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The synergistic action of accessory enzymes enhances the hydrolytic potential of a “cellulase mixture”… Hu, Jinguang; Arantes, Valdeir; Pribowo, Amadeus; Saddler, Jack N Aug 3, 2013

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RESEARCH Open AccessThe synergistic action of accessory enzymesenhances the hydrolytic potential of a “cellulasemixture” but is highly substrate specificJinguang Hu, Valdeir Arantes, Amadeus Pribowo and Jack N Saddler*AbstractBackground: Currently, the amount of protein/enzyme required to achieve effective cellulose hydrolysis is still toohigh. One way to reduce the amount of protein/enzyme required is to formulate a more efficient enzyme cocktailby adding so-called accessory enzymes such as xylanase, lytic polysaccharide monooxygenase (AA9, formerlyknown as GH61), etc., to the cellulase mixture. Previous work has shown the strong synergism that can occurbetween cellulase and xylanase mixtures during the hydrolysis of steam pretreated corn stover, requiring lowerprotein loading to achieve effective hydrolysis. However, relatively high loadings of xylanases were required. Whenfamily 10 and 11 endo-xylanases and family 5 xyloglucanase were supplemented to a commercial cellulase mixturevarying degrees of improved hydrolysis over a range of pretreated, lignocellulosic substrates were observed.Results: The potential synergistic interactions between cellulase monocomponents and hemicellulases from family10 and 11 endo-xylanases (GH10 EX and GH11 EX) and family 5 xyloglucanase (GH5 XG), during hydrolysis ofvarious steam pretreated lignocellulosic substrates, were assessed. It was apparent that the hydrolytic activity ofcellulase monocomponents was enhanced by the addition of accessory enzymes although the “boosting” effectwas highly substrate specific. The GH10 EX and GH5 XG both exhibited broad substrate specificity and showedstrong synergistic interaction with the cellulases when added individually. The GH10 EX was more effective onsteam pretreated agriculture residues and hardwood substrates whereas GH5 XG addition was more effective onsoftwood substrates. The synergistic interaction between GH10 EX and GH5 XG when added together furtherenhanced the hydrolytic activity of the cellulase enzymes over a range of pretreated lignocellulosic substrates.GH10 EX addition could also stimulate further cellulose hydrolysis when added to the hydrolysis reactions when therate of hydrolysis had levelled off.Conclusions: Endo-xylanases and xyloglucanases interacted synergistically with cellulases to improve the hydrolysisof a range of pretreated lignocellulosic substrates. However, the extent of improved hydrolysis was highly substratedependent. It appears that those accessory enzymes, such as GH10 EX and GH5 XG, with broader substratespecificities promoted the greatest improvements in the hydrolytic performance of the cellulase mixture on all ofthe pretreated biomass substrates.* Correspondence: jack.saddler@ubc.caForestry Products Biotechnology/Bioenergy Group, Wood ScienceDepartment, University of British Columbia, 2424 Main Mall, Vancouver, BCV6T 1Z4, Canada© 2013 Hu 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.Hu et al. Biotechnology for Biofuels 2013, 6:112http://www.biotechnologyforbiofuels.com/content/6/1/112BackgroundAchieving good sugar yields from pretreated lignocellulosicsubstrates when using low enzyme loadings continues tobe a challenge for biochemically based biomass-to-biofuelsprocesses [1,2]. One approach that various groups haveassessed is to formulate more efficient enzyme cocktails byadding so-called accessory enzymes, such as hemicellulasesand lytic polysaccharide monooxygenase (AA9, formerlyknown as GH61) [3-5], and/or non-hydrolytic, chain separ-ating proteins such as swollenin and expansin [6-8] to theenzyme mixture. Recent work has shown that, when family11 endo-xylanases were added to commercial cellulasepreparations, their synergistic cooperation not only sub-stantially enhanced the hydrolysis extent of both theglucan and xylan present in steam pretreated corn sto-ver, it also dramatically reduced the required cellulasedosage (about 7 times) needed to achieve reasonablecellulose hydrolysis yields (>70%) [3]. Even thoughhigher amount of xylanase was still required to achievebetter hydrolysis, the overall total protein loading eitherdid not change or was slightly lower.Based on their amino acid sequence similarities, most xy-lan hydrolyzing enzymes belong to either glycoside hydro-lase family 10 (GH10) or family 11 (GH11) [9-11]. Family10 endo-xylanases (GH10 EX) have been shown to pre-dominantly attack glycosidic linkages next to a substitutedxylose residue, requiring at least two unsubstituted xyloseresidues and it tends to exhibit broad catalytic versatility[9,12]. In contrast, family 11 endo-xylanases (GH11 EX)require at least three consecutive unsubstituted xyloseresidues. GH11 EX cannot cleave glycosidic linkages nextto a branch [10,13], and they are sometimes characterizedas “true” xylanases because of their exclusive activity on D-xylose containing substrates [14].Although there is a considerable amount of informa-tion known about the differences between these twomajor families of endo-xylanases regarding their struc-ture, catalytic mechanisms and specific activities on“model” xylanolytic substrates [12,15,16], there has beenlittle work carried out regarding their hydrolytic poten-tial or their interactions with cellulases during hydrolysisof industrially relevant pretreated lignocellulosic substrates[15-17]. However, previous work has shown that GH10 EXaddition was more effective on corn fibre and hydrother-mally pretreated wheat straw [16,17] while GH11 EX wasmore hydrolytic on destarched wheat bran arabinoxylan[15]. Although synergistic cooperation between GH10 EXand GH11 EX was observed [16,18] during the hydrolysisof corn fiber, a similar level of synergism was not observed[15] during hydrolysis of arabinoxylan obtained from wheatbran. A related study [19] showed that the addition of bothGH10 EX and GH11 EX along with other enzymes resultedin a more effective enzyme cocktail when hydrolysing thecellulosic component of ammonia fiber expansion (AFEX)pretreated corn stover. This earlier work suggested thatGH10 EX had broad catalytic specificity and that this en-zyme might prove to be a better candidate for enhancingthe hydrolysis of more realistic biomass substrates.Xyloglucanase addition has also been shown to enhancethe hydrolyzability of various lignocellulosic substrateswhen added to a cellulase mixture [20,21]. Xyloglucanaseis known to hydrolyze xyloglucan, which is one of themajor components of the primary cell wall of higherplants and it has been shown to limit the accessibility ofcellulase enzymes to the cellulosic component [20,22-24].Benko et al., [20] showed that a xyloglucanase fromTrichoderma reesei enhanced the catalytic performance ofa cellulase mixture during hydrolysis of several lignocel-lulosic substrates. Related work also showed that thedepolymerization and re-arrangement of the linkages inxyloglucan by hydrolases or transferases was an essentialstep in plant cell wall expansion and deposition during cellgrowth [25]. This suggested that the cleavage of glycosidiclinkages within the xyloglucan resulted in the swelling ofthe cellulose microfibrils [22,24] which has been shown toincrease cellulose accessibility and, consequently, effective-ness of enzyme hydrolysis [22].The work reported here has looked at the potentialsynergistic interaction between cellulases and severalhemicellulosic hydrolyzing enzymes (GH11 EX, GH10EX), and a family 5 xyloglucanase (GH5 XG) during theenzymatic hydrolysis of various steam pretreated lignocel-lulosic substrates. As well as adjusting the “cocktails” at thebeginning on the hydrolysis we also tried to “re-start” hy-drolysis by the addition of further enzymes when the rateof hydrolysis had leveled off. As is explained later, althoughGH11 EX exhibited higher activity on “model” xylanolyticsubstrates, GH10 EX addition was better able to boost thehydrolytic potential of the cellulase monocomponentsduring hydrolysis of pretreated lignocellulosic substrates.However, the observed improvements in hydrolysis yieldswere highly substrate dependent.ResultsChemical composition of pretreated substratesOf the three pretreated agricultural residues (corn stover,sweet sorghum bagasse, and corn fiber), the steampretreated sorghum bagasse (SPSB) and the steampretreated corn stover (SPCS) showed only slight differ-ences in their xylan content (9.8% and 7.0%, respect-ively) (Table 1). As expected, the steam pretreated cornfiber (SPCF) had a much higher xylan content (15.3%)with the highly branched xylan backbone resulting in asignificant amount of arabinosyl (6.9%), galactosyl(2.8%), and mannosyl (2.2%) residues as compared tothe SPCS and SPSB substrates (Table 1). When the pop-lar chips were steam pretreated at increasing severities,the cellulosic rich, water insoluble fractions had a xylanHu et al. Biotechnology for Biofuels 2013, 6:112 Page 2 of 12http://www.biotechnologyforbiofuels.com/content/6/1/112content of 6.6% (SP180) and 3.7% (SP200) respectively.These substrates were subsequently used to try to assesshow the relative amount of residual hemicellulose mightinfluence the benefit of adding accessory enzymes, suchas xylanases, on the overall ease of cellulose hydrolysis.The steam pretreated lodgepole pine (SPLP) containedundetectable levels of mannan, typically found in soft-wood hemicelluloses (Table 1).Determination of enzymatic activities using “model”substratesThe xylanase, xyloglucanase, endo-glucanase, β-glucosidaseand β-xylosidase activities of the purified enzymes weredetermined as detailed in the material and methods section(Table 2). As expected, both family endo-xylanases wereable to effectively hydrolyze all of the xylan “model”substrates, while the other enzymes showed very low or un-detectable levels of activity. The GH11 EX showed substan-tially higher hydrolytic activity (190–230 U/mg) on all ofthe xylan substrates than did GH10 EX (100–160 U/mg).This difference in activity was also observed previously [26]when using thermostable recombinant xylanases fromNonomuraea flexuosa and Thermoascus aurantiacus.When various p-nitrophenyl (pNP) substrates were used toassess any differences between the two xylanases, the GH10EX showed detectable activities on pNPC, pNPG, andpNPX, suggesting that GH10 EX had a broader catalyticversatility and may also have higher hydrolysis efficiency to-wards short xylooligomers (p-NPX activity) as compared toGH11 EX. In contrast, the family 5 xyloglucanase (GH5XG) was the only enzyme that showed significant activitytowards xyloglucan (146 U/mg) (Table 2). It also displayednoticeable hydrolytic activity towards CMC (3.7 U/mg). Ofthe various cellulase monocomponents that were assessed,only Cel7A showed any activity on the p-NPC substrateand it also showed detectable levels of activity on other“model” substrates such as CMC, xylan (birch wood, beechwood, oat spelts) and xyloglucan (Table 2). As expected, β-glucosidase was the only enzyme that displayed notable ac-tivity on the p-NPG substrate (0.4 U/mg).Interaction of GH11 EX with cellulase monocomponentsEarlier work had shown the strong synergistic cooperationbetween a commercial cellulase mixture (NovozymesCelluclast 1.5 L) and a commercial xylanase mixture(Genencor Multifect Xylanase) during hydrolysis of SPCS[3]. This synergism resulted in a 7-fold reduction in thetotal amount of cellulase loading required to achieve asimilar extent of hydrolysis [3]. As the Multifect Xylanasepreparation used for this earlier work was enriched in sev-eral xylanases, we were not able to identify which proteinor proteins acted synergistically with the cellulases. To tryto identify which of the proteins resulted in this significantsynergistic interaction, we purified the major cellulasemonocomponents present in Celluclast 1.5 L and themajor xylanase present in Multifect Xylanases.The major protein within the Multifect Xylanase wasfound to be GH11 EX, as shown by tandem mass spec-trometry, with this protein constituting more than 80%of the total protein present in the mixture. As expected,it had a relatively low molecular weight of about 20 kDa(Figure 1). The major cellulase monocomponents withinCelluclast 1.5L, on a protein weight basis, were T. reeseiCel7A, Cel6A, Cel7B, and Cel5A (Figure 1), whichTable 1 Steam pretreatment conditions and chemical composition of pretreated lignocellulosic substratesSubstrate PretreatmentconditionsComposition of pretreated feedstocks AbbreviationAra Gal Glu Xyl Man AILCorn fiber 190°C, 5 min, 3% SO2 6.9 2.8 38.2 15.3 2.2 12.6 SPCFSweet sorghum bagasse 190°C, 5 min, 3% SO2 0.6 0.8 54.3 9.8 1.0 25.8 SPSBCorn stover 190°C, 5 min, 3% SO2 1.0 0.7 56.1 7.0 1.1 27.0 SPCSPoplar 180°C, 5 min, 4% SO2 0.3 0.8 59.8 6.6 1.3 30.4 SPP180200°C, 5 min, 4% SO2 0.3 0.8 59.3 3.7 1.2 33.9 SPP200Lodgepole pine 200°C, 5 min, 4% SO2 bdl bdl 46.4 bdl bdl 45.0 SPLPAra Arabinan, Xyl Xylan, Glu Glucan, Gal Galactan, Man Mannan, AIL Acid Insoluble Lignin, bdl below detectable level.Table 2 Specific activities (U/mg) of the purified enzymes assessed on “model” substratesEnzymes Birch wood xylan Beech wood xylan Oat spelts xylan Xyloglucan CMC p-NPC p-NPG p-NPXGH11 EX 193.2 191.5 229.3 n/a n/a n/a n/a n/aGH10 EX 103.6 119.2 162.9 n/a n/a <0.2 <0.1 0.5GH5 XG n/a n/a n/a 145.6 3.7 n/a n/a n/aCel7A <0.2 0.2 <0.2 1.24 <0.2 0.3 n/a n/aGH3 BG n/a 0.2 0.35 n/a n/a <0.1 0.4 n/an/a - negligible activity was detected.Hu et al. Biotechnology for Biofuels 2013, 6:112 Page 3 of 12http://www.biotechnologyforbiofuels.com/content/6/1/112comprise about 56%, 12%, 5%, and 6% of the total pro-tein respectively. These values were similar to the pro-portion of protein concentrations reported earlier byother workers [27,28].The possible synergistic interaction between the GH11EX and the cellulase monocomponents, Cel7A, Cel6A,and Cel5A was first assessed using both a “model” cellu-losic substrate (dissolving pulp, DP) and a pretreatedbiomass substrate (SPCS). As Cel7B had previously beenshown to exhibit significant levels of endo-xylanase ac-tivity [29,30], its interaction with GH11 EX was notassessed. When dissolving pulp was used as the sub-strate, the addition of GH11 EX enhanced the hydrolyticactivity of both of the exo-type cellulases (Cel7A andCel6A) but not the endo-type cellulase (Cel7B), as deter-mined by the amount of glucose released (Table 3). Whenthe enzyme interactions were assessed on the SPCS sub-strate, the addition of GH11 EX enhanced the hydrolyticpotential of all of the cellulase monocomponents (Cel7A,Cel6A, and Cel5A). In all cases, the highest enhanced cel-lulolytic activity promoted by GH11 EX was observed withCel7A (Table 3). Although the BSA protein control, usedto substitute for GH11 EX, also improved the hydrolyticactivity of the cellulase enzymes (Table 3), the slight in-crease in hydrolysis due to BSA addition was substantiallylower than the benefit observed after GH11 EX addition.This beneficial action combined with the previous obser-vation that GH11EX alone showed no hydrolytic activitytowards either the DP or SPCS substrates indicated thatthe greater hydrolytic potential exhibited by the cellulasemonocomponents after GH11 EX addition was the re-sult of the synergistic interaction of the GH11 xylanaseand cellulase monocomponents. As Cel7A displayed thehighest degree of synergism with GH11 EX on both theDP and SPCS substrates, it was used to try to better de-fine the mechanisms behind the observed synergisticinteraction between the enzymes.Enhancement of the hydrolytic activity of Cel7A onvarious lignocellulosic substrates by the addition ofaccessory enzymesThe potential of the hemicellulose-hydrolyzing enzymesGH10 EX, GH11 EX, and GH5 XG to enhance thehydrolytic activity of Cel7A was next assessed on thedissolving pulp and the various pretreated lignocellulosicsubstrates (Figure 2). It was apparent that both of theendo-xylanases (GH10 EX and GH11 EX) could effect-ively enhance the cellulolytic activity of Cel7A on all ofthe pretreated lignocellulosic substrates tested. Com-pared to GH11 EX, the addition of GH10 EX resulted inFigure 1 SDS-PAGE of purified enzymes: GH11 EX (lane 1), GH10 EX (lane 2), GH5 XG (lane 3), Cel7A (lane 4), Cel6A (lane 5), Cel7B(lane 6), Cel5A (lane 7), GH3 BG (lane 8) and marker (lane M). Proteins were identified by LC-MS/MS. Proteins are named according to theirglycoside hydrolase family.Table 3 Cellulose hydrolysis of dissolving pulp (DP) and steam pretreated corn stover (SPCS) by cellulasemonocomponents with or without supplemental GH11 EX after 72 hCel7A Cel7A + GH11 EX Cel7A + BSA Cel6A Cel6A + GH11 EX Cel6A + BSA Cel5A Cel5A + GH11 EX Cel5A + BSADP 41.4% 51.3% 48.4% 10.8% 12.1% 11.7% 9.6% 8.8% 8.6%SPCS 22.2% 30.1% 24.9% 3.4% 5.5% 3.9% 0.5% 1.5% 0.9%Control: BSA.Hu et al. Biotechnology for Biofuels 2013, 6:112 Page 4 of 12http://www.biotechnologyforbiofuels.com/content/6/1/112substantial increases in the hydrolytic action of Cel7A. Therange of improvement varied from 10 to 100% dependingon the substrate that was used. A greater increase in hy-drolysis due to GH10 EX addition was observed with therelatively higher xylan containing substrates such as SPCS(7.0%), SPSB (9.8%) and SPP180 (6.6%), resulting in ≥ 80%increase in the catalytic activity of the supplemented Cel7A(Figure 2). However, there was only a modest increase inthe hydrolytic activity of Cel7A when each of the endo-xylanases was supplemented during the hydrolysis of theSPCF substrate, despite its high xylan (15%) content(Table 1). Endo-xylanases also significantly improved thehydrolytic activity of Cel7A when added to substratescontaining very low or virtually no xylan such as the DPand SPLP substrates (Table 2). Although the addition ofthe BSA controls resulted in improvements in the range of0.5-20% (Figure 2), these increases in the hydrolytic poten-tial of Cel7A were again, substantially lower than thoseachieved after GH10 EX and GH11 EX supplementation.We next used the two steam pretreated poplar sampleswith differing hemicellulose content (SPP180 (6.6% xy-lan) and SPP200 (3.7% xylan)) to assess the possibleinfluence of the xylan on the hemicellulose-depolymerizingenzyme’s ability to improve the cellulolytic activity ofCel7A. The addition of the GH11 EX resulted in similarhydrolysis improvements (about 20%) on both substratesdespite the almost 2-fold higher xylan content in theSPP180 substrate as compared to the SPP200 substrate(Table 2). In contrast, GH10 EX addition improved theCel7A catalytic activity 4-fold when applied to the SPP180substrate (95%) compared to the hydrolysis yield observedwith the SPP200 substrate (24%).The trends observed in Figure 2 showed that GH5 XGaddition slightly improved Cel7A activity when acting onthe SPCF and SPP180 substrates whereas a significant im-provement (about 20%) was observed on the SPLP sub-strate. It appears that the greatest improvements in Cel7Ahydrolytic activity were observed when the supplementalenzymes were added to the hydrolysis of the pretreatedwood substrates (SPP180, SPP200, and SPLP).To try to assess the possible synergistic cooperationbetween cellulases and hemicellulases as a function ofhemicellulases loading, the SPCS was hydrolyzed withcellulases supplemented with varying amounts of GH10Figure 2 Relative improvement in cellulose hydrolysis by supplementation of accessory enzymes (GH11 EX, GH10 EX and GH5 XG) toCel7A during hydrolysis of various pretreated lignocellulosic substrates (SPCS, SPCF, SPSB, SPLP, SPP180 and SPP200) after 72 h.Substrate control: dissolving pulp (DP). Protein control: BSA.Hu et al. Biotechnology for Biofuels 2013, 6:112 Page 5 of 12http://www.biotechnologyforbiofuels.com/content/6/1/112EX. The titration curve indicated that the cellulose hy-drolysis boosting effect of adding xylanases decreasedslightly (from ~90% to ~85%) when GH10 EX loadingwas decreased by half (5 mg/g glucan). Further decreas-ing the GH10 EX enzyme loading to 3 and 1 mg/gglucan could still improve cellulose hydrolysis by ~50%and ~18%, respectively.Enhancement of the hydrolytic activity of Cel7A onvarious lignocellulosic substrates by the addition ofbinary mixtures of accessory enzymesTo assess the possible synergistic interaction betweenthe accessory enzymes and Cel7A, binary mixtures ofGH10 EX/GH11 EX, GH10 EX/GH5 XG, and GH11EX/GH5 XG were formulated and compared with thehydrolysis obtained when Cel7A was supplemented withGH10 EX alone (Figure 3). With the exception of thebinary mixture GH11 EX/GH5 XG, all of the otherbinary mixtures resulted in a similar or an increase inhydrolytic activity as compared to when Cel7A wassupplemented with GH10 EX alone. The greatest im-provement was observed with binary mixture GH10 EX/GH5 XG, which enhanced the glucose yields obtainedfrom the SPCS, SPP200, DP and SPLP substrates by 120,60, 60 and 20% respectively (Figure 3). As GH5 XG hadpreviously exhibited detectable CMCase activity (Table 2),we next substituted GH5 XG in the binary mixture GH10EX/GH5 XG with a purified endo-glucanase (Cel5A) toassess the interaction of GH10 EX with a “true”endoglucanase enzyme. Although the addition of theGH10 EX/Cel5A mixture resulted in the improvedhydrolytic activity of the Cel7A on the SPLP andSPP200 substrates as compared to the addition of GH10 EX alone, these improvements were similar (onSPLP) or lower (on DP, SPCS and SPP200) to those ob-served with the GH10 EX/GH5 XG mixture. This sug-gested that the detected endo-glucanase activity of GH5XG did not explain the strong synergistic interactionobserved with the binary mixture GH10 EX/GH5 XG.Subsequent GH10 EX addition releases bound Cel7A andrestarts hydrolysis after glucose yields have levelled offTo try to further determine the possible mechanisms in-volved in the observed improvements in the cellulolyticFigure 3 Relative improvement in cellulose hydrolysis by supplementation of binary mixtures of accessory enzymes (GH11EX/GH10EX,GH11EX/GH5XG and GH10EX/GH5XG) to Cel7A during the hydrolysis of various pretreated lignocellulosic substrates (SPCS, SPLP andSPP200) at 72 h. Substrate control: dissolving pulp (DP). Enzyme control: GH10EX and GH10EX/Cel5A.Hu et al. Biotechnology for Biofuels 2013, 6:112 Page 6 of 12http://www.biotechnologyforbiofuels.com/content/6/1/112activity of Cel7A due to GH10 EX addition, hydrolysisexperiments were carried out where the SPCS substratewas initially hydrolyzed with Cel7A followed by additionof GH10 EX (or other protein control) at a time whencellulose hydrolysis was observed to start levelling off.The hydrolysis of SPCS by Cel7A followed a typical hy-drolysis profile (as shown in Figure 4) where, after aninitial rapid rate of hydrolysis, the rate gradually de-creased. The addition of more Cel7A after 24 h to theprehydrolyzed substrate resulted in an increase in hy-drolysis, from 21 to 28% after 48 h, and from 28 to 32%after 72 h. However, the addition of GH10 EX ratherthan the Cel7A to the 24 h-prehydrolyzed SPCS resultedin significantly better cellulose hydrolysis, with the yieldsincreased from 21 to 33% after 48 h and from 33 to 38%after 72 h (Figure 4). Controls where BSA and GH3 BGwere each added after 24 h to the prehydrolyzed sub-strate did not promote a significant increase in cellulosehydrolysis.We next tried to quantify the adsorption/desorptionprofile of Cel7A after 24 h hydrolysis in the presenceand absence of GH10 EX or the protein control (BSA)(Figure 5). In general, when Cel7A alone was added, itsadsorption profile did not change during the following48 h, other than a slight decrease in the amount ofprotein detected in the supernatant after this additional48 h. However, the addition of GH10 EX resulted in adifferent Cel7A adsorption/desorption profile with al-most 40% of the Cel7A desorbed within 60 seconds afterthe addition of GH10 EX. After a further 10 min, theamount of Cel7A in solution started to gradually de-crease, indicating the re-adsorption of the Cel7A ontothe SPCS substrate.DiscussionIt is recognised that one of the ongoing challenges forany biomass-to-sugars process is to use minimum pro-tein loadings to achieve good sugar yields on heteroge-neous lignocellulosic substrates. Recent work [3,4] hasshown how important the role that so-called accessoryenzymes (xylanases, AA9, etc.) can play in achieving thisgoal. Although all of the xylanases assessed in the currentwork acted synergistically with cellulases to enhance thehydrolysis of the cellulose present in a range of pretreatedlignocellulosic substrates, the extent of improvement washighly dependent on the substrate used as well as on theFigure 4 Time course of hydrolysis of steam pretreated corn stover (SPCS) by (▲) 15 mg Cel7A with the addition of (■) 10 mg GH10EX, (●) 10 mg Cel7A, (♦) 10 mg GH3 BG and (○) 10mg BSA at 24 h.Hu et al. Biotechnology for Biofuels 2013, 6:112 Page 7 of 12http://www.biotechnologyforbiofuels.com/content/6/1/112nature of the accessory enzyme added. In general, theendo-xylanases interacted synergistically with the cellu-lases in the hydrolysis of the steam pretreated agricultureresidues (SPCS, SPSB) and hardwood (SPP180, SPP200).These substrates had a relatively high xylan content. Incontrast, as was previously observed by Benko et al., [20],the greatest hydrolysis boosting effect obtained withxyloglucanase addition was observed during hydrolysis ofsteam pretreated softwood (SPLP), which contained virtu-ally no xylan (Table 1). Interestingly, despite the muchhigher xylan content of the SPCF substrate (15.3%) ascompared to, SPCS (7%) and SPSB (9.8%), no significantenhancement in cellulose hydrolysis was observed afterendo-xylanases addition (Figure 2). This could have beendue to the highly branched xylan structure of the SPCFsubstrate which would be expected to restrict the accessi-bility of endo-xylanase towards the glycosidic bondswithin the xylan backbone [31,32].Previous work has suggested that GH11 EX can act asan important accessory enzyme capable of boosting bio-mass hydrolysis due to its ability to effectively hydrolyzevarious xylanolytic substrates [10,33]. Its relatively smallsize is also thought to facilitate access to the xylanbackbone in the complex cellulose-hemicellulose-ligninmatrix [9,34]. However, among the hemicellulases thatwere assessed, despite the higher xylanase activity ofGH11 EX when compared to GH10 EX, the GH10 EXresulted in a greater synergistic interaction than did theGH11 EX during the hydrolysis of all of the pretreatedlignocellulosic substrates. This observation agrees withprevious work [35] which suggested that “model” sub-strates used to detect enzyme activity do not necessarilypredict their hydrolytic performance on heterogeneouslignocellulosic materials. The greater boosting effect ofadding supplemental GH10 EX to cellulases was likelydue to its ability to extensively cleave xylan to smallerchain products [17,36]. It has long been suggested that,the longer the xylo-oligosaccharides, the greater the ex-tent of cellulases inhibition [37,38]. In addition, GH10EX has also been shown to have a higher affinity to thehighly branched xylan backbone [36,39] and to be moretolerant than GH11 EX to plant protein inhibitors suchas TAXI and XIP [40].Although earlier work [19] had shown cooperative inter-action between accessory enzymes such as GH11 EX andGH 10 EX to enhance glucose release during lignocelluloseFigure 5 Change of Cel7A level in the liquid phase of steam pretreated corn stover SPCS after 24 h hydrolysis (Δ) with and (□) withoutaddition of GH10 EX. Control: (*) addition of BSA.Hu et al. Biotechnology for Biofuels 2013, 6:112 Page 8 of 12http://www.biotechnologyforbiofuels.com/content/6/1/112hydrolysis, we found that the binary mixtures of these twofamily endo-xylanases mixture (GH11 EX/GH10 EX)resulted in equal or slightly lower improvements in cellu-lose hydrolysis yields than did the addition of GH10 EXalone to the cellulases. However, this earlier work [19] usedAFEX pretreated corn stover, in contrast to the SO2-cata-lyzed steam explosion pretreatment used in this study. It islikely that the AFEX treatment left a far higher amount ofresidual hemicellulose in association with the cellulose,likely requiring the cooperation of both family endo-xylanases to achieve effective cellulose hydrolysis.Although the GH11 EX/GH5 XG mixture did not ap-pear to offer any advantages in enhancing the hydrolyticactivity of Cel7A beyond the enhancement observedwith the individual enzymes, a strong synergistic co-operation was observed with the GH10 EX/GH5 XGmixture. Although the exact mechanisms behind thiscooperation have yet to be fully resolved, it is likely thataccessory enzymes with broader substrate specificities(such as GH10 EX and GH5 XG) result in strongercooperative interactions with cellulase enzymes andtherefore the greater hydrolysis yields observed with thepretreated biomass substrates.One of the ways that accessory enzymes are thoughtto aid in achieving better hydrolysis is by helping releaseor dislodge cellulases that seem to be stuck on thesubstrate. For example, Eriksson et al. [41] observed achange in the adsorption equilibrium of Cel7A uponaddition of Cel7B when the hydrolysis of steam pre-treated spruce had levelled off. They suggested this wasdue to competition between the two cellulases. It islikely that, in the work reported here, the observedchange in the adsorption equilibrium of Cel7A uponaddition of GH10 EX is also due to competitive adsorp-tion. It is also worth noting that, like Cel7B, GH10 EXhas a cellulose binding module with high affinity to cel-lulose [42,43]. Previous work has shown that Cel7A getsstuck on cellulose microfibrils during enzymatic cellu-lose hydrolysis [44], leading to a substantial decrease inthe rate of hydrolysis. It is possible that the GH10 EXhydrolyzed possible obstacles such as xylan that re-stricted the processive movement of the Cel7A.One of the main challenges in achieving effective cel-lulose hydrolysis is to overcome the still-not-well-under-stood gradual decrease in hydrolysis rate in the latterstages of hydrolysis. Various mechanisms are thought toplay a role such as the inactivation of cellulase enzymes(denaturation, product inhibition, unproductive binding)and an increase in the recalcitrance of the residual sub-strate [41,45]. The results presented here indicate thattwo of the major roles that accessory enzymes such asxylanases might play in enhancing the hydrolytic poten-tial of a “cellulase mixture” is to both help release cellu-lases that are stuck on the substrate while hydrolysingthe non-cellulose components of the substrate that re-strict access of the cellulases to the cellulose.MethodsLignocellulosic substrates pretreatment and compositionThree different agricultural residues (corn stover, sweetsorghum bagasse, and corn fiber), one hardwood (pop-lar), and one softwood (lodgepole pine) substrates weresteam pretreated as described earlier [46-48]. Pretreatmentconditions were a compromise, based on the previouswork to both maximise overall sugar recovery (hemicellu-lose and cellulose) while providing a cellulosic componentthat could be readily hydrolysed with relatively low enzymeloadings. A poplar sample (SPP180) was also pretreated atlow severity in order to maintain a relatively high hemicel-lulose/xylan content in the water insoluble cellulosicfraction. The chemical composition of the water insolublefraction after steam pretreatment was determined usingthe modified Klason lignin method derived from theTAPPI standard method T222 om-88, as previously de-scribed [49]. The pretreatment conditions and chemicalcomposition of the pretreated substrates are shown inTable 1. Dissolving pulp (DP) was used as an almost purecellulolytic substrate control (less than 0.5% lignin, lessthan 3% xylan).Enzymes purificationFour major cellulase components of T. reesei, Cel7A(CBHI), Cel6A (CBHII), Cel7B (EGI), and Cel5A (EGII)were purified from Celluclast 1.5 L (Novozymes,Franklington, NC) as previously reported [28,50-52].Family 11 endo-xylanase (GH11 EX) and family 3 β-glucosidase (GH3 BG) were purified from MultifectXylanases (Genencor US Inc., Palo Alto, CA) andNovozyme 188 (Novozymes A/S, Bagsvaerd, Denmark),respectively, as described by [53]. In brief, family 10endo-xylanase (GH10 EX) was purified from H-Tec(Novozymes, Franklington, NC) through two steps ofchromatography. First the enzyme mixture was injectedinto a size exclusion column (Hiload 16/60 Superdex 75prep grade column) with an isocratic flow of triethanolamine (TEA) buffer (20 mM, pH 7.0). The peakscontaining the majority of the endo-xylanase activity werecollected and further purified by ion exchange chromatog-raphy (using an ion exchange UNO Q1 column) with alinear gradient change of buffer stock A (20 mM TEA, pH7.0) to buffer stock B (20 mM TEA, 1 M Nacl, pH 7.0).The family 5 xyloglucanase (GH5 XG) derived fromPaenibacillus sp. was purchased from Megazyme anddesalted using a sodium acetate buffer (50 mM, pH 4.8).All purification procedures were performed in an auto-mated FPLC system (BioLogic Due-Flow). The buffers usedfor enzyme purification were prepared using nanopurewater and filtered through a 0.22 μm membrane filterHu et al. Biotechnology for Biofuels 2013, 6:112 Page 9 of 12http://www.biotechnologyforbiofuels.com/content/6/1/112(Millipore) followed by sonication for at least 30 min.The purity of the enzymes and lack of contamination byother cellulases and hemicellulases was confirmed bySDS-PAGE and Liquid chromatography–mass spec-trometry/mass spectrometry (LCMS/MS) as describedby Pribowo et al., [54].Enzymatic hydrolysisThe purified enzymes were used in different combinationsand the cellulase monocomponents (Cel7A, Cel6A, Cel7B,and Cel5A) were assessed at a loading of 15 mg/g cellulosewhile the accessory enzymes (GH11 EX, GH10 EX, andGH5 XG) were individually assessed at loading of 10 mg/gcellulose, or at a loading of 5 mg/g cellulose when used ina binary combination. All of the reconstituted enzymemixtures were supplemented with GH3 B-glucosidase at aloading of 2.5 mg/g cellulose.The hydrolysis assays were carried out at 2% (w/v)solids loading in sodium acetate buffer (50 mM, pH 4.8)in an 8 ml total volume. The reaction mixtures were mech-anically shaken in an orbital shaker incubator (Combi-D24hybridization incubator) at 50°C for up to 72 h. The restarthydrolysis experiments were carried out by incubatingsteam pretreated corn stover (SPCS) with Cel7A for 24 h.Thereafter, accessory enzymes and different protein con-trols were added to the pre-hydrolyzed mixture and incu-bated for a further 48 h.The hydrolysis was terminated by boiling the reactionmixture at 100°C for 10 min to inactivate the enzymes.The supernatants collected after centrifugation at 16000 gfor 10 min and stored at −20°C for further analyses. Sub-strate and enzyme blanks were run at the same time byincubating the substrates without enzymes and by incu-bating the enzymes without substrates, respectively.Analytical methodsThe specific activities of the purified enzymes are detailedin Table 2. The xylanase, xyloglucanase, and carboxymethylcellulose activities (CMCase) were assessed as described by[55]. The cellobiohydrolase, β-glucosidase and β-xylosidaseactivities were determined using p-nitrophenyl-β-D-cellobioside (p-NPC), p-nitrophenyl-β-D-glucopyranoside(p-NPG), and p-nitrophenyl-β-D-xylopyranoside (p-NPX)as substrates, respectively, according to [56]. The proteincontent was measured by the Ninhydrin assay using bovineserum albumin (BSA) as the protein standard [57].In the restart hydrolysis experiments, the adsorption/desorption profile of Cel7A after the addition of variousenzymes and BSA was determined using an immunoassayto specifically quantify the amount of Cel7A present inthe supernatant. Briefly, a monoclonal antibody (MAb)specific for Cel7A was used to distinguish this enzymefrom the other enzymes present in the supernatant. ACel7A polyclonal antibody (PAb) was then used to bindthe captured Cel7A. The amount of Cel7A was indir-ectly quantified by measuring the bound PAbs using athird antibody conjugated to alkaline phosphatase (AP,Biorad). The quantitation was achieved by adding p-nitrophenylphosphate (Bio-Rad), a substrate for alkalinephosphate, and the reaction was incubated at roomtemperature for 30 min. The reaction was stopped byadding 400 mM glycine-NaOH. The amount of Cel7Awas then indirectly quantified by measuring the absorb-ance of p-nitrophenyl at 405 nm.The quantitative analysis of chemical compositions ofvarious steam pretreated lignocellulosic substrates afterKlason procedure were performed by high performanceanion exchange chromatography (Dionex DX-3000,Sunnyvale, CA) as described earlier [58]. The quantita-tive analysis of glucose concentration in the hydrolysatewas performed by Glucose Oxidase Assay [59]. The ex-tent of cellulose hydrolysis of the pretreated substrateswas calculated as a percentage of the theoretical glucanavailable in the substrate. All hydrolysis experimentswere performed in duplicate and the mean values andstandard deviations are presented.AbbreviationsSPCS: Steam pretreated corn stover; SPCF: Corn fiber; SPSB: Sweet sorghumbagasse; SPLP: Lodgepole pine; SPP180: Poplar steam pretreated at 180°C;SPP200: Poplar steam pretreated at 200°C; DP: Dissolving pulp; BSA: Bovineserum albumin; GH11 EX: Glycoside hydrolase family 11 endo-xylanase; GH10EX: Family 10 endo-xylanase; GH5 XG: Family 5 xyloglucanase; GH3BG: Family 3 β-glucosidases.Competing interestsThe authors declare that they have no competing interests.Authors’ contributionsAll authors (JH, VA, AP and JNS) contributed jointly to all aspects of the workreported in the manuscript. All authors have read and approved the finalmanuscript.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 thankNovozymes and Genecor for their donations of the enzymes preparationsused in this study.JH gives his sincere thanks to the China Scholarship Council of the Ministryof Education of China for their financial support. All of the authors thank themembers of the UBC FPB/Bioenergy Group for invaluable discussions andhelp.Received: 20 May 2013 Accepted: 2 August 2013Published: 3 August 2013References1. Arantes V, Saddler JN: Cellulose accessibility limits the effectiveness ofminimum cellulase loading on the efficient hydrolysis of pretreatedlignocellulosic substrates. Biotechnology for Biofuels 2011, 4:3.2. 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Saha BC, Bothast RJ: Production, purification, and characterization of ahighly glucose-tolerant novel beta-glucosidase from Candida peltata.Appl Environ Microbiol 1996, 62(9):3165–3170.57. Starcher B: A ninhydrin-based assay to quantitate the total proteincontent of tissue samples. Anal Biochem 2001, 292(1):125–129.58. 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):284–289.59. Berlin A, Maximenko V, Bura R, Kang KY, Gilkes N, Saddler J: A rapidmicroassay to evaluate enzymatic hydrolysis of lignocellulosic substrates.Biotechnol Bioeng 2006, 93(5):880–886.doi:10.1186/1754-6834-6-112Cite this article as: Hu et al.: The synergistic action of accessoryenzymes enhances the hydrolytic potential of a “cellulase mixture” butis highly substrate specific. Biotechnology for Biofuels 2013 6:112.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 2013, 6:112 Page 12 of 12http://www.biotechnologyforbiofuels.com/content/6/1/112


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