Vol. 56, NO. 10

Subcellulosome Preparation with High Cellulase Activity fromClostridium thermocellum

T. KOBAYASHI,t M. P. M. ROMANIEC, U. FAUTH,4 AND A. L. DEMAIN*

Fermentation Microbiology Laboratory, Department of Biology, Massachusetts Institute of Technology,Cambridge, Massachusetts 02139

Received 23 March 1990/Accepted 3 July 1990

We have prepared a much simpler cellulase preparation than that of cellulosomes from the extracellularbroth of Clostridium thermocellum. This "subcellulosome" preparation from C. thermocellum was obtained bycolumn chromatography on CM-Bio-Gel A and then on a lectin-affinity material (Jacalin). The subcellulosomepreparation is a macromolecular complex, composed of six main protein subunits (molecular weight, 210,000to 58,000) revealed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The specific activities ofcarboxymethylcellulase (CMCase) and Avicelase are 15- and 8-fold-higher, respectively, than those of crudeextracellular cellulase. We could not further fractionate this preparation without denaturing it. The optimumpH and temperature of the subcellulosome preparation are 5.5 to 7.0 and 70°C for CMCase and 5.5 to 7.0 and65°C for Avicelase. The subcellulosome preparation acted on various types of carboxymethyl cellulose,cellulose, and p-nitrophenyl-jI-D-cellobioside but not on p-nitrophenyl-13-D-glucoside. Sulfiydryl reagents andN-bromosuccinimide inhibited both CMCase and Avicelase activities, whereas EDTA and o-phenanthrolineinhibited Avicelase activity only.

The anaerobic, thermophilic, cellulolytic, ethanol-produc-ing bacteria (5) have great potential for conversion of cellu-lose and hemicellulose into liquid fuel. The best-knownmember of this group is Clostridium thermocellum, whichdegrades both crystalline and amorphous cellulose (9).Present on the extracellular surface of C. thermocellumvegetative cells are protuberances which contain high-mo-lecular-weight, multienzyme, multifunctional complexescalled cellulosomes (13, 16); these are not only cell boundbut found free in the extracellular medium. Cellulosomeshave molecular weights of 2 x 106 to 6.5 x 106 (13, 15, 17,25). These aggregated macromolecular glycoprotein com-plexes are arranged into polycellulosomes with sizes of 5 x107 to 8 x 107 daltons (2, 17) which line the surfaceprotuberances (13). Several hundred cellulosomes appear tobe present in each protuberance (12).

Cellulosomes have been estimated to contain 14 to 50proteins, detectable by sodium dodecyl sulfate-polyacryla-mide gel electrophoresis (SDS-PAGE), ranging in molecularweight from 20,000 to 250,000 and, like the extracellularculture fluid, they exhibit true cellulase activity (completesolubilization of a microcrystalline cellulose such as Avicel),which is activated by Ca2+ and thiols and inhibited bycellobiose (7-9, 13, 17).

It has been extremely difficult to purify individual en-zymes from undenatured cellulosomes since these structureshave thus far been dissociable only by SDS (2, 16, 17, 25).The cloning of 18 or so individual genes (encoding cellulases,xylanases, and ,-glucosidases) from C. thermocellum intoEscherichia coli and Saccharomyces cerevisiae (6, 10, 19,22, 23) has been useful, but no such enzyme produced byrecombinant DNA technology degrades crystalline cellu-

* Corresponding author.t Permanent address: Kao Corp., Tochigi Research Laboratories,

Tochigi 321-34, Japan.t Permanent address: Universitat Tubingen, Institut fur Biologie

II, Lehrstuhl Mikrobiologie I, D7400 Tubingen, Federal Republic ofGermany.

lose. We (25, 26) showed that the combination of twocellulosome proteins, named SL and Ss, could completelysolubilize Avicel but only at very low rates, presumablysince the proteins were isolated under denaturing conditions.SL, the largest cellulosome component, is glycosylated andhighly antigenic and has a molecular weight of 210,000 to250,000 (13, 25); apparently, it is the same as S, in theterminology of Lamed and Bayer (13). It has been reportedto possess no enzymatic activity (13, 25) but allows theendo-p-glucanase S, to hydrolyze crystalline cellulose (25,26). It may be an anchor protein, holding the cellulosometogether, linking the cellulosome to the cell surface and/or tothe cellulose molecule (13, 25). SL contains 25 to 40%carbohydrate (13, 25) which is predominantly galactose (5)and binds strongly to Griffonia simplicifolia lectin GS-I andto its homotypic isolectin B4 (14).Due to the complexity of the cellulosome and the diffi-

culties involved in dissociating it without denaturing itscomponents, we have been interested in its simplification.With the aid of a lectin (Jacalin) preferentially bindinggalactosyl carbohydrates, we have isolated a simpler com-plex which we call the "subcellulosome" preparation. Thispaper describes its isolation from the extracellular broth ofC. thermocellum and some of its properties.

MATERIALS AND METHODSEnzyme source. A crude cellulase powder from C. thermo-

cellum was furnished by N. B. Afeyan. It was prepared fromthe extracellular fluid of C. thermocellum S7-19 grown in a200-liter fermentor on a Solka floc (SW 40) complex mediumat 60°C for 65 h (N. B. Afeyan, Ph.D. thesis, MassachusettsInstitute of Technology, Cambridge, 1987). The powder hadbeen prepared by sequential ultrafiltration and 67% acetoneprecipitation, followed by drying in a vacuum desiccator. C.thermocellum S7-19 is a mutant culture derived from ATCC27405 by a series of mutations and selections for increasedethanol tolerance (D. I. C. Wang, personal communication).Enzyme assays. Carboxymethyl cellulase (CMCase) activ-

ity was measured by the method of Wu et al. (26). One unit

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FIG. 1. CM-Bio-Gel A chromatography of crude cellulase. Bed volume was 2.6 by 38 cm. I, Elution with 640 ml of 10 mM succinate buffer(pH 4.5) containing 2.5 mM each CaCI2 and 2-ME; II, elution with 520 ml of same buffer supplemented with 0.5 M KCl; III, elution with 20mM Tris hydrochloride buffer (pH 7.0) containing 2.5 mM each CaCl2 and 2-ME. Flow rate was 26 ml/h per cm2. Fractions of 11 ml werecollected.

of CMCase activity is that amount of enzyme that liberatesreducing sugar equivalent to 1 ,umol of cellobiose per minunder standard assay conditions. Avicelase activity wasmeasured by the turbidimetric method of Johnson and De-main (7). One unit of Avicelase activity is that amount ofenzyme that reduces turbidity equivalent to liberation of 1nmol of cellobiose per min under standard assay conditions.

Protein determinations. Protein was determined by themethod of Bradford (1), with bovine serum albumin as thestandard. A280 was used for monitoring proteins in columneffluents.

Gel electrophoresis. Polyacrylamide slab gel electrophore-sis was done by the method of Davis (3), using a 3%polyacrylamide gel (Mini gel system; Pharmacia-LKB Bio-technology, Inc., Piscataway, N.J.). SDS-PAGE was doneby the method of Laemmli (11), using a 7.5% polyacrylamidegel. Both techniques were performed at 40 mA for about 1 hat room temperature. Gels were stained for protein withCoomassie brilliant blue R-250 and destained with 7% aceticacid.

Lectin blotting. Protein samples were resolved by SDS-PAGE and transferred onto nitrocellulose membranes (24).The protocol for the localization of glycoproteins was thatsupplied by Vector Laboratories, Inc., Burlingame, Calif.The membranes were blocked with TBST (10 mM Trishydrochloride [pH 8.0], 0.15 M NaCl, 0.05% Tween 20)supplemented with 1% (wt/vol) bovine serum albumin for 30min at room temperature. The glycoprotein band was de-tected with biotinylated Jacalin (Vector Laboratories) at 10,ug/ml in TBST and the Vectastain ABC reagent (VectorLaboratories).

Chemicals. Carboxymethyl cellulose (CMC) with a degreeof substitution range of 0.8 to 0.95 and a polymerization levelof 1,100 (type 9M31) was from Aqualon Co., Wilmington,Del. Avicel (FMC PH105; particle size, 20 ,um) was from

FMC Corp., Philadelphia, Pa. CM-Bio-Gel A was fromBio-Rad Laboratories, Richmond, Calif. Jacalin (agarose-bound) gel was from Vector Laboratories. According to themanufacturer, Jacalin is a lectin composed of four identicalsubunits of approximately 10 kilodaltons each, isolated fromArtocarpus integrifolia (Jackfruit) seeds. It binds only 0-gly-cosidically linked oligosaccharides, preferring the structuregalactosyl (0-1,3) N-acetylgalactosamine. The lectin columncould be regenerated and reused. Sulfhydryl reagents, metalchelators, and other enzyme inhibitors were from SigmaChemical Co., St. Louis, Mo. All other chemicals were ofthe highest grade commercially available.

RESULTS

Isolation of subcellulosome preparation. Preparation wascarried out at 0 to 5°C. The crude cellulase powder (800 mg)was dissolved in 35 ml of double-distilled water and appliedto a CM-Bio-Gel A column which had been equilibrated with10 mM succinate buffer (pH 4.5) containing 2.5 mM eachCaCI2 and 2-mercaptoethanol (2-ME). The column waseluted first with equilibration buffer, then with equilibrationbuffer supplemented with 0.5 M KCI, and finally with 20 mMTris hydrochloride buffer (pH 7.0) containing 2.5 mM eachCaCl2 and 2-ME at a flow rate of 26 ml/h per cm2 (Fig. 1).

Protein (A280) corresponded closely with CMCase activityin all three peaks. On the other hand, Avicelase activity wasdetected only in peaks I and III. Fractions were analyzed bySDS-PAGE, and SL was detected in peak III only. Fractions136 to 165 of peak III were pooled (285 ml), and 160 g ofammonium sulfate was added to the solution (80% satura-tion). The precipitate was collected by centrifugation (14,000x g, 20 min) and dissolved in a small amount of 10 mM Trishydrochloride buffer (pH 7.0) containing 2.5 mM each CaCl2and 2-ME. The concentrate was dialyzed overnight against

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Fraction numberFIG. 2. Jacalin gel chromatography of concentrated peak III.

Concentrated protein (2.6 mg) of peak III (Fig. 1) was applied to aJacalin (agarose-based) gel column (1.5 by 5 cm) equilibrated with150 mM Tris hydrochloride buffer (pH 7.0) containing 2.5 mM eachCaCl2 and 2-ME. The column was washed with 60 ml of equilibrat-ing buffer. Elution was with the same buffer supplemented with 0.8M galactose at a flow rate of 16 ml/h per CM2 . Fractions of 3 ml werecollected. For measurement of CMCase, the fractions were dialyzedagainst 20 mM Tris hydrochloride buffer (pH 7.0) containing 2.5 mMeach CaCl2 and 2-ME. AU, Avicelase activity units; CU, CMCaseactivity units.

two changes of buffer (1 liter). The retentate was centrifuged(14,000 x g, 20 min) to remove precipitates. An aliquot (2.6to 5.0 mg of protein) was applied to a Jacalin (agarose-bound) gel column which had been equilibrated with 150 mMTris hydrochloride buffer (pH 7.0) containing 2.5 mM eachCaCl2 and 2-ME. The column was washed with 60 ml ofequilibration buffer. Adsorbed protein was eluted with equil-ibration buffer supplemented with galactose. Figure 2 showsa representative elution profile. Washing with buffer re-moved a peak of protein containing a small amount ofCMCase activity. Both CMCase and Avicelase activitieswere detected in the peak eluted with 0.8 M galactose. Uponexamination of each fraction by SDS-PAGE, SL and fiveother protein bands were detected in the fractions elutedwith galactose.The galactose-eluted fractions were pooled, concentrated

by ultrafiltration (Nova cell 50 K; Pharmacia LKB Biotech-

-67

FIG. 3. Lectin blot of subcellulosome preparation (5.5 jig). LaneA, Lectin blot of SDS-PAGE gel; lane B, Coomassie brilliant blue

stain of SDS-PAGE gel. The indicated molecular weights were

determined with high-molecular-weight markers (Pharmacia LKB

Biotech).

nology) and sequentially diluted and ultrafiltered several

times with 10 mM Tris hydrochloride buffer (pH 7.0) con-

taining 2.5 mM each CaCl2 and 2-ME to remove galactose.The concentrate (Jacalin eluate) was subjected to further

purification methods, e.g., column chromatography on

DEAE-Bio-Gel A, hydroxyapatite, hydrophobic interaction

alkyl Superose HR 5/5, activated charcoal, and gel filtration

on Sepharose 6B in the presence of EDTA (10 mM), urea (6

M), or Triton X-100 (0.01%) after heat treatment (60'C, 20

min) of the concentrate with these reagents. However, we

were not able to separate the six main subunits (detected by

SDS-PAGE) by any of these treatments. Thus, the Jacalin

eluate was named the subcellulosome preparation.A summary describing the isolation of the subcellulosome

preparation is shown in Table 1. Avicelase specific activitywas increased by 8-fold, and CMCase activity was increased

by 15-fold. Recoveries were 2.1 and 4.0%, respectively. To

the final preparation was added glycerol to 25% (vol/vol) to

avoid inactivation during freezing. The preparation was

stored at -20'C.Figure 3 shows SDS-PAGE of the subcellulosome prepa-

ration, which contains six main components ranging in

molecular weight from 58,000 to 210,000. The largest com-

ponent (210,000 daltons) is subunit SL and the 80,000-dalton

TABLE 1. Summary of isolation of subcellulosome preparation

CMCase activity Avicelase activityStep ~~~~~ProteinStep (mg) Total Specific Yield Total Specific Yield

(CU)- (CU/mg) (%) (AU)b (AU/mg) (%)

Crude enzyme 409 856 2.1 100 5,327 13 100CM-Bio-Gel A chromatography 110 331 3.0 38.6 2,195 20 41.2Jacalin gel chromatography 3.4 NDc ND ND 345 100 6.5Ultrafiltration 1.1 34.6 31.5 4.0 110 100 2.1

a CU, Units of CMCase activity; see text.b AU, Units of Avicelase activity; see text.cND, Not determined.

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pHFIG. 4. Effect of pH on CMCase (A) and Avicelase (B) activities of the subcellulosome preparation. CMCase activity was measured in

a total volume of 1.8 ml containing 0.37 ,ug of the subcellulosome complex in the following 22 mM buffers: A, ,-alanine-hydrochloride; 0succinate; *, phosphate; 0, MOPS (morpholinepropanesulfonic acid); 0, Tris hydrochloride; A, glycine-NaOH; O, carbonate. Avicelaseactivity was measured in a total volume of 5 ml with 4.6 ,ug of the subcellulosome complex in the following 60 mM buffers: *, bis-Tris; othersymbols as above.

component is Ss (25, 26). When examined by PAGE on a 3%polyacrylamide gel, the subcellulosome fraction barely mi-grated into the gel. With Sepharose 6B gel filtration, theelution volume of the subcellulosome preparation was about40% of the column bed volume, the same elution volume asdetermined with the crude cellulosome preparation. Thesedata indicate that the subcellulosome preparation exists asan aggregated protein complex.A lectin blot (Fig. 3) indicated that the subcellulosome

preparation contains a single glycoprotein which corre-sponds to the highest-molecular-weight compound, namely,SL (210,000). According to the manufacturer of the lectinpreparation, Jacalin binding is thought to occur only to0-glycosidically linked oligosaccharides preferring the struc-ture galactosyl (13-1,3) N-acetylgalactosamine.

Effect of pH. The optimum pH values of the subcellulo-some preparation for degrading CMC and Avicel werebetween 5.5 and 7.0 in different buffers (Fig. 4). Bothactivities were very stable in the pH range of 6 to 9 whenincubated at 60°C for 1 h in various buffers. Above pH 9.0,Avicelase activity was more stable than CMCase activity(Fig. 5).

Effect of temperature. The optimum temperatures of CMCand Avicel degradation by the subcellulosome preparationwere 70 and 65°C, respectively (Fig. 6).

Activity on different cellulosic preparations. The subcellu-losome preparation displayed higher activity on CMC as thedegree of substitution decreased (Table 2). Avicel degrada-tion did not appear to depend on particle size. The influenceof the degree of CMC substitution was the same as seen by

Reese et al. (21) and Poulsen and Petersen (20). The subcel-lulosome preparation also displayed activity on p-nitrophe-nyl-13-D-cellobioside, but not on p-nitrophenyl-p-D-gluco-side.

Effect of various compounds. Both CMCase and Avicelaseactivities were inhibited by sulfhydryl reagents (Table 3),especially by p-chloromercuribenzoate. N-Bromosuccinim-ide (NBS), which can oxidize cysteine, tryptophan, tyrosine,histidine, and methionine residues of proteins and inhibittheir action (18), also inhibited both activities strongly.When dithiothreitol was included in the Avicelase assaymixture, the inhibition by these compounds was preventedor decreased. Phenylmethylsulfonyl fluoride at 1 mM, dieth-ylpyrocarbonate at 5 mM, trinitrobenzene sulfonic acid at 5mM, and phenylglyoxal at 5 mM inhibited neither enzumeactivity (data not shown). CMCase activity was not inhibitedby o-phenanthroline (OPA) at 5 mM, but Avicelase wasinhibited by OPA and EDTA. Inhibition of Avicelase byEDTA, but not that by OPA, was prevented by CaCl2.Table 4 shows the effect of pretreatment of the subcellu-

losome preparation with the above compounds. Sulfhydrylreagents and NBS dereased both activities. No effect afterpretreatment was observed with phenylmethylsulfonyl fluo-ride, diethylpyrocarbonate, trinitrobenzene sulfonic acid,phenylglyoxal, and 2-hydroxy-5-nitrobenzylbromide (5 mM)(data not shown). CMCase activity was not affected bypretreatment with metal chelators (EDTA and OPA). On theother hand, Avicelase activity was decreased by pretreat-ment with EDTA but not with OPA.

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pHFIG. 5. Effect of pH on CMCase and Avicelase stability of the

subcellulosome preparation. Subcellulosome preparation (6.0 p.g)was stored at 60°C for 1 h in the following 5 mM buffers: A,P-alanine-hydrochloride; 0, succinate; 0, Tris hydrochloride; A,glycine-NaOH. Residual activities were measured by standard assayconditions for CMCase (----; 0.4 ,ug of subcellulosome preparation)and Avicelase ( ; 5.0 jig).

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Temperature (Ic)

TABLE 2. Activity of subcellulosome preparation on differentcellulosic compoundsa

Degree of Relative activityCompound substitution or

particle size

CMC12M31 1.15-1.45 65CMC9M31 (control substrate) 0.8-0.95 100CMC7H 0.65-0.90 132CMC7H4 0.65-0.90 91CMC7L2C 0.65-0.90 135CMC4H1 0.38-0.55 227

Avicel PH 105 20 p.m 0.77Sigmacell 20 20 p.m 0.74Sigmacell 50 50 p.m 0.34Solka floc 40 100-140 p.m 0.22Solka floc 200 30-35 p.m 0.37Solka floc 300 NF 20-25 p.m 0.22Cellulose powder (Whatman 0.26CF11)

DEAE-cellulose (Sigma) 0.15CMC (Whatman CM23) 0.11PNP-cellobioside 3.1 (0.46 ,umol/

min per mg)PNP-glucoside NDb

a Assay mixtures contained 0.37 ,ug of the subcellulosome preparation for2 CMC or 4.6 pug for the other celluloses under standard assay conditions,

except that dithiothreitol was not present in the Avicelase assay mixture dueto interference with the reducing sugar determination. The activities weredetermined as micromoles of cellobiose/min per mg by assaying reducingsugars released and setting the hydrolysis rate for CMC (9M31) at 100%. Forp-nitrophenyl-,3-D (PNP)-cellobioside or PNP-glucoside hydrolysis, 1 U of theenzyme activity is that amount of enzyme that liberates 1 ,umol ofp-nitrophe-nol per min. The reaction mixture contained 100 mM succinate buffer (pH5.8), 4 mM substrate, and enzyme (0.12 to 0.82 pLg) in a final volume of 1.0 ml.The reaction was started by adding enzyme followed by incubation at 60°C for30 min. The reaction was stopped by adding 2 ml of 1.0 M Na2CO3. Thep-nitrophenol liberated was measured at 400 nm.

b ND, None detected.

DISCUSSIONC. thermocellum produces an extracellular macromolecu-

lar cellulase complex, the cellulosome, which is composed of14 to 50 subunits. A simpler preparation, the subcellulo-some, was prepared in the present study by CM-Bio-Gel Aand Jacalin gel column chromatography. It is composed ofsix main subunits and is a more potent cellulase complexthan the crude extracellular complex.CM-Bio-Gel A column chromatography gave three large

protein peaks from crude extracellular cellulase. Peak I waseluted with equilibration buffer and contained both CMCaseand Avicelase activities. SL was not detected in peak I bySDS-PAGE. This suggests that peak I contains Avicel-degrading enzymes not requiring SL and possibly includesthe enzyme recently described (Afeyan, Ph.D. thesis). Un-less CaCl2 and 2-ME (2.5 to 10 mM each) were included inthe equilibration buffer, both enzyme activities of peak Iwere very weak.The SL-containing fraction, presumably the cellulosome-

containing fraction, was eluted with 20 mM Tris hydrochlo-ride buffer (pH 7.0) as peak III. Even though CM-Bio-Gel A

FIG. 6. Effect of temperature on CMCase (0) and Avicelase (0)I activities of the subcellulosome preparation. The CMCase assay

80 90 was carried out at the indicated temperature for 15 min with 0.37 p.gof the subcellulosome preparation. The Avicelase assay was for 24to 48 h with 4.6 ,ug of the subcellulosome preparation.

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TABLE 3. Effect of potential inhibitors on cellulase activitiesof subcellulosome preparationa

Inhibition ofInhibi- Avicelase (%)

Compound ~Concn tion ofCompound (mM) CMCase Normal Less(%) rma dithio- Casasythreitol

Monoiodoacetate 5 12 0 39 NTN-Ethylmaleimide 5 19 0 83 NTp-Chloromercuribenzoate 0.01 10 0 67 NT

0.1 77 0 100 NTNBS 0.01 0 0 42 NT

0.1 83 23 100 NTOPA 5 0 43 37 34EDTA 0.1 0 0 0 13

S NTb 0 0 75

a Assay mixture contained 0.8 and 10 p.g of the subcellulosome preparationfor the CMCase and Avicelase assays, respectively.

b NT, Not tested.

is an ion-exchange medium, peak III could not be eluted bya high-salt concentration (up to 1.0 M KCl) in succinatebuffer at low pH (pH 4.5). However, the SL-containingfraction was eluted by a buffer with no KCl but with a pHabove 5.0.SL is thought to be the only glycoprotein of the cellulo-

some. Recently, the carbohydrate structure has been deter-mined by Gerwig et al. (5). Because of the galactose moietyin SL and binding of SL to GS-1 lectin (14), Jacalin gelcolumn chromatography was attempted; a preparation con-taining six main subunits, including SL and Ss, was obtained.No further separation was achieved without denaturation,suggesting that SL and the five other subunits constitute theprotein core of the cellulosome. Therefore, the Jacalin eluatewas named the subcellulosome preparation.The optimum pH values and temperatures for CMCase

and Avicelase activities of the subcellulosome preparationare similar to those previously reported for the crude cellu-lase (9). Both CMCase and Avicelase activities in the sub-cellulosome preparation were inhibited by sulfhydryl re-agents and NBS. It should be noted that Johnson andDemain (7) found no inhibition of CMCase (measured byviscosity) of the culture filtrate by 20 ,uM pchloromer-curibenzoate, whereas we observed 10% inhibition at 10 ,uMand 77% inhibition at 100 ,uM (by reducing sugar assay).Unfortunately, higher pchloromercuribenzoate concentra-tions were not tested in the earlier study. The NBS action is

probably due to its oxidation of cysteine residues in theenzyme(s), as suggested by the following observations: (i)NBS inhibition was reversed by dithiothreitol; (ii) diethyl-pyrocarbonate, a histidine-modifying agent, did not inhibitactivity; (iii) 2-hydroxy-5-nitrobenzylbromide, a specific in-hibitor of enzymes containing tryptophan in their activesites, failed to inhibit activity after preincubation of theinhibitor with the enzyme. This indicates that cysteine existsin the active sites of the subcellulosome preparation. Avice-lase, but not CMCase, activity was inhibited by the metalchelators EDTA and OPA when they were present in theassay mixtures; the EDTA inhibition was prevented byaddition of Ca2 . Pretreatment of the subcellulosome prep-

aration with EDTA also led to reduced activity but not withOPA.

Since OPA can chelate both Fe2+ and Zn2+, we wonderedwhich ion was important to the Avicelase activity. We notedthat the color of the subcellulosome preparation turnedbrown after OPA addition. Since OPA forms a coloredcomplex with Fe2+ but an uncolored complex with Zn2+, itappears that Fe2+ is the important ion. This is in agreementwith the work of Johnson and Demain (7), who found thatOPA inactivation of Avicelase activity of extracellular brothwas reversed by a mixture of Fe2+ and Fe3+. Since OPAchelates Fe3` only weakly but chelates Fe2+ strongly, we

favor the presence of Fe2+ in the subcellulosome prepara-tion.The inability of OPA pretreatment to inhibit Avicelase and

its ability to inhibit the activity when added directly to thereaction mixture lead us to speculate that the shape of thesubcellulosome complex in the absence of substrate pre-vents access of the chelating agent to the Fe2+; i.e., themetal may be on the inside of the enzyme complex. How-ever, when the subcellulosome contacts Avicel, the shapemay change, thus exposing the Fe2+ to chelation with OPA.If this is correct, then Ca2+ would be on the surface of thesubcellulosome structure since EDTA was inhibitory both as

a pretreatment and when added to the reaction mixture, botheffects being reversed by Ca2+ addition. Another reason forconsidering Ca2+ to be external is the finding by Lamed et al.(14) that, of more than 20 lectins tested, only G. simplicifolialectin GSL-1 interacted with cells of C. thermocellum.According to the manufacturer (Vector Laboratories), bind-ing of glycoproteins by this lectin requries a divalent cationsuch as Ca2+.We are interested in the synergistic Avicelase activity of

SL and Ss and hope to determine the contribution, if any, of

TABLE 4. Effect of pretreatmenta of subcellulosome preparation with potential inhibitors on cellulase activities

CMCase activity Avicelase activityConcn during Ihbto

Compound pretreatment Concn of Inhibition Concn of Inhibition (%)(mM) compound (%) compound Normal Less

(mM) (mM) assay dithiothreitol ess a

Monoiodoacetate 5 0.05 16 0.2 25 48 NTbN-Ethylmaleimide 5 0.05 32 0.2 67 78 NTp-Chloromercuribenzoate 0.5 0.005 72 0.02 50 100 NTNBS 0.1 0.001 18 0.004 0 0 NT

0.5 0.005 100 0.02 100 100 NTOPA 5 0.05 0 0.2 0 0 0EDTA 5 0.05 0 0.2 0 0 88

a The subcellulosome preparation (27 JLg of protein) was incubated with the compounds in 100 mM succinate buffer (pH 5.8) at 60°C for 15 min. The treatedpreparation was diluted with double-distilled water and added to the assay mixtures. The final subcellulosome concentrations were the same as in Table 3.

b NT, Not tested.

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the other four proteins of the subcellulosome preparation tosuch true cellulase activity.

ACKNOWLEDGMENTS

This work was funded by National Science Foundation grantEET-8711725 and U.S. Army Research Office contract DAAL03-88-K-0064. Support for T.K. was supplied by the Kao Corp.M.P.M.R. was a Merck Posdoctoral Fellow and U.F. was a scholarof the Deutsche Forschungsgemeinschaft.We sincerely appreciate the gift of crude cellulase powder and

encouragement from Noubar Afeyan. We thank Claus von derOsten and Anthony Sinksey as well as members of the BiotechProcess Engineering Center for use of their facilities.

LITERATURE CITED

1. Bradford, M. M. 1976. A rapid and sensitive method for thequantitation of microgram quantities of protein utilizing theprinciple of protein-dye binding. Anal. Biochem. 72:248-254.

2. Couglan, M. P., K. Hon-Nami, H. Hon-Nami, L. G. Ljungdahl,J. J. Paulin, and W. E. Rigsby. 1985. The cellulolytic enzymecomplex of Clostridium thermocellum is very large. Biochem.Biophys. Res. Commun. 130:904-909.

3. Davis, B. J. 1964. Disc electrophoresis. II. Method and applica-tion of human serum proteins. Ann. N.Y. Acad. Sci. 121:404-427.

4. Duong, T.-V. C., E. A. Johnson, and A. L. Demain. 1983.Thermophilic, anaerobic and cellulolytic bacteria. Topics En-zyme Ferment. Biotechnol. 7:156-195.

5. Gerwig, G. J., P. de Waard, J. P. Kamerling, J. F. G. Vliegent-hart, E. Morgenstern, R. Lamed, and E. A. Bayer. 1989. Novel0-linked carbohydrate chains in the cellulase complex (cellulo-some) of Clostridium thermocellum. J. Biol. Chem. 264:1027-1035.

6. Hazelwood, G. P., M. P. M. Romaniec, K. Davidson, 0. Grap-inet, P. Beguin, J. Millet, 0. Raynaud, and J.-P. Aubert. 1988. Acatalogue of Clostridium thermocellum endoglucanase, P-glu-cosidase and xylanase genes cloned in Escherichia coli. FEMSMicrobiol. Lett. 51:231-236.

7. Johnson, E. A., and A. L. Demain. 1984. Probable involvementof sulfhydryl groups and a metal as essential components of thecellulase of Clostridium thermocellum. Arch. Microbiol. 137:135-138.

8. Johnson, E. A., E. T. Reese, and A. L. Demain. 1982. Inhibitionof Clostridium thermocellum cellulase by end products of cel-lulolysis. J. Appl. Biochem. 4:64-71.

9. Johnson, E. A., M. Sakajoh, G. Halliwell, A. Madia, and A. L.Demain. 1982. Saccharification of complex cellulosic substratesby the cellulase system from Clostridium thermocellum. Appl.Environ. Microbiol. 43:1125-1132.

10. Kadam, S., and A. L. Demain. 1989. Addition of cloned P-glu-cosidase enhances the degradation of crystalline cellulose byClostridium thermocellum cellulase complex. Biochem.Biophys. Res. Commun. 161:706-711.

11. Laemmli, U. K. 1970. Cleavage of structural proteins during theassembly of the head of bacteriophage T4. Nature (London)

227:680-685.12. Lamed, R., and E. A. Bayer. 1986. Contact and cellulolysis in

Clostridium thermocellum via extensile surface organellas. Ex-perientia 42:72-73.

13. Lamed, R., and E. A. Bayer. 1988. The cellulosome of Clostrid-ium thermocellum. Adv. Appl. Microbiol. 33:1-46.

14. Lamed, R., J. Naimark, E. Morgenstern, and E. A. Bayer. 1987.Specialized cell surface structures in cellulolytic bacteria. J.Bacteriol. 169:3792-3800.

15. Lamed, R., E. Setter, and E. A. Bayer. 1983. Characterization ofa cellulose-binding, cellulase-containing complex in Clostridiumthermocellum. J. Bacteriol. 156:828-836.

16. Lamed, R., E. Setter, R. Kenig and E. A. Bayer. 1983. Thecellulosome-a discrete cell surface organelle of Clostridiumthermocellum which exhibits separate antigenic, cellulose-bind-ing and various cellulolytic activities. Biotechnol. Bioeng.Symp. 13:163-181.

17. Mayer, F., M. P. Coughlan, Y. Mori, and L. G. Ljungdahl. 1987.Macromolecular organization of cellulolytic enzyme complex ofClostridium thermocellum as revealed by electron microscopy.AppI. Environ. Microbiol. 53:2785-2792.

18. Means, G. E., and R. E. Feeney. 1971. Reducing and oxidizingreagents, p. 149-174. In G. E. Means (ed.), Chemical modifica-tion of proteins. Holden-Day, San Francisco.

19. Millet, J., D. Petre, P. Beguin, 0. Raynaud, and J.-P. Aubert.1985. Cloning of ten distinct DNA fragments of Clostridiumthermocellum coding for cellulases. FEMS Microbiol. Lett.29:145-149.

20. Poulsen, 0. M., and L. W. Petersen. 1985. A standard formulafor the determination of the initial rate of hydrolysis of car-boxymethylcellulose. Biotechnol. Bioeng. 27:409-414.

21. Reese, E. T., R. G. H. Siu, and H. S. Levinson. 1950. Thebiological degradation of soluble cellulose derivatives and itsrelationship to the mechanism of cellulose hydrolysis. J. Bacte-riol. 59:485-497.

22. Sacco, M., J. Millet, and J.-P. Aubert. 1984. Cloning andexpression in Saccharomyces cerevisiae of a cellulase genefrom Clostridium thermocellum. Ann. Microbiol. (Paris) 135A:485-488.

23. Schwarz, W. H., S. Schimming, K. P. Rucknagel, S. Burg-schwaiger, G. Kreil, and W. L. Staudenbauer. 1988. Nucleotidesequence of the celC gene encoding endoglucanase C of Clos-tridium thermocellum. Gene 63:23-30.

24. Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretictransfer of proteins from polyacrylamide gels to nitrocellulosesheets: procedure and some applications. Proc. Natl. Acad. Sci.USA 76:4350-4354.

25. Wu, J. H. D., and A. L. Demain. 1988. Proteins of Clostridiumthermocellurn cellulase complex responsible for degradation ofcrystalline cellulose. p. 117-131. In J.-P. Aubert, P. Beguin, andJ. Millet (ed.), Biochemistry and genetics of cellulose degrada-tion. Academic Press, Inc., New York.

26. Wu, J. H. D., W. H. Orme-Johnson, and A. L. Demain. 1988.Two components of an extracellular protein aggregate of Clos-tridium thermocellum together degrade crystalline cellulose.Biochemistry 27:1703-1709.

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