JOURNAL OF BACTERIOLOGY,0021-9193/00/$04.0010

Oct. 2000, p. 5676–5682 Vol. 182, No. 20

Copyright © 2000, American Society for Microbiology. All Rights Reserved.

Synergistic Hydrolysis of Carboxymethyl Cellulose and Acid-SwollenCellulose by Two Endoglucanases (CelZ and CelY)

from Erwinia chrysanthemi†SHENGDE ZHOU AND LONNIE O. INGRAM*

Institute of Food and Agricultural Sciences, Department of Microbiology and Cell Science,University of Florida, Gainesville, Florida 32611

Received 6 April 2000/Accepted 27 July 2000

Erwinia chrysanthemi produces a battery of hydrolases and lyases which are very effective in the macerationof plant cell walls. Although two endoglucanases (CelZ and CelY; formerly EGZ and EGY) are produced, CelZrepresents approximately 95% of the total carboxymethyl cellulase activity. In this study, we have examined theeffectiveness of CelY and CelZ alone and of combinations of both enzymes using carboxymethyl cellulose (CMC)and amorphous cellulose (acid-swollen cellulose) as substrates. Synergy was observed with both substrates.Maximal synergy (1.8-fold) was observed for combinations containing primarily CelZ; the ratio of enzymeactivities produced was similar to those produced by cultures of E. chrysanthemi. CelY and CelZ were quitedifferent in substrate preference. CelY was unable to hydrolyze soluble cellooligosaccharides (cellotetraose andcellopentaose) but hydrolyzed CMC to fragments averaging 10.7 glucosyl units. In contrast, CelZ readilyhydrolyzed cellotetraose, cellopentaose, and amorphous cellulose to produce cellobiose and cellotriose asdominant products. CelZ hydrolyzed CMC to fragments averaging 3.6 glucosyl units. In combination, CelZ andCelY hydrolyzed CMC to products averaging 2.3 glucosyl units. Synergy did not require the simultaneouspresence of both enzymes. Enzymatic modification of the substrate by CelY increased the rate and extent ofhydrolysis by CelZ. Full synergy was retained by the sequential hydrolysis of CMC, provided CelY was used asthe first enzyme. A general mechanism is proposed to explain the synergy between these two enzymes basedprimarily on differences in substrate preference.

The hydrolysis of cellulose into soluble sugars by microbialsystems offers the potential to provide a renewable feedstockfor the production of fuels and chemicals (10, 17, 22). How-ever, the crystalline structure and insoluble nature of celluloserepresents a formidable challenge for enzymatic hydrolysis.Interactions between different cellulase enzymes and sub-strates are quite complex (2, 5, 20, 24, 36). The solubilization ofcrystalline cellulose by the fungus Trichoderma longibranchia-tum has been extensively studied as a model primarily due to itscommercial utility. Cellulases produced by T. longibranchiatumcan be divided into three classes: endoglucanases (carboxy-methyl cellulases [CMCases]), which hydrolyze amorphous re-gions of cellulose; exoglucanases (cellobiohydrolases), whichprogressively cleave cellobiose units from the ends of crystal-line or amorphous cellulose; and b-glucosidases (cellobiases),which hydrolyze soluble cellooligosaccharides into glucose (5,20). Multiple enzymes of each type are produced by T. longi-branchiatum. Combinations of these fungal enzymes functionin a synergistic fashion (23, 24, 32, 35, 36). Bacteria also pro-duce multiple enzymes for cellulose hydrolysis (5, 21, 25).Synergy has been demonstrated for combinations of bacterialexoglucanases and endoglucanases (3, 12, 23, 28) and for com-binations of bacterial endoglucanases and fungal exogluca-nases (2, 18, 33). In nature, it is likely that enzymes from manydifferent organisms function together during cellulose hydro-lysis.

Our laboratory is developing recombinant strains of eth-

anologenic Escherichia coli and Klebsiella oxytoca that producebacterial cellulases and reduce the amount of fungal cellulaserequired for biofuel production (17). In previous studies, wehave expressed the celZ gene, which encodes the major endo-glucanase (CelZ; formerly EGZ) from Erwinia chrysanthemi, athigh levels in E. coli (39) and K. oxytoca (38). Expression andsecretion were facilitated in both recombinant hosts by addingthe out genes, which encode a type II protein transport systemfrom E. chrysanthemi (14, 38, 39).

E. chrysanthemi produces a battery of hydrolase and lyaseenzymes which are very effective in the maceration of planttissues (9, 29, 31). This organism produces two different endo-glucanases, CelY (formerly EGY) and CelZ (7, 8, 13). Withcarboxymethyl cellulose (CMC) as a substrate, 95% of the totalendoglucanase activity was attributed to CelZ while only 5% ofthe activity was attributed to CelY. Although the latter per-centage indicates a minor activity, the retention of both en-zymes during evolution suggests that the combination of CelYand CelZ is beneficial for the efficient hydrolysis of cellulose.Genes encoding both activities have been previously clonedand sequenced (7, 13). Based on their deduced amino acidsequences, CelY and CelZ have been assigned to differentfamilies of glycohydrolases, family 8 and family 5 (25), respec-tively. In recombinant E. coli, the celY gene from E. chrysan-themi was poorly expressed due to promoter structure (13).

In this study, we have constructed plasmids that expresshigher levels of CelY in recombinant E. coli. Surprisingly, 90%of the CelY activity was secreted as an extracellular product bya native E. coli secretion system. Using recombinant CelY andCelZ, the combined actions of both enzymes were investigatedusing CMC and acid-swollen cellulose as substrates. Synergywas observed with both substrates.

* Corresponding author. Mailing address: Dept. of Microbiology andCell Science, IFAS, P.O. Box 110700, University of Florida, Gaines-ville, FL 32611. Phone: (352) 392-8176. Fax: (352) 846-0969. E-mail:ingram@ufl.edu.

† Florida Agricultural Experiment Journal Series no. R-07249.

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MATERIALS AND METHODS

Bacteria, plasmids, and culture conditions. Bacterial strains and plasmidsused in this study are listed in Table 1. E. coli DH5a and TOPO10F9 were usedas hosts for plasmid constructions. The celZ gene was previously cloned in ourlaboratory from E. chrysanthemi P86021 (4). The celY gene was cloned byGuiseppi et al. (13) from E. chrysanthemi 3937. The out genes were cloned by Heet al. (14) from E. chrysanthemi EC16.

E. coli cultures were grown at 37°C in Luria-Bertani (LB) broth (1) containing,per liter, 10 g of Difco (Detroit, Mich.) tryptone, 5 g of Difco yeast extract, and5 g of sodium chloride or on solid LB medium containing agar (1.5%). Cloneswere screened for endoglucanase production using the Congo red method (34).Indicator plates were prepared by supplementing LB agar with low-viscosityCMC (0.3%). Ampicillin (50 mg/ml), kanamycin (50 mg/ml), and spectinomycin(100 mg/ml) were added as appropriate for selection.

Genetic methods. Standard methods were used for plasmid construction andanalyses (1). The coding region for celY was amplified by PCR using pMH18 asthe template with the following primer pair: N terminus 59CTGTTCCGTTACCAACAC39 and C terminus 59GTGAATGGGATCACGAGT39. The E. chry-santhemi out genes (pCPP2006) were transferred by conjugation using pRK2013for mobilization (39). DNA was sequenced by the dideoxy method using aLI-COR model 4000-L DNA sequencer and fluorescent primers.

Enzyme assay. Endoglucanase activity was determined in vitro using CMC asa substrate. Appropriate dilutions of cell-free culture broth (extracellular activ-ity) or broth containing cells that had been disrupted by ultrasound (total activ-ity) were assayed at 35°C in 50 mM citrate buffer (pH 5.2) containing low-viscosity CMC (20 g per liter). Reactions were terminated by heating in a boilingwater bath for 10 min. Reducing sugars were measured using 3,5-dinitrosalicylicacid reagent with glucose as a standard (34). Enzyme activity (CMCase) isexpressed as micromoles of reducing sugar released per minute (in internationalunits). Results are averages of two or more determinations.

Synergism. Stationary-phase cultures of DH5a(pLOI1620 plus pCPP2006)and DH5a(pLOI2311) were sonicated and centrifuged as previously described(39) as a source of CelZ and CelY, respectively. These were diluted as necessaryto provide equal CMCase activities. Mixtures of CelZ and CelY were tested forsynergy at 35°C in 50 mM citrate buffer (pH 5.2) containing CMC (20 g per liter)or acid-swollen cellulose (20 g per liter). For tests with Avicel (20 g per liter),enzyme preparations were mixed without prior dilution. Hydrolyzed samples ofacid-swollen cellulose and Avicel were centrifuged (10,000 3 g, 5 min) to removeinsoluble material prior to the determination of concentrations of reducingsugars.

The effects of sequential additions of CelZ and CelY were also investigated.Substrates were hydrolyzed with a single enzyme for 4 h and then inactivated byboiling for 20 min. After the hydrolysate cooled, the second enzyme was addedand incubated for an additional 4 h. Control experiments were conducted withboth enzymes together (4 h) and with each enzyme alone (4 h). Samples wereanalyzed for reducing sugar. In some cases, products were also analyzed bythin-layer chromatography.

The degree of synergism for enzyme mixtures was calculated as the observedactivity divided by the sum of predicted contributions from CelY alone and CelZalone (28).

Hydrolysis products from soluble cellooligosaccharides and cellulose. Hydro-lysis products from cellobiose, cellotriose, cellotetraose, cellopentaose, acid-swollen cellulose (34), and Avicel were analyzed by thin-layer chromatography.For tests with soluble cellooligosaccharides, 15 ml of a 1% substrate was mixedwith 45 ml of crude enzyme (0.07 IU) and incubated at 35°C for 2 h, and thereaction was terminated by heating in a boiling water bath. Avicel (2%, 48-h

incubation) and acid-swollen cellulose (2%, 6-h incubation) were digested withdifferent concentrations of endoglucanse, namely, 8 IU of CMCase/ml and 0.8 IUof CMCase/ml, respectively. Again, reactions were terminated by heating in aboiling water bath.

Hydrolysis products were separated for approximately 4 h using Whatman250-mm-thick Silica gel 150A plates with the solvent system described by Kim(19). By volume, this solvent contained 6 parts chloroform, 7 parts acetic acid,and 1 part water. Sugars were visualized by spraying with 6.5 mM N-(1-naph-thyl)ethylenediamine dihydrochloride and heating at 100°C for approximately 10min (6).

Materials and chemicals. Tryptone and yeast extract were products of Difco.Antibiotics, low-viscosity CMC, cellobiose, cellotriose, and cellotetraose wereobtained from the Sigma Chemical Co. (St. Louis, Mo.). Cellopentaose wasobtained from V-Lab (Covington, La.). Avicel was purchased from FlukaChemika (Buchs, Switzerland).

RESULTS

Production of CelY and CelZ by recombinant E. coli. Asreported previously (7, 13), low levels of CelY activity wereproduced by native E. chrysanthemi 3937 and by recombinantE. coli harboring plasmid pMH18. Poor expression from thehigh-copy-number plasmid in E. coli was attributed to pro-moter function and a putative requirement for a celY activatorprotein (13). A new clone was constructed to produce higherlevels of CelY for our investigations of synergy. The CelYcoding region (without promoter) was amplified by PCR andcloned behind the lac promoter in pCR2.1-TOPO. The result-ing plasmid, pLOI2311, was strongly positive on CMCase in-dicator plates. Replacement of the native promoter with thelac promoter increased celY expression by approximately 10-fold, from 165 to 1,800 IU/liter (Table 2). Approximately 90%of CelY activity was found in the extracellular milieu. Ex-pression of celZ was included for comparison (Table 2). Highlevels of CelZ were produced by E. coli harboring plasmidpLOI1620. Extracellular CelZ and total CelZ activities werefurther increased by addition of the E. chrysanthemi out genes(pCPP2006) as reported previously (39). Unlike CelZ activity,however, CelY activity was not affected by the presence of outgenes. Maximal CelY and CelZ activities were obtained from24-h cultures. The supernatants from disrupted cultures ofDH5a containing pLOI2311 or pLOI1620 and pCPP2006 (outgenes) were used as a source of CelY or CelZ, respectively, forfurther investigations.

Synergistic action of CelY and CelZ with CMC as a sub-strate. Initial experiments examining the combined actions ofCelY and CelZ were conducted with CMC (20 g per liter) fora single incubation time (Fig. 1A). Disrupted cell preparations

TABLE 1. Strains and plasmids used in this study

Strain or plasmid Description Source or reference

StrainsEscherichia coli

DH5a lacZDM15 recA Bethesda Research LaboratoryB Prototrophic ATCC 11303HB101 recA lacY recA ATCC 37159TOP10F9 This strain expresses the lac repressor (lacIq gene) from an F episome Invitrogen

PlasmidspCR2.1-TOPO TOPO TA cloning vector, Apr Kmr InvitrogenpRK2013 Kmr mobilizing helper plasmid (mob1) ATCCa

pCPP2006 Spr, ca. 40-kbp plasmid carrying the complete out genes from E. chrysanthemi EC16 14pLOI1620 Apr, celZ gene and its native promoter from E. chrysanthemi P86021 4pMH18 Apr, celY gene and its native promoter from E. chrysanthemi 3937 13pLOI2311 celY gene (without native promoter), cloned into pCR2.1-TOPO vector and oriented

for expression from the lac promoterThis study

a ATCC, American Type Culture Collection.

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containing CelY and CelZ were each diluted to equal activities(CMCase) and combined in different proportions to maintaina constant sum of individual activities. CelY and CelZ weretested individually as controls. Activities of CelY and CelZ inall mixtures were significantly higher than that of either en-zyme assayed alone, indicating a synergistic interaction. Thesynergistic effect increased with the proportion of CelZ. Max-imal synergy (1.42) was observed with ratios of CelZ to CelYactivities of 9 to 1 and 19 to 1.

Further experiments examined the effect of incubation timeusing CMC as the substrate and an activity ratio of 9 to 1 forCelZ and CelY, respectively (Fig. 1B). CelZ and CelY alonewere included as controls. The synergistic effect of combiningCelZ and CelY was clearly evident as increases in the rate andextent of hydrolysis. Calculated synergy increased with incuba-tion time. At the end of the incubation (4 h), the concentrationof reducing sugars was 1.8-fold higher in the mixed enzymepreparation than that predicted by the arithmetic sum of indi-vidual CelZ and CelY activities.

Effect of substrate (CMC) concentration on synergy. A pre-vious study has determined that synergy in other systems isaffected by substrate concentration (37). This was also truefor synergy between CelZ and CelY (Table 3). Increasingthe CMC concentration from 2.5 to 20 g per liter increasedthe observed synergy from 1.12 to 1.89. Based on the specificactivities of CelZ and CelY and a maximal synergism of 1.89,the enzyme turnover rate for the combination was 8-fold thatof purified CelY alone and 1.5-fold that of purified CelZ alone.

CelY was more sensitive to substrate concentration thanCelZ. Increasing the CMC concentration resulted in an eight-fold increase in reducing sugar products with CelY but onlya threefold increase with CelZ. Based on a double reciprocalplot of the data in Table 3, apparent Km values of 104, 12, and38 g per liter were estimated for CelY, CelZ, and the combi-nation of both enzymes (9 parts CelZ plus 1 part CelY), re-spectively. The higher apparent Km for CelY is consistent witha requirement for longer substrate molecules.

The extent of CMC hydrolysis was also examined by deter-mining the approximate sizes of hydrolysis products. CMC(1.25 g per liter) was incubated (4 h, 0.75 IU of CMCase/ml)with CelY, CelZ, and a combination of both enzymes (9 partsCelZ plus 1 part CelY). Chain length was estimated based onresults of the reducing sugar assay before (250 glucosyl units)and after incubation. The average chain length was substan-tially reduced by all three enzyme preparations. CelZ was moreeffective in reducing chain length than CelY, with CelZ reduc-

ing chain length to 3.6 glucosyl residues, versus 10.7 with CelY.The combination of both enzymes resulted in a synergisticaction. Simultaneous hydrolysis with both enzymes reduced theaverage size of the hydrolysis products to 2.3 glucosyl residues,which is 36% smaller than with CelZ alone and 79% smallerthan with CelY alone. These results confirm that CelZ readilyhydrolyzes both large CMC polymers and smaller saccharides.The action of CelY appears more limited in that it hydrolyzesprimarily large polymers with greater than 10 glucosyl units.

Sequential and simultaneous hydrolysis of CMC with CelZand CelY. The mechanism of synergistic action between CelZand CelY was further investigated by comparing the effects ofsequential hydrolysis with individual endoglucanases to thoseof simultaneous hydrolysis by a mixture of both enzymes (Ta-ble 4). Again, synergy was observed for the simultaneous ac-tions of both enzymes. No synergy was observed for the se-quential hydrolysis of CMC when CelZ was used as the firstenzyme and CelY was used as the second enzyme (after heat in-activation of CelZ). In contrast, full synergy was retained whenCMC was first treated with CelY and then with CelZ (afterheat inactivation of CelY). These results indicate that synergycan be achieved by the independent activities of CelY and CelZ.Enzymatic modification of the substrate by CelY increased therate and extent of subsequent hydrolysis by CelZ. These resultsprovide further evidence that CelY and CelZ function quitedifferently. CelY appears primarily to reduce the chain lengthsof large polymers, while CelZ appears to act more randomly,hydrolyzing both large and small substrate molecules.

Synergistic action on acid-swollen and crystalline cellulose.Potential synergy was investigated using acid-swollen celluloseas the substrate and a 9 to 1 ratio of CelZ to CelY based onCMCase activities (Fig. 1C). Since the activities of CelZ andCelY with acid-swollen cellulose are lower than those withCMC (7), enzyme loading (1.5 IU) and incubation times wereincreased. When assayed individually with acid-swollen cellu-lose, CelY was approximately one-third as active as CelZ.However, the combination of these two enzymes was signifi-cantly more active than was predicted by the arithmetic sum ofindividual activities at all time points. The degree of synergywas essentially constant (1.36 6 0.17) during the 36-h period ofincubation.

The hydrolysis products from acid-swollen cellulose (6 h)were analyzed by thin-layer chromatography (Fig. 2A and B).No soluble saccharides were observed after incubation withCelY alone. Cellobiose and cellotriose were the primary prod-ucts from hydrolysis with CelZ alone and a combination of

TABLE 2. Effects of E. chrysanthemi out genes on the expression and secretion of celY and celZ in E. coli DH5a

Enzymeexpressed Promoter Growth

(h)

Without out genes With out genes (1 pCCP2006)

ExtracellularCMCasea

(IU per liter)

TotalCMCase

(IU per liter)

Apparentsecretion

(%)

ExtracellularCMCase

(IU per liter)

TotalCMCase

(IU per liter)

Apparentsecretion

(%)

CelY Native promoter (pMH18) 24 136 165 82 136 180 76

lac promoter (pLOI2311) 8 208 266 78 NDb ND ND16 1,420 1,590 90 ND ND ND24 1,650 1,800 90 1,360 1,510 90

CelZ Native plus lac promoter(pLOI1620)

8 130 1,320 10 6,710 7,460 9016 1,200 9,030 13 13,400 19,700 6824 1,800 12,500 14 23,600 36,800 64

a CMCase activity secreted or released in the culture supernatant.b ND, not determined.

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CelY and CelZ. With the combination of both enzymes, higherproduct levels were evident as darker and larger spots, con-firming a synergistic action.

The synergistic action of CelZ and CelY was also investi-gated with Avicel (Fig. 2C), a highly crystalline cellulose. Smallamounts of cellobiose and cellotriose were observed as hydro-lysis products with CelZ alone and with the mixture of CelYand CelZ. Due to low activity with Avicel, large loadings (10ml) were required on thin-layer plates to visualize products.Note that this additional salt increased the relative migrationsof oligosaccharide products in comparison to those of the stan-dards. No cellooligosaccharide spots were observed with CelYalone. Again, synergism was evident with the combination ofCelY and CelZ. We observed larger and more intense spotscorresponding to cellobiose, cellotriose, and cellotetraose withthe enzymes combined than with CelZ alone. The low activitywith Avicel as a substrate and the relatively low levels ofproducts are consistent with the hydrolysis of the amorphousrather than the crystalline regions of Avicel. These resultsindicate that the synergistic action of CelZ and CelY is notlimited to a model substrate such as CMC. Synergistic hydro-lysis was also observed for acid-swollen cellulose and the amor-phous regions of Avicel.

Hydrolysis of cellooligosaccharides. The substrate specifici-ties of CelZ and CelY were further investigated using soluble

TABLE 3. Effect of substrate concentration on synergy

Amt of CMCsubstrate(g/liter)

Concn of reducing sugarreleased (mmol/ml)a Avg synergyb

6 SDCelZ CelY CelZ 1 CelY

20 3.98 6 0.04 3.83 6 0.04 7.51 6 0.07 1.89 6 0.0210 4.53 6 0.01 2.91 6 0.07 5.38 6 0.04 1.25 6 0.01

5.0 2.87 6 0.01 1.18 6 0.04 2.92 6 0.04 1.08 6 0.022.5 1.42 6 0.01 0.50 6 0.04 1.49 6 0.01 1.12 6 0.01

a Averages 6 standard deviations. CelZ and CelY were diluted to equalCMCase activities. Reaction mixtures (0.15 IU/ml) with both CelZ and CelYcontained 9 parts CelZ and 1 part CelY. As controls, CelZ (0.15 IU/ml) andCelY (0.15 IU/ml) were each tested individually.

b Synergy was calculated as the observed activity divided by the sum of pre-dicted contributions from CelY alone (10%) plus CelZ alone (90%).

TABLE 4. Sequential and simultaneous hydrolysis ofCMC by CelZ and CelY

Enzyme(relative proportion)a

Measuredreducing

sugarreleased

(mmol/ml)b

Predicted activityfrom the arith-metic sum of

CelY and CelZ(mmol/ml)c

Synergyb,d

CelZ (10 parts) 1 CelY (0 parts) 4.65 6 0.08 4.65 1.00 6 0.02CelZ (0 parts) 1 CelY (10 parts) 4.14 6 0.04 4.14 1.00 6 0.01CelZ (9 parts) 1 CelY (1 part)

(simultaneously)8.28 6 0.08 4.60 1.80 6 0.02

CelZ (9 parts) and then CelY(1 part) (sequentially)

4.86 6 0.23 4.60 1.06 6 0.05

CelY (1 part) and then CelZ(9 parts) (sequentially)

8.75 6 0.14 4.60 1.90 6 0.03

a CelZ and CelY were diluted to equal CMCase activities. Both simultaneousand sequential hydrolysis reactions (0.15 IU/ml) were investigated using 9 partsCelZ and 1 part CelY. In the sequential hydrolysis experiments, the first enzymewas incubated with the substrate for 4 h and inactivated by boiling for 20 min.After cooling, the second enzyme was added and incubated for an additional 4 h.All reactions were terminated by boiling.

b Averages 6 standard deviations (three experiments).c Calculated sum of individual CelY and CelZ activities.d Synergy was calculated as the observed activity divided by the sum of pre-

dicted contributions from CelY alone (10%) plus CelZ alone (90%).

FIG. 1. Synergistic action of CelY and CelZ. Both enzymes were diluted toequal CMCase activities (1.5 IU/ml). Calculated synergies are shown in paren-theses. (A) Effect of enzyme ratios on synergy. Different amounts of CelY andCelZ were combined to maintain a constant predicted activity (0.15 IU/ml) basedon the contributions of individual enzymes. Assays were incubated with CMC for1 h at 35°C, and reactions were terminated by boiling. Numbers on the x axesindicate the proportions of CelZ and CelY. Synergy is shown above each bar. (B)Hydrolysis of CMC by CelZ and CelY, alone and in combination (9 parts CelZto 1 part CelY). All assay mixtures contained equal total activities (0.15 IU/ml)based on the sum of individual CelY and CelZ activities. Synergy is shown aboveeach point for the combination of both enzymes. (C) Hydrolysis of acid-swollencellulose by CelZ and CelY, alone and in combination. A 9-to-1 ratio of CelZ toCelY was used for the combined enzyme reaction. All assay mixtures contained1.5 IU/ml based on the sum of individual CelY and CelZ activities. Synergy isshown above each point for the combination of both enzymes.

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cellooligosaccharides (cellobiose, cellotriose, cellotetraose,and cellopentaose). Hydrolysis products were analyzed by thin-layer chromatography (Fig. 3). Cellobiose was not hydrolyzedby CelY, CelZ, or a combination of both enzymes (data notshown). None of the cellooligosaccharides was hydrolyzed byCelY alone (Fig. 3B). In contrast, CelZ hydrolyzed cellote-traose and cellopentaose but not cellotriose (Fig. 3C). CelZhydrolysis products from cellotetraose were primarily cellobi-ose, with lesser amounts of cellotriose and glucose. With cello-pentaose as the substrate, CelZ produced approximately equalamounts of cellobiose and cellotriose, indicating a preferentialattack on the second or third glycosidic bond. This conclusionwas further confirmed by examining samples at various timesduring the incubation of cellopentaose with CelZ (Fig. 3D).Cellobiose and cellotriose progressively accumulated duringincubation, with a corresponding reduction in cellopentaose.Thus, unlike with CelY, which requires large substrates, CelZhydrolyzes soluble cellooligosaccharides containing 4 or moreglucosyl units.

DISCUSSION

E. chrysanthemi CelZ and CelY are typical endoglucanasesin that both have high activities with CMC as a substrate andlittle activity with crystalline cellulose (7). However, the struc-tures of these enzymes are quite different, with minimal se-quence identity (13). Each has been assigned to a differentglycohydrolase family (25), and only CelZ contains a cellu-lose-binding domain (25). In E. chrysanthemi, 95% of thetotal endoglucanase activity (CMCase) is attributed to CelZand 5% is attributed to CelY (7, 13). To be effective duringthe maceration of plant cell walls, these enzymes must besecreted into the extracellular milieu. CelZ is secreted usinga type II secretion system which requires the sec and outgenes (14, 39). In recombinant E. coli containing the outgenes, approximately half of the total CelZ activity was re-covered in the culture supernatant. In contrast, 90% of CelYwas secreted as an extracellular product in recombinantE. coli and this secretion was not affected by the presenceof out genes. This gene also contains an N-terminal leadersequence (13) and is presumed to utilize a type IV secretionsystem (16), similar to that proposed for the CelL endoglu-canase in Pseudomonas solanacearum (15). The use of twodifferent routes for the extracellular secretion of E. chrysan-themi endoglucanases may facilitate higher levels of endo-glucanase production.

CelZ and CelY act synergistically during the hydrolysis ofamorphous cellulose and CMC. This result was unanticipated,since a prior study with these enzymes failed to observe synergy(7). Since no quantitative results or details were provided, thisdiscrepancy is attributed to differences in methodology. Inour experiments, the extent of synergy was dependent uponthe ratio of the two enzymes, substrate concentration, and theperiod of incubation. Maximal synergy was observed for en-zyme mixtures containing 90 to 95% CelZ (CMCase activitybasis). Based on the specific activities for purified CelZ (200mmol/mg of protein; molecular weight, 45,000) and CelY(33 mmol/mg of protein; molecular weight, 35,000) with CMCas a substrate (7), the 9:1 and 19:1 mixtures of CelZ to CelYcorrespond to molar enzyme ratios of 1.2 to 1 and 2.4 to 1,respectively.

Synergy has been extensively documented for many combi-nations of endoglucanase with exoglucanase (24, 35, 36) andfor combinations of exoglucanases (3, 12, 23, 28). However,synergy between two endoglucanases is unusual. Two previous

FIG. 2. Thin-layer chromatography analysis of the hydrolysis products fromacid-swollen cellulose and Avicel. Abbreviations for y axis: G1, glucose; G2,cellobiose; G3, cellotriose; G4, cellotetraose; and G5, cellopentaose. Lanes: S,mixed-cellooligosaccharide standard; C, control lacking enzyme; Z, CelZ; Y,CelY; and Y 1 Z, CelY plus CelZ. (A) Acid-swollen cellulose (6-h incubation,1-ml loading); (B) acid-swollen cellulose (6-h incubation, 2-ml loading); (C)Avicel (48-h incubation, 10-ml loading).

FIG. 3. Hydrolysis of cellooligosaccharides by CelZ and CelY. Each test contained approximately 0.07 IU of CMCase per ml (2-h incubation, 35°C). Abbreviations:S, mixed-cellooligosaccharide standard; G1, glucose; G2, cellobiose; G3, cellotriose; G4, cellotetraose; and G5, cellopentaose. (A) Before hydrolysis; (B) afterincubation with CelY; (C) after incubation with CelZ; (D) CelZ hydrolysis of cellopentaose after different periods of incubation (0, 5, 10, and 25 min).

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reports of increased activity with mixtures of endoglucanaseshave been attributed to possible contamination with an exo-glucanase (20, 27). Contamination was very unlikely in ourstudy due to the use of recombinant enzyme preparations. Athird recent report has demonstrated synergy between endo-glucanases from two different species of Gloeophyllum, G. tra-beum and G. sepiarium (23). With softwood-dissolving pulp asa substrate, the combined activities of both enzymes was 108%of that predicted by the sum of individual activities.

The synergistic action of E. chrysanthemi CelY and CelZ wasmuch more pronounced than that observed with the Gloeo-phyllum endoglucanases (23). In both cases, synergy appears toresult from differences in substrate preferences and modes ofaction. CelY failed to hydrolyze soluble cellooligosaccharidesand produced no soluble products during the hydrolysis ofamorphous cellulose. With this enzyme, hydrolysis productsfrom CMC averaged 10 glucosyl units. CelY exhibited a nine-fold-higher apparent Km for CMC than CelZ. In contrast, CelZhydrolyzed both long-chain substrates and soluble cellooligo-saccharides (4 or more glucosyl residues). Cellobiose and cel-lotriose were produced as primary products from the hydroly-sis of amorphous cellulose by CelZ.

Equal levels of synergy were observed when both enzymeswere present simultaneously and after the sequential additionof CelY and CelZ (after heat inactivation of CelY). No synergywas observed when CelZ was used as the first enzyme. Thus,only CelY can independently modify the substrate to increasedigestibility. This action by CelY is consistent with a prefer-ence for long substrate molecules in converting CMC or amor-phous cellulose into a modified substrate containing fragmentsof intermediate lengths rather than a random assortmentof sizes. The smaller products from CMC are soluble and areobserved as an increase in the concentration of reducing sugar.The low apparent activity of CelY with acid-swollen cellulose isconsistent with the removal of longer products (6 or moreglucosyl units) as insoluble material during centrifugation. Theincrease in the concentration of soluble reducing sugar ob-served as synergy with amorphous cellulose is proposed toresult from an increase in soluble products from the CelZ-mediated hydrolysis of intermediate-length cellooligosacchar-ides (CelY products), which produces diffusable substrates thatare further hydrolyzed by CelZ. Analogous activities also in-crease the efficiency of CMC depolymerization. The enhancedactivity of CelZ on CelY-modified substrates is thus proposedto result from increases in the rate of production and effectiveconcentration of small, rapidly diffusing substrate molecules.

Both CelY and CelZ have been retained during the evolu-tion of E. chrysanthemi and are presumed to have unique fea-tures which contribute to the success of this organism amongthe biota. Our results establish that these two enzymes havedifferent but complementary requirements for substrate lengthwhich result in synergistic hydrolysis of acid-swollen cellulose(amorphous) and CMC. Maximal synergy was observed withenzyme mixtures containing small amounts of CelY activityrelative to that of CelZ (1 to 19, similar to the ratio producedby E. chrysanthemi in nature [7, 13]). The synergistic action ofCelY and CelZ resulting from complementary differences insubstrate preference may have provided an important evolu-tionary advantage for the retention of both endoglucanaseenzymes. Previous investigators (26, 30) have proposed analo-gous differences in substrate preferences as a rationale for theretention of multiple pectate lyase (pel) genes by E. chrysan-themi.

Figure 4 shows a cartoon model for the digestion of amor-phous cellulose by E. chrysanthemi. This organism appears touse a combination of three glucosidase enzymes (CelY, CelZ,

and phospho-b-glucosidase). Nicks are inserted into amor-phous cellulose at relatively long intervals by CelY to reducethe average chain length and thus minimize the number ofCelZ-catalyzed events required to create soluble fragments of2 to 6 glucosyl units. Resulting soluble fragments are furtherhydrolyzed by CelZ to dimers and trimers. Dimers (and pre-sumably trimers also) are then transported into the cell by thephosphoenolpyruvate-dependent phosphotransferase systemfor cellobiose (11) and hydrolyzed by the cytoplasmic phospho-b-glucosidase. The resulting products, glucose and glucose-6-phosphate, enter glycolysis for further metabolism. Comparedto the number of organisms able to use glucose, relatively feworganisms are capable of cellobiose uptake and direct intra-cellular metabolism. By avoiding complete extracellular hydro-lysis to glucose through the actions of CelY, CelZ, and an

FIG. 4. Model illustrating the utilization of amorphous cellulose by E. chry-santhemi. Three glucosidases are used for the catabolism of amorphous cellulose.Two of these, CelY and CelZ, are extracellular endoglucanases which functiontogether in a synergistic fashion. CelY requires large substrate molecules andhydrolyzes these into shorter, insoluble fragments. CelY does not hydrolyzesoluble cellooligosaccharides (2 to 5 glucosyl residues). CelZ readily hydrolyzessoluble cellooligosaccharides (cellopentaose and cellotetraose) and amorphousfragments of intermediate lengths to produce cellobiose and cellotriose. Cello-biose (G-G) and cellotriose (G-G-G) are phosphorylated (P) during cellularuptake by a phosphoenolpyruvate-dependent phosphotransferase system (PTS).Hydrolysis is completed intracellularly by a third enzyme, phospho-b-glucosi-dase. Resulting monomeric products (glucose and glucose-6-phosphate) are me-tabolized by glycolysis.

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active transport system for cellobiose and cellotriose, E. chry-santhemi has evolved to minimize the availability of cellulose-derived products to competing organisms in the environment.

ACKNOWLEDGMENTS

We thank F. Barras for sharing plasmid pMH18, which contains thecelY gene from E. chrysanthemi 3937, and A. Collmer for sharing plas-mid pCPP2006, which contains the out genes from E. chrysanthemiEC16.

This research was supported in part by grants from the U.S. Depart-ment of Agriculture, National Research Initiative (98-35504-6177);the U.S. Department of Energy, Office of Basic Energy Science(FG02-96ER20222); and the Florida Agricultural Experiment Station,University of Florida.

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