Optimization of enzyme complexes for lignocellulose hydrolysis

ARTICLE

Optimization of Enzyme Complexesfor Lignocellulose Hydrolysis

Alex Berlin, Vera Maximenko, Neil Gilkes, Jack Saddler

Forest Products Biotechnology, Department of Wood Science, Faculty of Forestry,

University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4;

telephone: (604) 827-5005; fax: (604) 822-9159; e-mail: [email protected]

Received 7 April 2006; accepted 2 October 2006

Published online 20 October 2006 in Wiley InterScience (www.interscience.wiley.com
). DOI 10.1002/bit.21238
ABSTRACT: The ability of a commercial Trichoderma reeseicellulase preparation (Celluclast 1.5L), to hydrolyze thecellulose and xylan components of pretreated corn stover(PCS) was significantly improved by supplementation withthree types of crude commercial enzyme preparations nom-inally enriched in xylanase, pectinase, and b-glucosidaseactivity. Although the well-documented relief of productinhibition by b-glucosidase contributed to the observedimprovement in cellulase performance, significant benefitscould also be attributed to enzymes components that hydro-lyze non-cellulosic polysaccharides. It is suggested that so-called ‘‘accessory’’ enzymes such as xylanase and pectinasestimulate cellulose hydrolysis by removing non-cellulosicpolysaccharides that coat cellulose fibers. A high-throughputmicroassay, in combination with response surface metho-dology, enabled production of an optimally supplementedenzyme mixture. This mixture allowed for a �twofoldreduction in the total protein required to reach glucan toglucose and xylan to xylose hydrolysis targets (99% and 88%conversion, respectively), thereby validating this approachtowards enzyme improvement and process cost reductionfor lignocellulose hydrolysis.

Biotechnol. Bioeng. 2007;97: 287–296.

� 2006 Wiley Periodicals, Inc.

KEYWORDS: bioconversion; lignocellulose; cellulose; hemi-cellulose; xylan; cellulase

Introduction

Plant biomass contains large amounts of cellulose and otherpolysaccharides that can be hydrolyzed to glucose andvarious other simple sugars for subsequent fermentation tofuel ethanol or used in the production of other industrialchemicals. In nature, the biodegradation of plant biomass isa slow process because lignin and substrate crystallinity

Correspondence to: A. Berlin

Contract grant sponsor: Natural Science and Engineering Research Council of Canada

(NSERC)

Contract grant sponsor: Natural Resources Canada (NRCAN)

� 2006 Wiley Periodicals, Inc.

greatly restrict the access of hydrolytic enzymes to thepolysaccharide components. However, raw biomass can bepretreated and partially fractionated, using processes thattypically involve elevated temperature and pressure com-bined with acid or base catalysis, to yield lignocellulosicmaterials that are much more susceptible to enzyme attack(Kim and Lee, 2005; Mais et al., 2002; Palonen et al., 2004).Nevertheless, although the economics of ethanol productionfrom lignocellulose continues to improve (GenencorInternational, Inc., 2003), the costs for biomass pretreat-ment and enzyme-catalyzed hydrolysis continue to deterwidespread commercialization of this process.

Hydrolysis of the pretreated lignocellulose to simplesugars typically uses a complex of secreted enzymes derivedfrom filamentous fungi, particularly Trichoderma sp. Suchenzyme complexes contain high levels of cellulases(endoglucanases and cellobiohydrolases), together withlower amounts of enzymes that attack non-cellulosicpolysaccharides such as hemicellulose and pectin. Attemptsto improve the hydrolytic efficiency of such enzymecomplexes have traditionally focused on their componentcellulases because cellulose is the most abundant poly-saccharide component in lignocellulose. However, it is nowrecognized that the hydrolytic efficiency of fungal cellulasecomplexes determined using a model cellulosic substratedoes not provide a reliable indication of its performance onpretreated lignocellulose (Berlin et al., 2005a, 2006; Kabelet al., 2005). Evidently, other components in pretreatedbiomass, particularly hemicellulose and lignin, exertsignificant restraints on cellulose hydrolysis. For example,one mechanism whereby lignin seems to reduce hydrolyticperformance is by binding enzyme components non-productively. Consequently enzyme mixtures with similarcellulase activity may show differences in performance onlignocellulose if they differ in affinity for lignin (Berlin et al.,2005). Similarly, it is probable that hemicelluloses restrict

Biotechnology and Bioengineering, Vol. 97, No. 2, June 1, 2007 287

the access of cellulolytic enzymes by coating cellulose fibers.In some lignocelluloses, pectin could exert a similar effect.Consequently, enzyme mixtures with similar cellulaseactivity may show differences in performance on lignocel-lulose if they differ in hemicellulase composition (Berlinet al., 2005, 2006). However, it should be possible tocompensate for deficiencies in these so-called ‘‘accessory’’enzymes by supplementation of cellulase mixtures withappropriate activities.

As a first step towards evaluating this approach forenzyme improvement, we sought to increase the hydrolyticactivity of a commercial Trichoderma reesei cellulasepreparation towards corn stover, pretreated using a diluteacid process, by supplementation with enzyme preparationsenriched in xylanase and pectinase activity. The effect ofsupplementation with a b-glucosidase preparation wasexamined in parallel. This study also evaluates whethersupplementation offers any advantage over simply increas-ing the dose of unsupplemented T. reesei cellulase. In orderto examine a broad range of enzyme permutations, we haveemployed a high-throughput microassay system recentlydeveloped for this purpose (Berlin et al., 2005c) andanalyzed the data using response surface methodology.

It should be noted that a diverse range of plant biomasswill be needed to satisfy projected demands for fuel ethanol(Perlack andWright, 2005). However, biomass derived fromagricultural residues such as corn stover or cereal stalks,purpose-grown crops like switchgrass or hybrid poplar, andsoftwoods such as spruce and pine from boreal forests showsignificant quantitative and qualitative differences in theirnon-cellulosic polysaccharide components. Further differ-ences in composition are introduced according to thepretreatment technology employed. Therefore, it has beensuggested that enzyme mixtures could be customized forparticular feedstocks and pretreatments in order to optimizehydrolysis (Berlin et al., 2005c; Eggeman and Elander, 2005).Consequently, this study provides preliminary data forsubsequent studies that seek to examine this approachtowards improving process economics.

Materials and Methods

Enzyme Preparations

Celluclast 1.5L, a commercial T. reesei cellulase preparation,and Novozym 188, a commercial Aspergillus niger (Asp.niger) b-glucosidase, were provided by Novozymes (Frank-linton, NC). Multifect Xylanase, a commercial xylanasederived from a genetically modified strain of T. reesei, andMultifect Pectinase, a commercial pectinase complexderived from a selected strain of Asp. niger, were providedby Genencor International (Palo Alto, CA).

Lowmolecular weight compounds were removed from allenzyme preparations by gel filtration on a HiPrep 26/10desalting column (Amersham Biosciences, Uppsala, Swe-den) prior to use. Protein concentrations were determined

288 Biotechnology and Bioengineering, Vol. 97, No. 2, June 1, 2007

using the BCA protein assay microplate procedure (PierceChemical Co., Rockford, IL), according to the manufac-turer’s recommendations. Enzyme activities were assayed aspreviously described (Berlin et al., 2005).

Substrate Preparation

Dilute acid-pretreated corn stover (PCS) was provided bythe US National Renewable Energy Laboratory (Golden,CO). The stover sample (from Kramer farm,Wray, CO) wasmilled with a Mitts and Merrill knife mill to pass 0.625 cmscreen. Milled stover (�9% (w/w) moisture content) waspretreated by dilute acid for 1.5 min in a Sunds verticalreactor in ‘‘flow through mode’’ at 1808C using 0.03 gsulfuric acid/g dry feedstock and 25% insoluble solids. Afterpretreatment, the sample was washed several times with tapwater to remove soluble components and filtered to removeexcess moisture. The washed PCS was stored in sealed plasticbags at 48C for up to 2 weeks, or at �188C if storage for alonger period was required. The PCS was then formed intohandsheets for use in the high-throughput microassayprocedure, as described below.

Chemical Analysis of Substrates

The carbohydrate and lignin contents of milled corn stover,PCS pulp, and handsheets were determined using amodifiedKlason lignin method derived from the TAPPI standardmethod T222 om-88, as previously described (Bura et al.,2002). Monosaccharides were analyzed by HPLC withfucose as internal standard, as previously described(Boussaid et al., 1999). All analyses were performed intriplicate. Substrate composition data are presented inFigure 1.

Assay of Substrate Hydrolysis

Enzymatic hydrolysis of PCS was performed according tothe microassay procedure previously described (Berlin et al.,2005c). In brief, PCS disks (�1.4 mg) were dispensed into96-well microplates at �0.6% (w/w) and hydrolyzed usingthe cellulase preparation (Celluclast 1.5L), either alone orsupplemented with various combinations of three differentenzyme preparations: Multifect Xylanase, Multifect Pecti-nase, and Novozym 188. The characteristic specific activitiesof Celluclast 1.5L and the three accessory enzymes arepresented in Table I.

Optimization of the enzyme mixture (Celluclast1.5Lþ accessory enzyme), targeting maximum glucoserelease from PCS in 24 h (99% glucan conversion), wasperformed by response surface methodology (RSM) usingSAS v.9.0 software (SAS Institute, Cary, NC) and a centralcomposite uniform precision experimental design com-prises 3 factors (X1, X2, X3), corresponding to each of theaccessory enzymes, 20 runs, and 6 central points. Models

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Figure 1. Chemical composition of raw corn stover and dilute acid-pretreated corn stover (PCS). The composition of the PCS is shown before and after formation into

handsheets for use in the high-throughput microassay used in subsequent experiments.

were fitted to a second-order polynomial equation.Sensitivity analysis was performed to determine the relativeimportance of each accessory enzyme. The experimentaldesign involved a preliminary enzymatic microhydrolysis ofPCS for 24 h using three Celluclast 1.5L loadings (0.025,0.123, 0.246 g protein/g glucan) at 508C, pH 4.8 (Fig. 2),followed by the determination of the saturation limits foreach accessory enzyme (Figs. 3–5). Celluclast 1.5L wasloaded at 0.025 g protein/g glucan for the RSM study and theaccessory enzymes were loaded as described in Figures 6Aand B and 7A and B. The release of glucose and xylose wasmonitored by HPLC as previously described (Boussaid et al.,1999). Results of the RSM study are illustrated in Figures 6Aand B and 7A and B. The saturation limit of the T. reeseicellulase supplemented by the same enzyme was alsodetermined (Fig. 8). Further confirmation of the maximum

Table I. Specific activities (U/mg protein) of Celluclast 1.5L and the three comm

substrates.

Enzyme preparation FPA� b-glucosidase Man

Celluclast 1.5L (Novozymes) 0.9 0.2 0

Novozym 188 (Novozymes) 0.1 1.1 0

Multifect Xylanase (Genencor) 0.1 0.6 0

Multifect Pectinase (Genencor) 0.1 3.3 3

�FPA, filter paper activity.

found by the RSM study was performed by PCS hydrolysisand sampling the hydrolysate at 3, 6, 10, and 24 h (Fig. 9). Allexperiments were run in triplicate.

Results

Chemical Composition of Substrates

The chemical compositions of untreated corn stover, PCSpulp, and PCS handsheets, as determined by Klason analysis,are shown in Figure 1. The percent lignin content (Klasonlignin plus acid-soluble lignin) of PCS pulp (25% w/w) wasincreased 1.7-fold relative to untreated corn stover (15% w/w), with a concomitant 1.6-fold increase in percent glucancontent (from 36% to 59%, estimated as glucose). The xylan

ercial enzyme preparations used in supplementation experiments onmodel

nanase Avicelase CMCase Pectinase Xylanase

.1 2.2 14.0 0.1 3.5

.2 0 0.3 0.1 0.4

.5 0.5 1.3 0.6 282.7

.0 0.3 2.0 25.1 8.7

Berlin et al.: Optimization of Enzyme Complexes 289

Biotechnology and Bioengineering. DOI 10.1002/bit

Figure 2. Cellulose hydrolysis of dilute acid-PCS by Celluclast 1.5L at three different levels of total protein. Data were determined using the high-throughput

microassay.

Figure 3. Percent glucan-to-glucose conversion at 24 h by Celluclast 1.5L cellulase supplemented with increasing levels of crude b-glucosidase (Novozym 188).


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Figure 4. Percent glucan-to-glucose conversion at 24 h by Celluclast 1.5L cellulase supplemented with increasing levels of xylanase (Multifect Xylanase).

content in PCS was significantly reduced in the PCS pulp(from 21% to 9%). A similar result was obtained forarabinan with a threefold reduction in PCS (from 3% to1%). The ash content was also reduced in PCS (4%).

Figure 5. Percent glucan-to-glucose conversion at 24 h by Celluclast 1.5L cel

There were no significant differences observed betweenthe lignin and monosaccharide compositions of the PCSpulp and the PCS handsheets (Fig. 1), indicating thathandsheet preparation did not significantly alter the

lulase supplemented with increasing levels of pectinase (Multifect Pectinase).



Figure 6. Percent glucan-to-glucose conversion at 24 h by Celluclast 1.5L

supplemented with various combinations of crude b-glucosidase, xylanase, and

pectinase (Novozym 188, Multifect Xylanase, and Multifect Pectinase, respectively).

A: fixed concentration of b-glucosidase (5.14 mg); B: fixed concentration of Multifect

Pectinase (10.28 mg).

Figure 7. Percent xylan-to-xylose conversion at 24 h by Celluclast 1.5L

supplemented with various combinations of crude b-glucosidase, xylanase, and

pectinase (Novozym 188, Multifect Xylanase, and Multifect Pectinase, respectively).

A: fixed concentration of b-glucosidase (5.14 mg); B: fixed concentration of Multifect

Pectinase (10.28 mg).

chemical composition of the lignocellulosic substrate usedfor microassay. This result agrees with previous resultsobtained for handsheets prepared from other pretreatedlignocellulosic substrates (Berlin et al., 2005c; Maximenkoand Berlin, 2005).

Enzyme Activities

The activities of the various enzyme preparations showedsignificant differences (Table I). Celluclast 1.5L contained


high specific cellulase activities: filter paper activity (FPA)(0.9 U/mg protein), Avicelase (2.2 U/mg protein), andcarboxymethylcellulase (CMCase) (14.0 U/mg protein),relative to the accessory enzymes. Novozym 188 containedmainly b-glucosidase activity (1.1 U/mg protein), with lowlevels of other activities, such as mannanase (0.2 U/mg) andFPA (0.1 U/mg; Table I). Multifect Pectinase showed highlevels of pectinase (25.1 U /mg protein) and mannanase (3.0U/mg protein). Multifect Pectinase also contained highlevels of b-glucosidase (3.3 U/mg protein) relative to

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Figure 8. Percent glucan-to-glucose conversion at 24 h by increasing levels of Celluclast 1.5L cellulase.

Figure 9. Evaluation of cellulose hydrolysis in PCS by Celluclast 1.5L supplemented with optimized combination of crude enzymes (T. reesei and OptEMix protein loading: 0.025

and 0.219 g protein/g glucan, respectively).



Novozym 188 (1.1 U/mg; Table I). Multifect Xylanase wascharacterized by high levels of xylanase activity (282.7 U/mgprotein) with moderate to low levels of other activities(Table I).

Saturation Limits for the Supplementation WithAccessory Enzymes

Supplementation of Celluclast 1.5L with each of the threeenzyme preparations resulted in significant improvement ofPCS hydrolysis, as determined by the conversion of glucanto glucose (Figs. 6A and B and 9) and xylan to xylose (Fig. 7Aand B). The most significant effect at low supplementaryprotein loading was observed when the cellulase complex(�20 mg loading) was supplemented with MultifectPectinase. Addition of <5 mg protein (<20% total protein)to the cellulase increased the glucose yield by�40% (Fig. 5).A higher level of improvement (�62%) was seen whenCelluclast 1.5L was supplemented with Multifect Xylanase(Fig. 4); however, a much higher level of total protein(>150 mg) was required. Novozym 188 also improvedglucose yield but the effect was less significant (�37%)compared to Multifect Xylanase and Multifect Pectinase(Fig. 3). The latter can be improved by further addition ofNovozym 188. A biphasic character is then observed.However, this further improvement in glucan hydrolysisseems to be attributed to the xylanolytic activity present inthis enzyme preparation (data not shown).

Optimization of the Enzyme Mixture

Following determination of saturation limits for each of thethree enzyme supplements (Figs. 3–5), optimization of theenzyme mixture was performed using response surfacemethodology. The microhydrolysis assay allowed determi-nation of the optimal enzymemixture for maximum glucoseyield (Fig. 6A and B), maximum xylose yield (Fig. 7A and B),and the total protein loading required to reach chosentargets for glucan-to-glucose conversion (99% at 24 h) andxylan-to-xylose conversion (88% at 24 h). Approximately400 mg Celluclast 1.5L was required to achieve the glucan-to-glucose conversion target, while only �200 mg proteinwas needed using the optimized enzymemixture (OptEMix)(Figs. 8 and 9). The conversion of xylan to xylose was alsoimproved significantly by supplementation with accessoryenzymes (Fig. 7A and B). It is remarkable that in order toachieve a lower glucan-to-glucose conversion target, �80%at 24 h for instance, about equal amounts (�50 mg) ofaccessory enzymes and additional Celluclast 1.5L wererequired (Figs. 6A and B and 8). This result together withthe previous observation indicates that the OptEMixis particularity effective, compared to Celluclast 1.5L, inovercoming the substrate recalcitrance associated with theconversion to glucose of the remaining glucan at the end of


hydrolysis (>80% glucan-to-glucose conversion). Sensitiv-ity analysis demonstrated that supplementation of thecellulase with Multifect Pectinase was more critical than forthe other two enzyme supplements (data not shown). Inparticular, the glucan-to-glucose yield was not significantlyaffected by supplementation with Novozym 188 when allthree accessory enzymes were supplemented simultaneously.

It is noteworthy that supplementation of Celluclast 1.5Lby Multifect Xylanase and Pectinase showed a parabolicconcentration dependence of the glucan-to-glucose con-version with distinct maxima (Fig. 6A and B). A similarconcentration dependence was observed for the xylan-to-xylose conversion (Fig. 7A and B).

Response surface methodology also allowed derivation ofthe following equations relating glucan-to-glucose conver-sion and xylan-to-xylose conversion to the levels of enzymesupplements: PCS glucan-to-glucose (Y1) and PCS xylan-to-xylose (Y2) conversion predictive model equations

Y
1¼ 72.87547þ 0.205735�X2þ 1.326516�X3� 0.000575�X2�X2� 0.055326�X3�X3 (r2¼ 73.29%)
Y
2¼ 43.00974þ 3.033077�X1þ 0.266672�X2þ 2.366963�X3� 0.40228�X1�X1þ 0.137267�X1�X3� 0.000748�X2�X2� 0.142687�X3�X3 (r2¼ 89.84%)
where Y1 is predicted glucan-to-glucose conversion (%); Y2is predicted xylan-to-xylose conversion (%); X1 is weight ofNovozym 188 (mg); X2 is weight of Multifect Xylanase (mg);X3 is weight of Multifect Pectinase (mg).

Discussion

Most studies on the enzymatic hydrolysis of lignocellulosesfor bioconversion involve cellulase complexes secreted bythe filamentous fungus T. reesei. This fungus is among thebest protein secretors known, achieving levels of 40–100 gtotal protein/L (Janas et al., 2002) which makes it attractivefor industrial applications. However, it is well documentedthat T. reesei cellulase complexes are typically low in b-glucosidase activity. As a result cellobiose, produced by thecombined actions of endo- and exo-b-1,4-glucanases,accumulates to levels that result in significant productinhibition during cellulose hydrolysis in vitro (Ballesteroset al., 2000; Teymouri et al., 2004). In accordance withnumerous previous reports, the data presented in Figure 3demonstrate that addition of further b-glucosidase toCelluclast 1.5L improves glucan-to-glucose conversion inPCS by up to �38%.

It is also well known that cellulase complexes produced byT. reesei and other cellulolytic fungi contain variousglycanases responsible for the hydrolysis of non-cellulosicpolysaccharides present in plant biomass, notably hemi-

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cellulases and pectinases. Since hemicellulose, and possiblypectin, is thought to restrict the access of cellulases to theirsubstrate in pretreated lignocellulose, we examined thepossibility that further improvements in hydrolysis could beachieved by manipulating the levels of these enzymes usingcommercially available xylanase and pectinase-enrichedpreparations. Positive effects on glucan hydrolysis and xylanhydrolysis were observed in both cases.

It is not possible to interpret the effects of supplementa-tion precisely because all three enzyme supplements used inthis study contain significant levels of other enzymes, inaddition to their nominal activities. For example, MultifectPectinase and Multifect Xylanase both contain significantlevels of b-glucosidase activity (Table I). However, theimprovements in hydrolysis demonstrated in Figures 3–5are evidently not simply the result of b-glucosidase activityin these preparations. For example, the addition of b-glucosidase, as Novozym 188, in Figure 6B is without effect(presumably because saturating levels are already providedby the fixed amount ofMultifect Pectinase), but the additionof Multifect Xylanase stimulates hydrolysis if supplementedin amounts below the optimum. Analyses aimed at furtherrefinement of enzyme composition will require the use ofpurified enzyme preparations with more defined activities.

These data clearly demonstrate the effectiveness ofcellulase supplementation using accessory enzymes. A keyfinding is that the optimized enzyme mixture, incorporatingall three enzyme supplements, catalyzed the targeted degreeof glucan-to-glucose conversion at approximately half thetotal protein loading required for unsupplemented Cellu-clast 1.5L. A similar result was obtained for hemicellulosehydrolysis, as indicated by the xylan-to-xylose conversion.These results are significant because the cost of producingbulk enzyme mixtures for large-scale industrial applications,such as bioconversion, can be reduced largely to the cost ofprotein production. Consequently, enzyme optimizationprocedures that reduce net protein requirements shouldsignificantly reduce process costs. Perhaps, ‘‘accessory’’enzymes with higher specific activities and lower productioncosts should be developed in the future in order to make theapplication of the observed hydrolytic boosting effectseconomically feasible. It should be further noted thatnegative effects on hydrolysis were observed at higher levelsof supplementation when three accessory enzymes wereadded simultaneously (Figs. 6A and B and 7A and B). Asimilar effect is seen when Multifect Xylanase is supple-mented separately and the experimental results are fitted to aparabolic curve (data not shown). However, parabolicfitting was not chosen since the results obtained for thedetermination of the saturation limit fitted better ahyperbolic dependence (Fig. 4). The parabolic coefficientof determination (R-Square) for Multifect Xylanase was0.936 (P< 0.0001). The value of R-Square in the case of theparabolic fitting was lower compared to the determinationcoefficient obtained when results were fitted to a hyperbolicdependence (R-Square¼ 0.992, Fig. 4). Supposedly, thisnegative effect seen is a result of competition for productive

binding sites between Multifect Xylanase and the Tricho-derma sp. cellulases. The mechanism underlying thisphenomenon is currently unknown but these resultsindicate that a strategy involving addition of supplementaryenzymes in excess is to be avoided, even if their productioncosts can be ignored. Detailed experiments will be requiredto establish whether the quantitative and qualitative optimafor enzyme supplementation are similar for all types oflignocellulosic substrates or whether attempts to customizeenzyme mixtures for individual substrates are justified. Anadditional potential benefit arising from the supplementa-tion of T. reseei cellulase with accessory activities is thepossibility of hydrolyzing lignocellulosic substrates pre-treated at milder conditions. The latter could result in ahigher overall fermentable carbohydrate recovery and loweryeast inhibition during fermentation of liquid streamsobtained after pretreatment of lignocellulosic substrates.Further experiments should be performed in order toconfirm this hypothesis.

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