried out by incubating theSPCS with xylanase for 24 h. Thereafter, cellulases wereadded to the pre-hydrolyzed mixture and incubated for48 h. Separate and simultaneous hydrolyzed were carriedout over 72 h.In all of the hydrolysis assays, b-glucosidase was sup-plemented at a cellobiase units to filter paper unitsactivity ratio of 1:2 to limit end-product inhibition. Theaddition of xylanase was based on the total cellulosecontent, as the overall goal of xylanase addition is toenhance cellulose hydrolysis. Protein controls of BSAwere used to assess the effect of the addition of a non-hydrolytic protein when compared to the addition ofxylanase.At the end of hydrolysis, samples were heated at 100°Cfor 10 min to inactivate the enzymes. Supernatants werecollected after centrifugation at 13,000 rpm for 10 min.The concentration of glucose and xylose in the superna-tants were measured using HPLC (Dionex DX-3000,Sunnyvale, CA) as described elsewhere [48]. The hydroly-sis yields of the pretreated substrates were calculatedfrom the cellulose and xylan content as a percentage ofthe theoretical cellulose and xylan available in the sub-strates. All hydrolysis experiments were performed induplicate and mean values and standard deviations arepresented.The following equation was used to calculate thedegree of synergism between cellulase enzymes andxylanase during SPCS hydrolysis:DS =GCmixture∑GCindividualwhere GCmixture is the cellulose hydrolysis achievedwith cellulases and xylanase added together, and ∑GCin-dividual is the sum of cellulose hydrolysis achieved withthe individual enzymes.Assessment of fiber gross characteristicsTo assess changes in fiber characteristics during hydro-lysis, a FQA (LDA02; OpTest Equipment, Inc., Hawkes-bury, ON, Canada) was used to monitor fiber lengthand width according to the procedure described pre-viously [49]. The settings on the FQA were adjusted tomeasure particles down to 0.07 mm, and the fiberlength distribution and average fiber width were mea-sured as described previously [19].The changes in available surface area were determinedusing a modified version of the Simons’ staining techniquepreviously used to evaluate cellulose accessibility to cellu-lase enzymes [19,50]. The increase in cellulose accessibilitywas expressed as an increase in the DO to DB ratio.List of abbreviationsBSA: bovine serum albumin; CMCase: carboxymethyl cellulase; DB: blue dye;DO: orange dye; DS: degree of synergism; FQA: fiber quality analyzer; HPLC:high performance liquid chromatography; SPCS: steam pretreated cornstover; SPLP: steam pretreated lodgepole pine; SPSB: steam pretreated sweetsorghum bagasse.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 would like to thank Novozymesand Genecor for the donations of the enzymes preparations.JH gives his sincere appreciation to the China Scholarship Council on behalfof the Ministry of Education of China for the financial support and all authorsthank Amadeus Pribowo, Keith Gourlay, Richard Chandra and all the membersof the UBC FPB/Bioenergy Group for invaluable discussions and help.Authors’ contributionsAll authors contributed jointly to all aspects of the work reported in themanuscript. JH carried out much of the laboratory work, contributed toHu et al. Biotechnology for Biofuels 2011, 4:36http://www.biotechnologyforbiofuels.com/content/4/1/36Page 11 of 13planning, interpretation of results and drafting of the paper. VA contributedto the planning, interpretation and drafting. JS contributed to the planning,interpretation and writing of the manuscript. 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Starcher B: A ninhydrin-based assay to quantitate the total proteincontent of tissue samples. Anal Biochem 2001, 292(1):125-129.48. Boussaid A, Robinson J, Cai YJ, Gregg DJ, Saddler JR: Fermentability of thehemicellulose-derived sugars from steam-exploded softwood (Douglasfir). Biotechnol Bioeng 1999, 64(3):284-289.49. Robertson G, Olson J, Allen P, Chan B, Seth R: Measurement of fiberlength, coarseness, and shape with the fiber quality analyzer. Tappi J1999, 82(10):93-98.50. Chandra R, Ewanick S, Hsieh C, Saddler JN: The characterization ofpretreated lignocellulosic substrates prior to enzymatic hydrolysis, part1: a modified Simons’ staining technique. Biotechnol Prog 2008,24(5):1178-1185.doi:10.1186/1754-6834-4-36Cite this article as: Hu et al.: The enhancement of enzymatic hydrolysisof lignocellulosic substrates by the addition of accessory enzymes suchas xylanase: is it an additive or synergistic effect? Biotechnology forBiofuels 2011 4:36.Submit your next manuscript to BioMed Centraland take full advantage of: • Convenient online submission• Thorough peer review• No space constraints or color figure charges• Immediate publication on acceptance• Inclusion in PubMed, CAS, Scopus and Google Scholar• Research which is freely available for redistributionSubmit your manuscript at www.biomedcentral.com/submitHu et al. Biotechnology for Biofuels 2011, 4:36http://www.biotechnologyforbiofuels.com/content/4/1/36Page 13 of 13

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