Enzymatic Degradation ofCarboxymethyl CelluloseHydrolyzed by theEndoglucanases Cel5A,Cel7B, and Cel45A fromHumicola insolens and Cel7B,Cel12A and Cel45Acore fromTrichoderma reesei

Johan Karlsson1

Dane Momcilovic2

Bengt Wittgren3

Martin Schulein4

Folke Tjerneld1

Gunnar Brinkmalm3

1 Department of Biochemistry,Lund University,

P. O. Box 124,S-221 00 Lund, Sweden

2 Technical AnalyticalChemistry,

Lund University,P. O. Box 124,

S-221 00 Lund, Sweden

3 AstraZeneca R&D Molndal,S-431 83 Molndal, Sweden

4 Novozymes,Smørmosevej 25,

DK-2880 Bagsværd,Denmark

Received 26 January 2001;accepted 28 June 2001

Abstract: Enzymatic hydrolysis of carboxymethyl cellulose (CMC) has been studied with purifiedendoglucanases Hi Cel5A (EG II), Hi Cel7B (EG I), and Hi Cel45A (EG V) from Humicola insolens,and Tr Cel7B (EG I), Tr Cel12A (EG III), and Tr Cel45Acore (EG V) from Trichoderma reesei. TheCMC, with a degree of substitution (DS) of 0.7, was hydrolyzed with a single enzyme until no furtherhydrolysis was observed. The hydrolysates were analyzed for production of substituted and non-substituted oligosaccharides with size exclusion chromatography (SEC) and with matrix-assistedlaser desorption/ionization mass spectrometry (MALDI-TOF-MS). Production of reducing ends andof nonsubstituted oligosaccharides was determined as well. The two most effective endoglucanasesfor CMC hydrolysis were Hi Cel5A and Tr Cel7B. These enzymes degraded CMC to lower molarmass fragments compared with the other endoglucanases. The products had the highest DSdetermined by MALDI-TOF-MS. Thus, Hi Cel5A and Tr Cel7B were less inhibited by the substitu-ents than the other endoglucanases. The endoglucanase with clearly the lowest activity on CMC wasTr Cel45Acore. It produced less than half of the amount of reducing ends compared to Tr Cel7B;furthermore, the products had significantly lower DS. By MALDI-TOF-MS, oligosaccharides withdifferent degree of polymerization (DP) and with different number of substituents could be sepa-rated and identified. The average oligosaccharide DS as function of DP could be measured for each

Correspondence to: Gunnar Brinkmalm; email: gunnar.brinkmalm@astrazeneca.com

Contract grant sponsor: Swedish Centre for Amphiphilic Poly-mers from Renewable ResourcesBiopolymers, Vol. 63, 32–40 (2002)© 2002 John Wiley & Sons, Inc.DOI 10.1002/bip.1060

32

enzyme after hydrolysis. The combination of techniques for analysis of product formation gaveinformation on average length of unsubstituted blocks of CMC. © 2002 John Wiley & Sons, Inc.Biopolymers 63: 32–40, 2002

Keywords: size exclusion chromatography; matrix-assisted laser desorption/ionization mass spec-trometry; carboxymethyl cellulose; substituent distribution; endoglucanases; enzymatic hydrolysis;Humicola insolens; Trichoderma reesei

INTRODUCTION

Carboxymethyl cellulose (CMC) is a cellulose deriv-ative with carboxymethyl groups bound to the hy-droxyl groups of the glucose unit. There can be up tothree substituents on each glucose unit, thus the max-imal degree of substitution (DS) is 3. CMC is solublein water when having a DS above 0.4; commercialCMC usually has a DS less than 1.5. The synthesis ofCMC is a random process even though some controlof the regioselectivity has been reported,1 e.g., syn-thesis of 6-O-CMC and of 2,3-O-CMC. CMC is anindustrially important cellulose derivative.2 Examplesof industrial applications are found in the food indus-try as thickener and binder, in the oil industry aslubricant for drilling, and in the cosmetic industry asstabilizer and binder.

The cellulolytic enzymes produced by fungi can bedivided into cellobiohydrolases and endoglucanases.The cellobiohydrolases are exoglucanases and de-grade the cellulose chain starting from the reducingend or the nonreducing end. The endoglucanasescleave the cellulose chain internally, thus producingshorter polymer fragments and oligosaccharides.CMC is a good substrate for endoglucanases, and isfrequently used as a standard substrate in assays ofendoglucanase activity.3 It is thus attractive to usepurified endoglucanases as tools in analytical charac-terization of CMC, and to develop techniques wherethese enzymes are used also for characterization ofother soluble cellulose derivatives.

The fungi Humicola insolens4 and Trichodermareesei5 both produce several endoglucanases. Wehave in this study adopted the glycoside hydrolasenomenclature suggested by Henrissat et al.,6 wherethe H. insolens endoglucanases are indicated with theprefix Hi and the T. reesei endoglucanases with theprefix Tr. We have used endoglucanases of H. inso-lens Hi Cel5A (EG II), Hi Cel7B (EG I), and HiCel45A (EG V),4 and T. reesei Tr Cel7B (EG I),7 TrCel12A (EG III),8,9 and Tr Cel45Acore (EG V).10 HiCel5A, Hi Cel45A, and Tr Cel7B have a two-domainstructure with a catalytic core domain and a cellulosebinding domain separated by a linker region. HiCel7B, Tr Cel12A, and Tr Cel45Acore consist of onlythe catalytic core domain. Tr Cel45Acore is a genet-

ically truncated enzyme where the linker and thecellulose binding domain have been deleted. Thethree-dimensional (3D) structure is known for thecatalytic core domain of Hi Cel7B,11 Hi Cel45A,12,13

and Tr Cel7B.14

Cellulases have earlier been used for studies ofcellulose derivatives; however, most studies havebeen performed with unpurified or unspecified en-zymes. The cellulases of Trichoderma viride wereable to hydrolyze 6-O-methylcellulose with DS 1.0but not 2,3-O-methylcellulose with DS 2.0.15–17 Ap-parently, at least one of the T. viride cellulases iscapable of hydrolysing a �-1,4-glycosidic bond with a6-O substituent on both adjacent glucose units. Fur-thermore, it was shown that the enzymes could hy-drolyze the unsubstituted parts of a highly substituted2,3-O-methylcellulose.16 A series of papers18–20 havebeen devoted to analysis of CMC after endoglucanasehydrolysis; however, the authors have not reportedwhich endoglucanase they worked with. Horner etal.18 have shown that a H. insolens endoglucanasewas able to hydrolyze the CMC adjacent to a 6-O-substituted glucose unit and produce 6-O-carboxy-methyl glucose. Heinze et al.19 showed that one of theH. insolens endoglucanases preferably hydrolyzed ar-eas on CMC with lower DS. Furthermore, CMC withsubstituents at O-2 and O-3 was hydrolyzed withsimilar efficiency as CMC with substituents at O-2and O-6. An Aspergillus endoglucanase was reportedto be inactive on CMC with DS above 1.6.20

We report here the enzymatic hydrolysis of CMCwith fungal endoglucanases. By using highly purifiedenzymes we were able to perform specific degradationof the cellulose derivative, i.e., each enzyme produceda different pattern of hydrolysis products. The hydro-lysates were analyzed with several techniques: sizeexclusion chromatography (SEC), analysis of reduc-ing ends, high performance liquid chromatography(HPLC) determination of soluble sugars, and matrix-assisted laser desorption/ionization mass spectrometry(MALDI-TOF-MS). While the other analysis tech-niques are well established, MALDI-TOF-MS israther novel in this area of application. Utilization ofMALDI-TOF-MS for analysis of a variety of syn-thetic polymers and carbohydrates has increased

Enzymatic Degradation of Carboxymethyl Cellulose 33

greatly in recent years.21,22 There are, however, ratherfew mass spectrometric studies made on cellulosederivatives and related compounds. Mischnick et al.have studied partial acid hydrolysates of amylosederivatives by fast atom bombardment mass spec-trometry (FAB-MS) and MALDI-TOF-MS,23 andArisz et al. have studied partial acid hydrolysates ofmethyl cellulose by FAB-MS.24 Enzymatic hydroly-sates of partially methylated pectin have been studiedby Korner/Limberg et al.25–27 and Daas et al.28 Re-cently native cellulose decomposed by hot-com-pressed water has been analyzed by MALDI-TOF-MS,29 and Jacobs and Dahlman have analyzed par-tially hydrolyzed CMC by MALDI-TOF-MS.30

Results of the analytical techniques we used werecombined to give further insight into the differencebetween the endoglucanases, as well as to gain infor-mation on the structure of CMC with regard to lengthof unsubstituted blocks.

MATERIALS AND METHODS

Substrates and Chemicals

Glucose was from Sigma (St. Louis, MO, USA), 2,5-dihy-droxybenzoic acid (DHB), and carboxymethylcellulose(CMC) from Aldrich (Steinheim, Germany). The CMC hada DS of 0.7 and the weight average molar mass (Mw) was250 kDa. Cellobiose, cellotriose, cellotetraose, and cello-pentaose were from Merck (Darmstadt, Germany). All otherchemicals were of analytical grade. All chemicals were usedwithout further purification.

Enzymes

Humicola insolens endoglucanases Hi Cel5A, Hi Cel7B,and Hi Cel45A were produced and purified according toSchou et al.31 The enzymes were heterologously expressedin noncellulolytic organisms and thus free of any contami-nating cellulase activity. Trichoderma reesei endoglucanaseTr Cel7B was purified according to Karlsson et al.32 TrCel12A was a kind gift from Dr. Michael Ward, Genencore,CA, USA. Tr Cel45Acore was purified as described bySiika-aho et al.33 Bovine serum albumin (BSA) was fromICN (Costa Mesa, CA, USA).

Enzymatic Hydrolysis of CMC

CMC, 10 g/L, in 50 mM sodium acetate pH 5.0 was hydro-lyzed with an excess of enzyme, 2 �M, for a prolongedhydrolysis time, 72 h. For all enzymes it was verified thathydrolysis had reached completion by adding a new aliquotof endoglucanase after 72 h and incubating for an additional24 h. The extra enzyme addition did not hydrolyze thesubstrate further for any of the endoglucanases used; thus

total hydrolysis was achieved after 72 h with 2 �M endo-glucanase. The hydrolysates were stored at �4°C. Thehydrolysis was performed at room temperature since someof the endoglucanases were inactivated at 40°C. Tr Cel12Awas inactivated when diluted, and 0.1 g/L BSA was addedto the hydrolysis solution to stabilize the endoglucanase.BSA was also added to the hydrolysis solution of Tr Cel7Band Tr Cel45Acore to avoid difficulties in comparing theresults for the T. reesei endoglucanases.

Size Exclusion Chromatography

SEC was performed in a FPLC system (Fast Protein LiquidChromatography, Pharmacia, Uppsala, Sweden). A Super-dex 75 HR 10/30 column was connected in series with aSuperdex 200 HR 10/30 column (Pharmacia, Uppsala, Swe-den). We used 200 mM ammonium acetate pH 5.0 as elutionbuffer at a flow rate of 0.5 mL/min and refractive indexdetector ERC-7515A (ERMA, Inc., Tokyo, Japan). Frac-tions of 1 mL were collected and analyzed further byMALDI-TOF-MS as described below. Dextran standardswith mean molar mass, Mw, of 80, 50, 25, 11.6, and 5.22kDa were used to calibrate the system.

Oligosaccharide Analysis

Soluble sugars, glucose, cellobiose, cellotriose, cellote-traose, and cellopentaose, were analyzed with an HPLCsystem with an anion exchange Carbopac PA100 columnand pulsed amperometric detection (Dionex, Sunnyvale,CA, USA). The oligosaccharides were eluted with isocratic50 mM sodium hydroxide and a gradient of sodium acetatefrom 50 to 300 mM. Glucose, cellobiose, cellotriose, cel-lotetraose, and cellopentaose were used to calibrate thesystem.

Reducing Ends Analysis

The reducing ends were analyzed using the dinitrosalicylicacid reagent.34 Glucose was used for the standard curve.

MALDI-TOF Mass Spectrometry

MALDI-TOF-MS was performed on a Voyager-DE STR(Applied Biosystems, Framingham, MA, USA) equippedwith an N2 laser, delayed extraction, reflector, and tandemcoupled microchannelplate detector in reflector mode. Afterattempting several combinations of matrix and solvents itwas concluded that (of the combinations attempted) reliabledata were obtained only with DHB as matrix and deionizedwater as solvent both for the CMC analyte and the matrix.After some initial experiments it was decided that a com-bination of four fractions from each SEC separation ofCMC hydrolysate (between 32 and 36 mL elution volume)well reflected the hydrolysate in a suitable molecular weightregion for MALDI-TOF-MS analysis of CMC (�5 kDa).

34 Karlsson et al.

The eluate from the SEC analysis was fractionated into1 mL fractions. The fractions were concentrated in a Speed-Vac concentrator for 48 h with heating (Savant, Farming-dale, NY, USA). The solvent had evaporated totally after48 h. The CMC fractions were then each dissolved in 400�L deionized (18 M�cm) H2O and vortexed a few min, andDHB was dissolved to 10 g/L in deionized H2O and vor-texed. For each CMC hydrolysate, the four fractions be-tween 32 and 36 mL elution volume were mixed (equalvolume) and these mixtures were then again separatelymixed 1:4 (v:v) with the DHB solution and vortexed. Onemicroliter of the mixture was then applied onto the stainlesssteel sample plate and dried in air. Unfortunately, DHBdissolved in H2O and dried in air give rather inhomoge-neous samples. Remaining salt in the analyte fractions alsocontributed to the sample inhomogeneity. To get good data,spectra were acquired from several positions of each samplespot and accumulated. Typically 400 laser shots from up to8 positions were recorded in each spectrum. All spectrawere recorded in negative ion mode using both delayedextraction and the reflector in order to get good enoughresolution to avoid overlapping signals.

RESULTS

Size Exclusion Chromatography

The CMC sample was analyzed with SEC before andafter enzymatic hydrolysis. The resulting elutioncurves are seen in Figure 1A for H. insolens and 1Bfor T. reesei. The elution volumes for the 50 and 5.2kDa dextran standards are used for a crude estimationof the molar mass of the hydrolyzed material. Clearly,all endoglucanases managed to hydrolyze CMC sig-nificantly, evidenced by the dramatic shift to higherelution volumes that is interpreted as a decrease inmolar mass. Hi Cel5A was the most efficient endo-glucanase of the H. insolens endoglucanases (Figure1A) and could hydrolyze CMC to a dextran-equiva-lent molar mass at about 10 kDa. Hi Cel45A wasslightly more efficient than Hi Cel7B. The CMC hy-drolysate of Hi Cel45A had a mean dextran-equiva-lent molar mass at around 20 kDa compared to HiCel7B where it was slightly higher. Tr Cel7B was themost efficient of the three endoglucanases of T. reesei,the dextran-equivalent molar mass of the product wasbetween 5 and 10 kDa (Figure 1B). Tr Cel12A wassignificantly more efficient than Tr Cel45Acore. Themean dextran-equivalent molar mass for the hydro-lyzed CMC was around 15 kDa for Tr Cel12A com-pared to around 50 kDa for Tr Cel45Acore. Hi Cel5Aand Tr Cel7B were very similar in their efficiency inhydrolyzing CMC.

Reducing Ends and Soluble SugarsProduction

The production of reducing ends and soluble sugarsafter hydrolysis was analyzed (Table I). The reducingend assay detects all reducing ends in the hydrolysate,whereas the HPLC analysis detects soluble nonsub-stituted sugars, from glucose to cellopentaose. In Ta-ble I, the glucose equivalents also have been calcu-lated for the soluble sugars detected by HPLC.

Tr Cel7B hydrolyzed CMC to a greater extent thanany other endoglucanase, 14.9% of the �-1,4 glyco-sidic bonds were hydrolyzed as seen in the reducingends analysis. Hi Cel5A was slightly less efficient,with 12% hydrolysis. Hi Cel7B and Tr Cel12A pro-duced similar amounts of reducing ends, 10.6%. Theleast efficient enzymes were Tr Cel45Acore followedby Hi Cel45A, which produced 6 and 8.5% reducingends respectively. The production of soluble sugarsfor Hi Cel7B was close to the production of reducingends; thus the majority of the reducing ends detectedwere soluble sugars. Tr Cel45Acore had the oppositehydrolytic behavior to Hi Cel7B—the production ofsoluble sugars was almost six times lower than theproduction of reducing ends. The behavior of HiCel45A was similar but not so pronounced as for TrCel45Acore. Hi Cel45A produced 3.5 times morereducing ends than soluble sugars. The other endo-glucanases (Hi Cel5A, Hi Cel7B, Tr Cel7B, and TrCel12A) produced about two times less soluble sugarsthan reducing ends. The amount of glucose equiva-lents were between 3.5 and 4.4 mM for all endoglu-canases except Hi Cel45A, 2.6 mM, and TrCel45Acore, 1.5 mM.

Analysis of Products UsingMALDI-TOF-MS

SEC analysis showed that products formed after en-zymatic hydrolysis of CMC had a wide range in molarmass (Figure 1). For MALDI-TOF-MS analysis theSEC eluates were fractionated and fractions contain-ing low molar mass polymers were collected. AMALDI-TOF mass spectrum is shown in Figure 2.The spectrum was averaged from four different HiCel5A hydrolysates. To be able to detect oligosaccha-rides shorter than cellopentaose in negative ion mode,fractions eluted after 36 mL had to be used. Unfortu-nately, when analysing the fraction of 36–37 mL nosignal was detectable for some of the hydrolysates.Moreover, when adding this fraction to the precedingones the previously detected signal now vanishedcompletely. The difficulty in analysis of the smalleroligosaccharides was probably due to salt interference

Enzymatic Degradation of Carboxymethyl Cellulose 35

from the hydrolysis buffer and thus fractions collectedafter 36 mL were omitted from this study for com-parison reasons. Earlier fractions did not present anyproblems of this kind, but when attempting to analyzepolymer distributions that are too wide severe dis-crimination effects appear, and therefore only fourfractions were selected for the experiment. From Fig-ure 2 can be seen that the produced oligosaccharides

with DP from 5 to 15 could be analyzed with respectto number of carboxymethyl substituents. The aver-age DSs for the oligosaccharides produced from thedifferent endoglucanase hydrolysates are shown inFigure 3. Measurements on acidically hydrolyzed cel-lulose derivatives (data not shown) suggest that theionization efficiency in the MALDI process is depen-dent on the number of substituents in the molecule,

FIGURE 1 Separation of hydrolyzed CMC on size exclusion chromatography. The amount of 0.5mL of CMC was injected on a Superdex 75 HR 10/30 column connected in series with a Superdex200 HR 10/30 column. The amount of 200 mM ammonium-acetate, pH 5.0, was used as elutionbuffer with a flow rate of 0.5 mL/min. The drop in detector signal at 38 mL elution volume comesfrom the hydrolysis buffer. The output from a refractive index detector is visualised in the figure.(A) The hydrolysate from H. insolens endoglucanases Hi Cel5A, Hi Cel7B, Hi Cel45A, andnonhydrolyzed CMC. (B) The hydrolysate from T. reesei endoglucanases Tr Cel7B, Tr Cel12A, TrCel45Acore, and nonhydrolyzed CMC.

36 Karlsson et al.

especially when there are few substituents present, asis this case where the degree of polymerization (DP)is low. It is therefore not possible to obtain exact

quantitative DS data. The obtained DS values hereshould still reflect the true values reasonably. Thus,the observed trend in Figure 3 is reliable: a hydroly-

Table I Production of Reducing Ends and Soluble Sugars on CMC DS 0.7 by Humicola insolens andTrichoderma reesei Endoglucanasesa

Substrate EnzymeReducingEnds (%)

Reducing Ends(mM)

Soluble Sugars(mM)

Glucose Equivalents(mM)

CMC Hi Cel5A 12.0 5.9 2.2 3.7CMC Hi Cel7B 10.6 5.3 3.8 3.8CMC Hi Cel45A 8.5 4.2 1.2 2.6CMC Tr Cel7B 14.9 7.3 3.8 4.4CMC Tr Cel12A 10.6 5.2 2.8 3.5CMC Tr Cel45Acore 6.0 2.9 0.5 1.5

a One hundred percent reducing ends indicates that all CMC has been hydrolyzed to monosaccharides. Soluble sugars is the sumof glucose, cellobiose, cellotriose, cellotetraose, and cellopentaose. Glucose equivalents is the sum of glucose units in the solublesugars, i.e., 1 cellobiose � 2 glucose, 1 cellotriose � 3 glucose, 1 cellotetraose � 4 glucose and 1 cellopentaose � 5 glucose.

FIGURE 2 A MALDI-TOF mass spectrum of the fractions at 32–36 mL SEC elution volumefrom the Hi Cel5A hydrolysate. The spectrum is averaged from spectra from eight different samplespots, acquired from four different hydrolysates of Hi Cel5A (two samples of each was analyzed),in order to minimize statistical (and other) fluctuations. Four hundred laser shots from up to eightpositions were recorded in each spectrum. Peaks with the same DP are indicated with symbols ofthe same kind that also are attached with lines and the DP is given by the large figures. The numberof substituents for the most intense peak of each DP are given by the superscripts, e.g., 64 denotescellohexaose with 4 carboxymethyl substituents and the adjacent peaks (�) are 63 and 65.

Enzymatic Degradation of Carboxymethyl Cellulose 37

sates with a higher measured DS than another also hasa higher true DS. The product from Tr Cel7B hadsignificantly higher DS compared to the other endo-glucanases, i.e., Tr Cel7B was able to hydrolyze oli-gosaccharides with higher DS than what the otherendoglucanases were able to. The products from HiCel5A had lower DS than Tr Cel7B but higher than HiCel7B, Hi Cel45A, and Tr Cel12A. The product for-mation by Hi Cel7B and Hi Cel45A had a similar DS.Tr Cel45Acore produced oligosaccharides with sig-nificantly lower DS than any of the other endoglu-canases. The average DS was 0.7 or higher for allenzymes except Tr Cel45Acore where it was between0.55 and 0.65, i.e., the DS for the products were lowerthan the DS for the substrate. The tendency for lowerDS at lower DP can be attributed to the nature of thehydrolysis, which greatly limits (depending on theenzyme) the number of substituents at positions closeto ends of the hydrolysate products, thus reducing theDS of the oligomers. The tendency for lower DS athigher DP, on the other hand, is most likely an effectof the fraction selection. Oligomers of the same DPand with few substituents will elute later than thosewith many substituents. Thus, two adjacent SEC frac-tions may both contain oligomers of the same DP buthaving different substituent distributions. Hence,there will be a drop in DS for the larger moleculessince a limited number of fractions were selected.

DISCUSSION

Our results clearly show a difference in the capabilityof the endoglucanases in hydrolyzing CMC. The en-doglucanases could roughly be separated in threegroups: First, the two most efficient enzymes HiCel5A and Tr Cel7B; secondly, the middle group HiCel7B, Hi Cel45A, and Tr Cel12A; and thirdly TrCel45Acore, which had the lowest activity on CMC.Furthermore, the different analysis methods werequite conclusive, e.g., Tr Cel7B was the most effec-tive enzyme according to the SEC analysis and thereducing end analysis and its products had highestDS.

Among the Humicola endoglucanases, Hi Cel5Awas more efficient than both Hi Cel7B and Hi Cel45Aas seen by the SEC and MALDI-TOF-MS analysis.Thus, Hi Cel5A is less sensitive to substituents on thesubstrate. Hi Cel45A was slightly more efficient thanHi Cel7B according to the SEC analysis (Figure 1A).Hi Cel7B produced more reducing ends than HiCel45A, but if one takes into account the part of thereducing ends that is soluble sugars, it is clear that HiCel45A was more efficient in hydrolyzing the sub-strate (Table I). The products had similar DS, slightlyhigher for Hi Cel45A (Figure 3). We cannot with theMALDI-TOF-MS analysis determine where the sub-stituents are located on the end products. Therefore,we can only draw limited conclusions about the activesite on the enzymes. Even though the DS for theproducts analyzed by MALDI-TOF-MS were similarfor Hi Cel7B and Hi Cel45A, the active sites for theenzymes might differ. However, the substituents seemto cause a similar obstacle for both Hi Cel7B and HiCel45A when hydrolyzing CMC.

The two Trichoderma endoglucanases that weremost different in their capability to hydrolyze CMCwere Tr Cel7B and Tr Cel45Acore. The obtained DSfor Tr Cel7B products ranged from 0.78 to 0.88 com-pared to Tr Cel45Acore were the DS ranged from0.56 to 0.66 (Figure 3). Evidently Tr Cel45Acore ismore sensitive to substituents on the substrate than TrCel7B. We have previously shown that Tr Cel45Adoes not hydrolyze cellopentaose even if it hydrolyzesdifferent cellulose materials.32 Apparently Tr Cel45Aneeds to bind a longer part of the cellulose chain,more than five glucose units, to be able to catalyze thehydrolysis. Furthermore, the hydrolytic efficiency forTr Cel12A was between Tr Cel7B and TrCel45Acore. This is also in agreement with our pre-vious results were Tr Cel7B, but not Tr Cel12A, wasable to hydrolyze cellotriose, which indicates differ-ences in the active site.32 Comparison of the different

FIGURE 3 The average DS of the oligomers from SECfractions eluted from 32 to 36 mL of the CMC hydrolysates.The horizontal axis shows the DP (i.e., the length of theoligomer backbone) and the vertical axis shows the DS foreach DP. Each curve is the average from three or fourseparate hydrolysates and for each hydrolysate twoMALDI-TOF-MS analyzes have been performed. Symbolsare as follows: � corresponds to Hi Cel5A, Œ to Hi Cel7B,F to Hi Cel45A, � to Tr Cel7B, ‚ to Tr Cel12A, and E toTr Cel45Acore.

38 Karlsson et al.

endoglucanases show that Tr Cel7B was the mosteffective endoglucanse in hydrolyzing CMC.

The structure of the catalytic core has been solvedfor Hi Cel7B, Hi Cel45A, and Tr Cel7B.11–14 The HiCel45A is the only structure that has been solved withsubstrate in the active site. There are no structurespublished with a substrate in the active site for HiCel7B and Tr Cel7B, thus the substrate–enzyme bind-ing is not known. However, it has been reported thatC-2 and C-6 at the �1 subsite of Hi Cel7B are notinvolved in the substrate–enzyme binding.35 Thus, itmight be possible with a substituent at either of thosetwo positions. Hi Cel45A is able to bind seven glu-cose units in the cellulose chain; it has seven substratebinding subsites, numbered from �4 to �3. Thecleavage of the substrate is between the �1 and �1subsite. However, it is not essential for the enzyme tobind at all subsites for catalysis; the enzyme showsactivity against cellotetraose, cellopentaose, and cel-lohexaose.31 The active site is in a groove with aflexible loop that is a part of the substrate binding.The substrate makes a tight binding to the enzyme,mostly via hydrogen bonds. No substrate has beenobserved in the �1 subsite; therefore no informationis available of the substrate–enzyme interaction at thisposition. All hydroxyls on the substrate are involvedin the substrate–enzyme binding except the C-2 andC-3 at the �4 subsite, C-2 at the �3 subsite, C-2 andC-3 at the �2 subsite, and C-6 at the �3 subsite. It isunlikely that the enzyme will accept a substituent onany of the hydroxyl groups that are involved in sub-strate–enzyme binding, at least not for the subsitescloser to the cleavage position. From the 3D structureit is plausible that a substituent will fit on the C-3 on�4 subsite, C-2 on �2 subsite and on C-6 on the �3subsite without causing any major hindrance for thesubstrate–enzyme binding. However, a substituent ona hydroxyl group not involved in binding might verywell cause a sterical hindrance for a correct substrate–enzyme binding.

The glucose units in CMC can be mono-, di-, andtrisubstituted and the substituents have three possiblepositions. Thus, parts of the CMC will lack substitu-ents and parts will be more substituted. HPLC anal-ysis of the amounts of produced unsubstituted oligo-saccharides was used to calculate the correspondingamount of glucose equivalents (Table I). The enzy-matic hydrolysis will be absent at the regions wherethe substituents are abundant, whereas the regionswithout substituents will be hydrolyzed. By analyzingthe amount of unsubstituted glucose units producedby the enzyme, it is possible to calculate the relativesize of blocks without substituents. The different en-zymes will not produce the same amount of unsubsti-

tuted sugars. The endoglucanases must have a certainsize of the substrate to be able to catalyze the hydro-lysis, e.g., Hi Cel7B is able to hydrolyze cellotriosewhereas Hi Cel45A is not.31 Furthermore, dependingon active site structure, the enzyme will be more orless hindered by substituents on neighboring glucoseunits. With the exception of the two Cel45A enzymes,the production of glucose equivalents were between3.5 and 4.4 mM, which corresponds to 7.0–8.8% ofthe CMC (Table I). Thus, we conclude that approxi-mately 8% of the CMC consists of blocks of unsub-stituted glucose units. Two different CMC samples,with DS 0.80 and 0.68, were reported to contain 37and 44.5% unsubstituted glucose respectively.36 If weassume that our CMC preparation contains about 40%unsubstituted glucose units, then this would mean thatabout one fifth of these exists in larger blocks. Thus,the majority of the unsubstituted glucose units areclose to a substituted glucose. The endoglucanase isable to hydrolyze at some of these latter positions.However, when cleavage occurs at a site with only afew unsubstituted glucose units, the enzyme will notproduce soluble unsubstituted sugars.

CONCLUSIONS

We have here shown that the endoglucanases of H.insolens and T. reesei hydrolyze CMC in a specificmanner. The enzymes clearly differ in their capacityin hydrolyzing CMC and in product formation ana-lyzed with MALDI-TOF-MS. Some endoglucanases(e.g., Hi Cel5A and Tr Cel7B) were able to hydrolyzethe substrate significantly whereas others (e.g., TrCel45Acore) were more affected by the substituents.It is thus possible to use pure endoglucanases forselective cleavage of a cellulose derivative. At themoment it is not known which substituents the differ-ent endoglucanases can accept close to the activesite—thus we cannot determine where the endoglu-canase is cutting in the cellulose derivative. In thefuture, with more knowledge of the active sites of theenzymes in combination with analytical techniques(e.g., MS, SEC, and NMR), this question will beanswered. With increasing knowledge about the struc-ture of the enzymes it will also be possible to producemutants with acceptance for a specific substituent thatthe wild type enzyme does not accept. The use of pureendoglucanase preparations will be an efficient tool inthe characterization of cellulose derivatives.

This work was supported by the Swedish Centre for Am-phiphilic Polymers from renewable resources (CAP). Anna-

Enzymatic Degradation of Carboxymethyl Cellulose 39

Karin Wihlborg is greatly acknowledged for excellent lab-oratory assistance.

REFERENCES

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