APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1990, p. 43-480099-2240/90/010043-06$02.00/0Copyright C 1990, American Society for Microbiology

Supernatant Protein and Cellulase Activities of the AnaerobicRuminal Fungus Neocallimastix frontalis EB188

ELENA M. BARICHIEVICH AND ROGER E. CALZA*

Department ofAnimal Sciences, Washington State University, Pullman, Washington 99164-6332

Received 5 October 1989/Accepted 10 October 1989

Protein and cellulase activities were measured in culture supernatants of the anaerobic ruminal fungusNeocallimastix frontalis EB188 established in glucose medium and switched to either glucose, cellobiose, or

cellulose media. Polyacrylamide gel electrophoresis was used to show differences caused by changing mediumcarbon source. Culture supernatants contained proteins with molecular weights ranging from greater than116,000 to about 19,000. Low levels of cellulase activity were evident in glucose-grown cultures. Increasedamounts of slowly migrating cellulase activities appeared in the supernatants of glucose-grown culturesswitched to cellulose. Cellulase activities which reacted differentially during colorimetric and in situ assays were

produced. Isoelectric points of celiulase activities varied from 3.7 to 8.3, and activities possessed optimal pHsof between 5.9 and 6.5.

Ruminal fungi have been shown to solubilize plant cellwalls in vitro (1, 12, 25, 29, 32, 34) and in vivo (2, 7, 9). Thesefungi, including Neocallimastix frontalis, Neocallimastixpatriciarium, Piromonas communis, Sphaeromonas com-munis, and Caecomyces equi, may serve key functionsduring cellulose solubilization reactions within ruminantherbivores (2, 7, 9) and others (8). In vitro, ruminal fungiferment certain soluble sugars and complex carbohydrates(3, 18, 19, 27).A complete understanding of the cellulase enzyme system

from ruminal fungi must await a detailed study of purifiedenzyme components. Crude ceilulase activity from Neocal-limastix spp. possesses optimal pHs of between 5 and 7 (11,18, 23). The cellulases may function as a large enzymecomplex (35). Reducing sugars are rapidly released fromhighly ordered cellulose, and the crude enzymes likelyinclude exoglucanase activity (36). Cellulase induction oc-curs after N. frontalis cultures (and others) are transferred tomedia containing cellulose (25, 32). Soluble sugars were lesseffective than cellulose in the induction of cellulases.We report the first molecular details and the separation of

the supernatant proteins and cellulases produced by theruminal fungus Neocallimastix strain EB188. Electrophore-sis was used to compare the production (or induction)patterns of cultures grown in glucose medium and switchedto either glucose, cellobiose, or cellulose media. The appear-ance of distinct gel banding patterns for protein (or cellulaseactivities) suggested a specific cellular response.

MATERIALS AND METHODSChemicals. All reagents for polyacrylamide gel electropho-

resis were purchased from International Biotechnologies,Inc. (New Haven, Conn.). All other chemicals, unless oth-erwise stated, were purchased from Sigma Chemical Co. (St.Louis, Mo.) or Aldrich Chemical Co. (Milwaukee, Wis.) andwere reagent or analytical grade.

Culture source. The anaerobic ruminal fungus (strainEB188) was isolated from the rumen of a cow fed a diet highin fiber (Medicago sativa) by the methods of Joblin (14). Thisstrain possessed characteristics consistent with ruminalfungi of the order Spizellomycetales reported by Heath et al.

* Corresponding author.

(10). Strain EB188 produced spherical zoospores which were

polyflagellated. The sporangia possessed well-defined septaat the base. Mycelial elongation was filamentous and fre-quently branched.

Culture conditions. The Hungate technique for anaerobicgrowth and transfer (13) was used for tube or 200-ml-flask(round bottom) cultures in liquid broth media. Mediumcomposition was similar to that described by Lowe et al.(20), except that ruminal fluid was not used and the concen-tration of yeast extract and Bacto-Tryptone (Difco Labora-tories, Inc., Detroit, Mich.) was halved. Antibiotics were

added as described by Joblin (14). The final concentration ofcarbon sources in all media was 0.2% (wt/vol) unless other-wise stated. The growth temperature was maintained at 39 +

1°C. To start cultures (200 ml), media were inoculated with200 zoospores per ml and were established in glucose mediagenerally for 48 h. If the carbon source was to be switched or

cultures were to be repeatedly sampled, the culture mediumwas decanted (reserving the mycelia which had tightlyattached to the vessel walls) and fresh medium containingeither glucose, cellobiose, or cellulose was added. Freshmedium was preheated to 39°C before addition to establishedcultures. Samples (5 ml) were taken periodically (usually for108 h), and supernatants were separated from culture solidsby centrifugation. Cultures intended for long-term storagewere frozen by the method of Phillips and Gordon (27).Enzyme methods. Culture supernatants were clarified be-

fore assay by centrifugation at 10,000 x g for 10 min. Whennecessary, proteins in media supernatants were concen-trated to approximately 500 pug/ml by ultrafiltration with PM2or PM10 membranes in a tangential flow cell (Amicon Corp.,Danvers, Mass.). This apparatus was also used, when nec-

essary, to remove soluble carbon source from the mediasupernatants. Recoveries of protein and cellulase activitiesranged between 95 and 98%. Protein was determined by themethod of Bradford (5) with bovine serum albumin as

standard. Cellulase activity was quantified as the generationof reducing sugars from carboxymethyl cellulose (CMC).The CMC had medium viscosity (400 to 800 cP) with a 0.7degree of substitution (DS) and an approximate molecularweight of 250,000. Reducing sugars were measured by thetetrazolium blue chloride (TZ) method of Jue and Lipke (15).Glucose was used as a standard. One international unit of

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44 BARICHIEVICH AND CALZA

cellulase activity has been defined as 1 ,umol of reducingsugar produced per min. Enzyme (generally 10 to 100 ng ofprotein per assay) was added to 50 ,ul of either 0.5% (wt/vol)CMC or 0.5% (wt/vol) cellulose for the colorimetric assay ofreducing sugar. The buffer system used (PST) was 10 mMPIPES [piperazine-N,N'-bis-(2-ethanesulfonic acid)], pH6.5, containing 5 mM NaCl and 0.01% (vol/vol) Triton X-100.Buffers at 50 mM for determining cellulase pH optimaincluded phosphate citrate (pH range, 2.2 to 7.9), sodiumacetate (pH range, 4 to 5.5), N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (pH range, 7 to 8), 2-(N-morpholi-no)ethanesulfonic acid (pH range, 5 to 6), phosphate (pHrange, 5.5 to 8.5) and PIPES (pH range, 6 to 7). All enzymereactions were performed within their linear responseranges.Chromatography methods. Sephacryl 300 (Pharmacia,

Uppsala, Sweden) matrix used for determining native cellu-lase molecular weights was suspended in PST buffer con-taining either 0.1 or 1% (vol/vol) Triton X-100 and 1 M NaCl.Matrix was packed into a column (1 by 20 cm or 0.8 by 40cm). The flow rate was controlled by using a peristaltic pump(Bio-Rad Laboratories, Richmond, Calif.) and maintained atapproximately 0.1 ml/min.

Thin-layer chromatography of soluble sugars was per-formed on silica gel plates (20 by 20 cm; Eastman KodakCo., Rochester, N.Y.) in a solvent development system ofabsolute ethanol-n-butanol-pyridine-water-acetic acid (10:1:1:3:0.3). Reducing sugars on plates were detected by thesilver nitrate method (30).

Electrophoresis methods. Sodium dodecyl sulfate-poly-acrylamide gel electrophoresis (SDS-PAGE) was performedon slabs by using a 10% separation gel (30% acrylamide-1%bisacrylamide) with a 4.5% stacking gel (30% acrylamide-1%bisacrylamide). Running conditions were as described byLaemmli (16). Samples were concentrated for electrophore-sis by using trichloroacetic acid (final concentration, 10%[wt/vol]) precipitation. Samples were boiled for 5 min inloading buffer before being loaded. Electrophoresis wascarried out at 7.5 or 25 mA until the bromphenol blue dyereached the bottom of gel. Proteins were located by staining(after fixation with 50% [vol/vol] methanol-10% [vol/vol]acetic acid) with Coomassie brilliant blue R-250. The densi-tometer gel scanner used was a model R-115 from BeckmanInstruments, Inc. (Fullerton, Calif.). Molecular weight stan-dards were ,3-galactosidase (116,000), phosphorylase b(97,400), bovine serum albumin (66,000), chicken egg oval-bumin (45,000), and carbonic anhydrase (29,000).

Isoelectric focusing gel electrophoresis was performed intubes by the method of O'Farrell (24) except that urea wasomitted and ampholines (pH range, 3 to 10) were added to2% (wt/vol). The gels were prerun as described previously(24), except that after samples were loaded, gels were run at400 V for 5 h and then at 500 V for 18 h. After electropho-resis, gel slices (2.5 mm each) were soaked in 1 ml of PSTovernight and finally assayed for cellulase activity (TZmethod with CMC as substrate).Nondenaturing polyacrylamide gel electrophoresis was

performed on slabs with a 7.5% separation gel (30% acryl-amide-1% bisacrylamide) and a 2.5% stack gel (10% acryl-amide-2.5% bisacrylamide). Running conditions were asdescribed by Maurer (22) and Calza et al. (6). The anionicbuffer system was used. The upper buffer (pH 8.6) contained5.16 g of Tris and 3.48 g of glycine per liter, and the lowerbuffer (pH 7.9) contained 14.5 g of Tris per liter and 0.06 NHCl. Electrophoresis was carried out at 25 mA until the dyereached the bottom of the gel. Extraction of cellulase from

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Time (hr8)FIG. 1. Measurements of supernatant cellulase production in

glucose and cellulose medium-switched cultures. Cellulase activity(TZ assay with CMC) is plotted against time of sampling. Symbols:0, cellulose culture; 0, glucose culture.

native-gel slices were accomplished by smashing each sec-tion (containing about 75 RI of liquid) in 400 [lI of PST bufferand then shaking the mixture at 4°C for 8 to 10 h. More than90% of the cellulase activity (TZ method with CMC) appliedto the gel was recovered. Negative staining of CMC withCongo Red dye as an in situ gel cellulase assay was per-formed with overlays of native gels as described previously(4, 28), with several modifications. The overlay contained0.5% (wt/vol) CMC in 50 mM PIPES buffer (pH 6.5), 5 mMNaCl, 0.01% (vol/vol) Triton X-100, and 0.7% (wt/vol)agarose (FMC Bioproducts, Rockland, Maine). After moltenoverlay (at approximately 46°C) had solidified on the gel, itwas carefully covered with plastic wrap and incubated at39°C for various times.

RESULTSProduction of supernatant cellulases as a function of time

in cultures established in glucose and switched to eitherglucose or cellulose medium (both at 0.2% [wt/vol]) wasmonitored. Cultures were sampled before the medium re-placement and then twice a day. Supernatant cellulaseactivity in the cellulose culture produced time-dependentincreases up to 72 h (Fig. 1). The glucose culture possessedsignificant but low (about 0.04 IU/ml) levels of cellulaseactivity.The effects of cellulose concentrations in medium on the

production of supernatant cellulase were tested. Glucose-established cultures were switched to cellulose media andsampled periodically for 144 h. Maximum cellulase activitiesoccurred in all cultures by 94 h after the addition of freshcellulose media. Cellulase activities (expressed as percent-ages of cellulase activity in the control at 94 h, i.e., 100% wasused for the 0.2% [wt/vol] cellulose sample) were 28.7% for0.05% (wt/vol) cellulose, 49.5% for 0.1% (wt/vol) cellulose,and 127.9% for 0.3% (wt/vol) cellulose. Higher concentra-tions of cellulose were not tested. The concentration ofculture medium glucose (identity confirmed by thin-layerchromatography) was less than 20 jig/ml in all cultures at 94h.

Supernatant protein and cellulase activities in culturesestablished on glucose and switched to either glucose, cel-

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EFFECTS OF CARBON SOURCE CHANGES ON EB188 45

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FIG. 2. Densitometry gel tracing of SDS-PAGE of supematantprotein samples from glucose (G)-, cellobiose (C)-, and cellulose(SC)-switched cultures. Equal amounts of supernatant protein (13,ug) were loaded. Arrowheads indicate areas of particular interest.Protein migration plotted against molecular weight is also shown,and molecular weights of markers (M) are indicated.

lobiose, or cellulose medium were measured. At the time ofmedium change, culture protein (total) was determined to belogarithmically increasing. Samples at 96 hours after mediumswitch (at maximum production) contained 15.4, 13.8, and25.2 ,ug of supernatant protein (averages of triplicate cul-tures) per ml for glucose, cellobiose, and cellulose cultures,respectively. Cellulase activities were also found in allculture supernatants and determined to be 0.058, 0.082, and0.330 IU/ml for glucose, cellobiose, and cellulose cultures,respectively. Cellulase-specific activities were therefore3.77, 5.94, and 13.10 IU/mg for glucose, cellobiose, andcellulose cultures, respectively.

Supernatant proteins of cultures established on glucosemedium and finally switched to either glucose, cellobiose, or

cellulose medium were compared by SDS-PAGE. Equalamounts of proteins from cultures 96 h after the switch were

collected by precipitation (trichloroacetic acid) and sepa-

rated by electrophoresis. Distinct protein band stainingpatterns were evident, and they were recorded by usingdensitometry tracing of gels (Fig. 2). Proteins ranged inmolecular weight from greater than 116,000 to about 19,000(at dye front). Compared with other samples, the cellulosesamples contained fewer low-molecular-weight proteins andwere rich in high-molecular-weight proteins. Several minorand major bands present (Fig. 2, arrowheads) in the cellulosecultures (at molecular weights of 52,000, 76,000, 85,000,approximately 120,000, and approximately 135,000) were not

present, or were fainter, in the glucose and cellobiosecultures. The glucose and cellobiose samples gave similarresults, although some differences (including band intensi-ties) were found. Such clarified supernatant samples held at20°C for 24 h or at 50°C for 2 h (with or without 1% SDS)before electrophoresis possessed identical banding patterns.Similar banding patterns were also obtained from 16 to 88 hafter the cellulose medium switch, although protein concen-tration in the supernatant increased with time.To identify which protein bands from SDS-PAGE gels

possessed cellulase activity, the CMC-Congo Red overlayprocedure was attempted. This was unsuccessful, since SDSdramatically inhibited cellulase activity (by about 95%).Activity was not recovered when SDS was removed fromgels with Triton X-100 or isopropanol. We opted to usenative PAGE separation methods coupled with overlaymethods. Supernatant culture samples from glucose, cello-biose, and cellulose medium-switched cultures were concen-trated by ultrafiltration and electrophoresed under nativeconditions. Gels were overlayed with agarose containingCMC and stained. Several bands of activity with various Rfvalues were evident (Fig. 3). The cellulose samples showedevidence of activity after a short incubation (3 h) (Fig. 3B).Longer exposure (18 h) of the gel to the overlay revealed thatthe supernatant samples from glucose- and cellobiose-switched cultures also possessed distinct cellulase activitybands (Fig. 3A). Differential response to overlay exposuretime was predicted on the basis of differences in samplespecific activities. A close examination of the banding pat-terns revealed that most activity bands were represented inall cultures. Several bands were apparently unique, how-ever, to the cellulose cultures (Fig. 3A, arrowheads). Cellu-lase activity banding patterns for cellulose-switched cultureswere unaltered when samples were stored for several days at4°C or held at 39°C for 12 h before electrophoresis.We attempted densitometry tracing to quantify banding

patterns of cellulases from cellulose-switched cultures. Thefragility of the overlay material, however, made such anal-ysis impossible. Activity was finally quantified by colorimet-ric assay methods after native PAGE and sectioning of thegel. Several peaks were evident (Fig. 4). The differences incellulase enzymes and detection methods likely accountedfor the differences (i.e., at RAs 0.60 and 0.31) of in situ gelactivity bands (Fig. 3A) and colorimetric activity peaks (Fig.4). Overlapping cellulase enzymes and differences in specificactivities could further complicate such analysis.

Gel matrix exclusion chromatography was used to esti-mate the nondenatured molecular weights of the supernatantcellulases from cellulose medium-switched cultures. TheSephacryl-300 matrix adequately separated several markerproteins, including P-galactosidase, bovine serum albumin,carbonic anhydrase, and cytochrome c. Cellulase activityeluted from the void volume through the low-molecular-weight elution volumes. Buffer containing Triton X-100 at1% (wt/vol) significantly altered the profile and yielded lessactivity at the low-molecular-weight elution volume.

Isoelectric focusing was used to determine the range ofisofocusing values of supernatant cellulase activities fromcellulose medium-switched cultures. Samples were collectedat 68 h after medium change. Cellulase activity was evidentthroughout the 3.7 to 8.3 pH range of the gel, and severalpeaks (or shoulders of peaks) were evident (Fig. 5). Simul-taneously run marker proteins (i.e., hemoglobin and cata-lase) tightly focused at their expected isoelectric focusingvalues of 6.65 (6.80) and 5.35, respectively. Coomassiestaining of the gel confirmed that the isofocusing worked

LogMW, 103daltons, I,,,. ,stack

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46 BARICHIEVICH AND CALZA

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Equal amounts (4 ,ug) of supernatant protein from glucose (G), cellobiose (C), or cellulose (SC) cultures were loaded. Relative migration (Rf)values are indicated. Arrowheads mark bands of particular interest. Incubation was for 18 (A) or 3 (B) h.

well, since tightly banded proteins were located throughoutthe gel (data not shown). Broad cellulase peaks thereforeprobably represented a mixture of different enzymes.

Activity pH optima of supernatant cellulases from culturesswitched to either glucose, cellobiose, or cellulose mediumwere determined. All samples possessed activity over a

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broad range of pHs, with optima of about 5.9 to 6.5. In thephosphate-citrate buffer system, the glucose sample pos-sessed significant activity (about 12%) at pH 2.75, whereasthe cellobiose and the cellulose samples possessed none.Alternative buffer systems tested (see Materials and Meth-ods) with supernatant cellulase from cellulose cultures

10 20 30 40 50 60 70 80 90 100 110 120

Fraction NumberFIG. 4. Supernatant cellulase activity from cellulose-switched cultures as a function of native PAGE gel slice. Supernatant protein (2.5 ,ug)

from a cellulose culture was used. Cellulase activity (TZ assay with CMC) is plotted against migration (Rf value) and fraction number. Thestack of gel ended at fraction 3. The bromphenol blue dye was included in fractions 101 to 103.

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EFFECTS OF CARBON SOURCE CHANGES ON EB188 47

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FIG. 5. Isoelectric focusing pH values of supemratant cellulase

activity from cellulose-switched cultures. Approximately 1 jig of

superatant protein from a cellulose culture was loaded. Cellulase

activity (0) is plotted against gel pH (LI) and slice number. Marker

proteins hemoglobin (hemo) and catalase (cat) are indicated.

yielded activity values between 85 and 100% of the activityvalues obtained with the phosphate-citrate buffer.

DISCUSSION

We provide the first report of the electrophoretic separa-

tion of several cellulase enzymes from a ruminal fungusisolated from beef cattle. Such evidence made it possible to

demonstrate cellulase enzyme diversity. We also found that

cellulase specific activity and amounts of excreted proteinincreased in cellulose-switched or cellulose-grown cultures,

as others have found (32, 33). Cultures producing cellulase

were viable and logarithmically growing, and we thereforebelieve that supernatant protein excretion was not predom-inately due to cell lysis. Supernatant cellulase activity in-creased approximately sixfold in cultures established on

glucose media and switched to cellulose-containing media.Maximum induction of activity occurred at 94 h with cellu-lose at 0.3% (wt/vol). Cellulase activity of strain EB188 wasdetermined at 0.33 IU/ml (with 0.2% [wt/vol] cellulose). Thisvalue was similar to that found by Hebraud and Fevre (11)but 6.5 times higher than that found by Pearce and Bauchop(26). Strain differences likely accounted for the differences inactivities recorded.The appearance of distinct families of supernatant proteins

(demonstrated by SDS-PAGE) in various cultures suggestedthat protein production was at least partially specific. Sev-eral cellulase activities were also evident (demonstrated bynative PAGE) only in cellulose-switched cultures. Thissupports the idea of a specific cellular response to carbonsources in media. We were unable to assign molecularweights to cellulase activities, but the cellulases did possessa range of molecular weights (demonstrated by exclusionchromatography) and may be associated with a high-molec-ular-weight complex. Recently, workers have shown thatcrude cellulase from N. frontalis elutes from similar columnsas high-molecular-weight complexes (35). Individual molec-ular weights of cellulases may be determined by usingSDS-PAGE methods with extracted native PAGE activitybands.

Low-level cellulase production was evident in glucose-switched cultures of EB188. Glucose repression of cellulaseinduction in other ruminal fungi has been reported (23).Residue cellulase production may form "effector" cellulosedegradation products and sensitize the cell to the presence ofinsoluble carbon sources in media.

Protease has been detected in the supernatants of culturesof ruminal fungi (31). We have, however, no evidence forprotease modification of supernatant protein or cellulasebanding patterns. Similar supernatant cellulase activity orprotein banding patterns were found in early and late stagesof production. Proteolysis modification of cellulase en-zymes, however, could be vigorously tested by using mono-specific antisera to compare cellulase relatedness. Proteoly-sis may be a normal characteristic of ruminal fungi andeliminated only with protease-negative strains. It has beensuggested that proteolysis may increase supernatant enzymediversity (6).The pH activity profiles of cellulases from strain EB188

were similar to others reported for ruminal fungi (18, 23).Isoelectric focusing pH values ranged between 3.7 and 8.3and were similar to those reported for Trichoderma cellu-lases (17, 21). In a manner similar to that of Trichodermastrains, EB188 exhibited inducible cellulase activity and maywork as a complex. Molecular weights of Trichodermacellulases ranged between 25,000 and 94,000 and may besimilar to those of ruminal fungi strain EB188. Trichodermacellulase enzymes have been shown to be both endogluca-nases and exoglucanases. It will be necessary to performviscometric and substrate hydrolysis experiments to deter-mine whether electrophoretically separated enzymes fromstrain EB188 are either endoglucanases or exoglucanases.Both types of cellulases are thought to be produced by N.frontalis (36).

ACKNOWLEDGMENTS

We acknowledge operational financial support from the Agricul-tural Research Center at Washington State University and generoussupport from the Washington Dairy Commission. E.M.B. receivedstipend support from the Washington State University Grant-in-Aidprogram.We gratefully acknowledge Jill Sheer for manuscript preparation

and colleagues for critical review.

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