Pertanika 5(2), 255-262 (982)

Cellulase production by the Thermophilic Fungus,Thermoascus aurantiacus

CHOW CHIN TO G' and ANTHONY L.J. COLE 2

Department of Biochemistry and Microbiology, Universiti Pert{[Tlian Malaysia,Serdang, Se/angor, Malaysia.

Key words: Cellulase; Thermascu$ aurantiacus; Thermophilic fungus; Isolation; Properties.

RINGKASAN

Thermoascus aurantiacus merupakan pellgeluar sellilase yang paling oktif daripada beberapa kulattermofil yang lelah diuji. Suhu optima pertumbuhan unruk T. aurantiacus dt dolam medium cecair fa/ah450C. Suhu optima unruk aktiviti (3-g1ukosidase dan karbosil-merilselulase didapati pada 700 e semen tarasuhu optima untuk aktiviti enzim penghancuran kertas turasan berlaku pada 6S°C. Aktiviti maksimadidapati pada pH 5.0 untuk enzim penghancuran kertas turasan dan ~glukosidase dan pH 4.3 untuk aktivitikarbosilme tilselulase.

SUMMARY

Thermoascus aurantiacus was the most active cellulase producer of several thermphilic fungi tested.The optimum growth temperature for T. aurantiacus in liquid medium was 45° C and maximum cellulaseproduction from filter paper occurred at 40°C. The optimum temperatue for {3-glucosidase and carboxy­methylcellulase activity was 70° C; for filter paper degrading activity it was 65°C. Maximum activity wasfound at pH 5.0 for the filter paper degrading enzyme and ~glucosidase and pH 4.3 for carboxymethyl­cellulase activity.

INTRODUCTION

The crystalline cellulose that occurs in natureis highly resistant to both chemical and microbialdegradation. In view of the projected energyshortage there is considerable interest in organismscapable of degrading cellulose. Thermophilic(Ballamy, 1974; Hajny er al. 1951;Stutzenberger,1972' Su and Paulavicius, 1976) bacteria and(Wak;man er al. 1939) fungi (Barnes er al. 19'72;Coulls and Smith, 1976; Eriksen and Goksoyr,1976; Rosenberg, 1978) offer considerable advan­tages as potential cellulose degraders since theelevated temperature of growth (and lower pHwith thermophilic fungi) reduces contaminationproblems and pennits a higher rate of cellulosedegradation.

The role of TlJermoascus aurantiacus as acellulose degrader has been confused by the claimof Fergus (1969) and Rosenberg (1978) that there

was no cellulase produced by this organism. This isin disagreement with the work of Tansey (1971)who demonstrated that T. aurantiacus was anactive cellulase producer. There is, however, apaucity of information on the hydrolytic break­down of cellulose or its derivatives by thisorganism. The purification of the cellulases fromT. aurantiaclls was reported earlier (Tong et al.1980) followed by another paper on the su bstratespecificity and mode of action of the cellulases(Shepherd er al. 1980). This report describes thegrowth conditions for optimal cellulase productionand the general properties of the enzymes fromculture filtrates of T. aurantiacus.

MATERIALS AND METHODS

Chemical and substratesWhatman No. 1 fIlter paper and Whatman

cellulose powder (CF 11) were obtained fromWhatman Ltd., Maidstone, Kent, U.K. CM-

1 To whom aU correspondence should be addressed.2 Botany Department, University ofCanterbu.ry, Christchurch, New Zealand.

Key to authors' names: C.C. Tong and A.L.J. Cole.

255

C.c. TONG AND A.LJ. COLE

cellulose type 7HF was purchased from Hercules,Wilmington, Delaware, U.S.A. Difco powderedyeast ex tract and DiCco Bacto peptone wereproducts of Difco Laboratories. Detroit, Michigan,U.S.A. Davis Bacteriological agar was purchasedfrom Davis Gelatine, Ltd., Christchurch. NewZealand. All other chemicals were of analyticalreagent grade.

Humicola lanuginosa, Myceliophthora thermophila,Penicillium thermophile and Thermoascus auran­tiacus.

The cellulolytic ability of these fungi wasteste d as described below and culture filtratesobtained from T. aurantiacus were most active inbreaking down filter paper and carboxymethyl­cellulose (CM-cellulose) (Table 1).

TABLE IDegradation of filter paper by culture filtrates of

thermophilic fungi

Choice of Thermoascus aurantiacusThe isolated thermophilic fungi were identi­

fied as Chaetomium thermophile var. coprophile,

Isolation of thermophilic fungiThermophilic fungi were isolated from samples

of sand and decomposed woody materials collectedon coastal beaches and compost heaps in Christ­church, New Zealand.

Two isolation techniques were used: (a) directhyphal isolation after soil or composting materialswere incubated at elevated temperatures to pro­mote the growth of thermophiles; and (b) thedirect inoculation method (Waksman et aL 1931;Waksman et al. 1939) in which samples weresprinkled lightly upon the surface of yeast glucoseagar (YGA) media prior to incubation.

The hyphal isolates were sub-cultured ontoYGA and yeast slarch (YpSs) agar (Cooney er al.1964) until pure cultures were obtained. The addi­tion of 30 units of streptomycin per mUlitre ofmedium reduced bacterial contamination.

A thermophilic fungus, as defined by Cooneyand Emerson (1964), is one that has a maximumtemperature for growth at or above 50° C and a

inimum temperature for growth at or above

Organism

T. auranrjacus (strain I)

T. aura1ltiacus (strain 11)

C thermophile var. coprophile

!If. lilermophifa

P. thennophi/e

H. lanuginosa

reducing equivalents(mglh/ml enzyme)

0.009

0.004

0.002

0.003

oo

InoculumThe inoculum for each growth experiment

was taken from cultures grown on YGA at sao Cfor 48 h. An agar-mycelium disc (0.8 cm in diam.)was cut from the perimeter of the colony with asterile cork borer, inverted and placed at thecentre of a 9 em diam. petri dish containing agarmedium. In liquid culture experiments, one agar­mycelium disc (0.8 em in diam.) was transferred toa medical flat (Wheaton C-16.500 ml) containing50 ml of yeast-glucose medium. For seeding liquidmedia, three agar-mycelium discs (0.8 em in diarn.)were placed into each medical flat containing60 ml of Fergus medium (1969).

Main tenance of stock cultureT. aurantiacus was routinely cultured on YGA

slopes which were incubated at SO°C for48 handstored at 2T C for no more than four weeks priorto further culturing. For long-term storage, freeze­drying was found to be effective.

Determination of Cellulolytic Ability of FungiThe isolated thermophilic fungi were initially

grown in Reese-Mandels medium (1963) with CM­cellulose as carbon source. Each medical flat con­taining 60 ml of medium was inoculated withthree agar-mycelium discs. After six days incuba­tion at 50° C, the medium was suction filteredthrough glass fibre paper (Whatman GF/C) and thefiltrate centrifuged at 10,000 g for 30 min. Theclear supernatant was assayed for enzyme actiVityagainst CM-cellulose by the viscometric technique.

The fungi were also grown in Fergus' mediumwith chopped filter paper as carbon source. Afterincubation at 50°C for 21 days, the filtrate wascollected as described above and assayed foractivity against fJ.lter paper and CM-cellulose.Cellulolytic activity is expressed in terms of totalreducing sugar (as glucose) produced, and isexpressed as mg reducing sugar/h/ml reactionmixture.

The reaction mixture and the method for estimatingreducing sugars are as described in Materials and Methods.Piltrates are from 21 day old cultures grown on Fergus'medium with chopped fLlter paper as the carbon source.

Determination of Optimum Temperature forGrowth

The optimum growth temperature was ascer­tained by measuring the diameter of colonies on

256

CELLULASE PRODUCTION BY THERMOASUS AURANTIACUS

YGA medium in 9 cm diam. petri dishes contain­ing 30 ml of agar medium. Each agar plate wasinoculated with one agar-mycelium disc as des­cribed earlier. Quadruplicate colonies were mea­sured after incubation at the temperatures shownin Fig. 2, for 24 h and the average diam. of thelargest and smallest colony was recorded. A beakerof distilled water was placed in each incubator toreduce dessication of the agar medium. Culturesincubated at 55° C and 60° C were placed inpolystyrene bags to further prevent dessication.

Effect of Temperature on Cellulase ProductionMedical flats containing 60 ml of Fergus'

medium were inoculated and incubated at tempe­ratures shown in Fig. 3. Two flats were removedfrom each temperature after the first, third andfifth day of incubation and then at regular inter­vals of five days up to 30 days. The contents of

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a 10 20 30

incubation period (day)

Influence of Temperature on CellulaseProduction. The culture filtrates weretested for activity on (a) filter paper and(b) CM-cellulose as described in Materialsand Methods. (e), 30°C; (0), 35°C; (_),40°C; (0), 45° C; (A), 50°C; (6), 55°C; (y),60° C.

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Mycelial dry weight from liquid cultures wasalso determined. Four medical flats each containing60 ml of yeast-glucose medium were inoculated\Vith one agar-mycelium disc and incu bated at thetemperatures shown in Fig. 2 for 24 h. Mycelialmats were hazvested by filtration through WhatmanNo. I filter paper, washed three times with dis­tilled water and dried to a constant weight at60°C.

E 0.18.:!Qi

~

257

C.C. TONG AND A.L.l. COLE

the flats were filtered and cellulase activitiesassayed on filter paper and CM-cellulose byreducing sugar liberation. The pH of each of theculture filtrates was also measured.

Enzyme Activity MeasurementsCellulase activity was measured by the

appearance of reducing end groups liberated fromfilter paper or CM-cellulose. The num ber of reduc­ing sugar groups created by hydrolysis of the cellu­losic substrates was measured spectrophotometri­cally using the Nelson-Somogyi procedure (Nelson,1964, Somogyi, 1952).

An absolute definition of a unit of cellulaseactivity is difficult. This is because in substratessuch as CM-cellulose, the glucose molecules aresubstituted with carboxymethyl groups, and theproducts of the enzyme reaction on filter paperand CM-cellulose are heterogeneous polymers; theeffect of this on the absorption coefficient ofreducing end groups is not known and thereforeusing a glucose standard to determine the num berof pmol of reducing end groups may not representan accurate estimate.

An indication of total cellulolytic activity wasobtained by determining filter paper (FP) degradingactivity. Standard reaction mixtures containing20 mg of filter paper (Whatman No.1), 0.9 mlcitrate-phosphate buffer (0.1 M citric acid, 0.2 Mdi-basic sodium phosphate) pH 5.0, 0.1 ml enzymesolution and one drop (10 pi) of toluene wereincubated at 50° C for 24 h. Mixtures were thenanalysed for the production of reducing sugar. Thetoluene added to prevent bacterial growth wasfound to have no effect on enzyme activity.Reaction mixtures were checked for contaminationby withdrawing samples and streaking ontonutrient agar plates and incubated at 37° C and50° C. A unit of activity is expressed as mg reducingsugar/h/ml enzyme.

In preliminary experiments designed to studythe production of cellulolytic enzymes by theisolated thermophilic fungi, carboxymethyl­cellulase (CM-cellulase) activity was assayed by amodification of the viscometric technique ofHorton and Keen (1966). CM-cellulose (0.75%(w/v» was dissolved in citrate phosphate buffer(0. 1 M citric acid; 0.2 M di- basic sodium phos­phate), pH 5.0 in a Waring blendor for 5 min.and at 4° C for three days, the viscous but clearsupernatant phase was removed and the fibrillarmatter at the bottom discarded. Five millilitresof the CM-cellulose solution were pipetted into aCannon-Fenske viscometer (Type BS/IP/CF size300) and equilibrated in a water bath at 50° C for

258

5 min. One ml of enzyme preparation was thenadded to the viscometer and mixed immediately.The viscosity of the reaction mixture was thendetermined at regular intervals.

eM-cellulase activity was also measured byreducing group estimation in reaction mixturescontaining 0.9 ml of 0.75% (w/v) CM-cellulose incitrate-phosphate buffer, pH 4.5 and 0.1 ml ofenzyme solution. Reaction'mixtures were incubatedfor 30 min. at 70° C and the rate of production ofredUcing sugar determined. A unit of activity isexpressed as mg reducing sugar/h/ml enzyme.

{3-Glucosidase activity was assayed by a modi­fication of the method of Umezurike (1969).Enzyme solution (0.1 ml) and 0.4 ml 0.001 Mp-nitrophenyl-glucoside in citrate phosphatebuffer (0.1 M citric acid; 0.2 M di-basic sodiumphosphate), pH 5.0, was as incubated for 30 min.at 70°C. After incubation, 1.0 ml I M sodiumcarbonate solution was added to 0.5 ml of thereaction mixture, diluted with 10 ml of distilledwater and the nitrophenol released estimatedfrom the absorbance at 420 nm. One unit {3­glucosidase activity is defined as that amount ofenzyme needed to liberate I pmol of p-nitrophenol per min. under the conditions of the assay.

RESULTS

Comparison of Cellulolytic Ability of IsolatedThermophilic Fungi

Initial experiments were designed to discoverwhich of the culture filtrates obtained from theisolated thermophilic fungi, namely C. thermophilevar. coprophile, H. lanuginosa, P. thermophile,M. thermophile and T. aurantiacus (strain I and II)were most active in degrading soluble CM-cellulose,as well as insoluble forms of cellulose, such asfilter paper. When grown in Reese-Mandelsmedium containing CM-cellulose as the main Csource, all the isolates except H. lanuginosa andP. thermophile grew well and the culture filtratescaused a dramatic decrease in the viscosity of a CM­cellulose solution (Fig. 1). Thermoascus aurantiacus(strain I) was found to be the most active cellulaseproducer.

When these organisms were grown in Fergus'medium with filter paper as a carbon source, asimilar pattern was observed. H. lanuginosa andP. thermophile failed to degrade filter paper whileC. thermophile var. coprophile, M. thermophilaand T. aurantiacus all grew well. The culturefiltrates when tested on filter paper showed T.aurantiacus to be the most active. Strain I showedalmost twice the activity of strain II (Table I).

CELLULASE PRODUCTION BY THERMOASUS AURANTIACUS

100

Effect of temperature on cellulase synthesisThe effect of varying growth temperature

on cellulase production was measured by deter-

Culture fultrates of C. thermophile var. coprophileand M. thermophile also degraded filter paper to alesser degree. No activity from culture filtrates ofH. lanuginosa andP. thermophile could be detected.

Effect of pH on (3-glucosidase and cellulaseactivities

Using a temperature of 70° C for (3-g1ucosidaseand CM-cellulase and 60° C for FP degradingenzyme, the pH optimum of the enzymes wasdetermined (Fig. 5). An optimum pH of 5.0 wasfound for {3-glucosidase and FP degrading enzymeand 4.3 for CM-cellulase. If the reaction time ofthe FP degrading activity was increased from 2 to24 h, there was a shift in the pH optimum from4.3 to 5.0.

The pH of the culture medium changed duringgrowth and these changes were similar for growthbetween 35 - 55° C. "Plere was a drop within thefirst day from the initial pH of 6.5 to pH 5.5,followed by a gradual rise to a final pH of between6.3 and 7.0. Main Cellulase production occurredwhen the pH was 6.3 - 6.8. The pH of the mediumwas 6.6 after growth at 40° C for 20 days.

Both {3-g1ucosidase and CM-cellulase showed atemperature optimum at 70° C. At higher tempera­tures, the activities decreased sharply with only30% of the maximum activity remaining at 80° C.The optimum temperature for FP degradingactivity was lower; after a 2 h reaction time, asopposed to the standard reaction time of 24 h,activity was greatest at 65° C compared with theoptimum temperature of 60° C for 24 h reactiontime. When the incubation temperature was loweredto 30° C, there was a 70 - 90% decrease of themaximum activity for all three enzymes.

Temperature optima for (3-glucosidase and cellulaseenzymes

A series of enzyme assays were carried out atpH 5.0 over the temperature range 30 - 80° C(Fig. 4).

The general pattern of enzymatic activityon both su bstrates was similar at the temperaturestested. Optimum temperature for the degradationof both CM-cellulose and filter paper was 40° C andmaximum enzyme activity was obtained after 20days growth. Increasing the incubation tempera­ture from 40 to 55° C resulted in a 90% decrease inthe filter paper degrading activity and 60% of theCM-cellulase activity. After 20 days, the integrityof the filter paper in the medium was lost com­pletely and a thin slurry was formed.

mrnlllg the ability of culture filtrates to degradeboth filter paper and CM-cellulose (Fig. 3).

b__1>__h.-----f,·__6£>-_-I::.6

10 20 30

Hydrolysis of CM-cellulose by CultureFiltrates of Thermophilic fungi. 5.0 ml of0.75% CM-cellulose (7.5 mg/ml, in O.lMcitric acid; 0.2M di-basic sodium phosphatebuffer, pH 5.0) and 1.0 ml of culturefiltrate were mixed and the efflux time ofthe mixture determined with a viscometerat 50° C at the times indicated. The per­centage reduction in the viscosity of theCM-cellu/C'Se solution after each incubationperiod was calculated as described byHorton and Keen (1966). The thermophilicfungi tested were: T. aurantiacus (strain I)(.); T. aurantiacus (strain II) (D); C.thermophile var. coprophile (-); M. ther­mophila (0); P. thermophile (A.); H. lanugi­nosa (6).

time (min)

Growing Thermoascus aurantianusOptimum temperature for growth on solid

medium was 50°C (Fig. 2a); in liquid mediumthe optimum was lower, at 45° C (Fig. 2b). Nogrowth occurred at 30°C on either media. At55°C, growth was retarded and at 60°C it wasceased. The fungus produced only 0.1 - 0.2 emof hyphal extension after one week at 30° C, butthere was no growth of the fungus at 60° C evenafter a prolonged incu bation period.

Fig. 1.

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259

C.C. TONG AND A.L.J. COLE

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Activities. Enzyme activities were measuredin the standard assay systems except thatthe buffer and the pH were varied. Buffersused were citrate-phosphate (0.1 M citricacid; 0.2M di-basic sodium phosphate)buffer (pH 3.0 - 7.0) and 0.2M Tris·HCl(pH 8.0, 9.0). The FP degrading enzymewas determined using two different reactiontime; (.), 24 hand (0), 2 h. Points showthe average of the determination.

2.0Q)

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over a pH range of 4.0 - 6.0. Cellulases exhibiteda wider pH range of stability. Thirty percent of thepH 5.0 activity of FP degrading activity and 37%

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Temperature Optimum for (3-glucosidaseand Cellulase Enzymes. Enzyme activitieswere assayed in the standard systems atpH 5.0 except that the temperatures werevaried as shown. The FP degrading enzymewas determined using two different reactiontimes; (.), 24 hand (0), 2h. Points shownare the average of duplicate determinations.

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Effect of pH changes on the stability of{3-glucosidase and cellulase activities

Figure 6 illustrates the effect of pH on thestability of {3-glucosidase and cellulases. The pH- ­stability curve for {3-glucosidase resembles itspH-activity curve (Fig. 5). The enzyme was stable

260

CELLULASE PRODUCTION BY THERMOASUS AURANTIACUS

of the maximum CM-cellulase activity still remainedat pH 9.0. Both CM-cellulase and (3-glucosidase arenot stable at pH 7.0 or higher.

phosphate (0.2M citric acid; OAM di-basicsodium phosphate) buffer, pH 5.0 and 4.3,respectively. The FP degrading activity wasassayed at 60° C in the same buffer at pH5. O. The pH of each assay mixture waschecked after mixing the componentsolutions. Points show the average of twodeterminations.

DISCUSSION

Reports on the ability of T. aurantiacus todegrade cellulose have been contradictory. Thisreport confirms the cellulolytic nature of T.aurantiacus and culture filtrates of this fungus arefound to be the most effective of the themophilicfungi isolated in degrading both filter paper(Table 1) and CM-cellulose (Fig. 1).

·f Q) 20 (a)

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The optimum growth temperature of T.aurantiacus on solid media occurred at 46 - 51°Cwhich is in agreement with that reported byRomanelli and co-workers (1975). However, theseresults differ somewhat from those given by Cooneyand Emerson (1964) where an optimum of 40 ­45° C is reported. The difference observed may beattributed to strain variation and the differentpH of the media used. The optimum temperaturefor cellulase production is lower than that forgrowth, a phenomenon that is commonly observedwith thermophilic microorganisms. This may bedue to thermal instability of the enzymes atelevated temperatures when long incubationperiods are used. This is supported by the observa­tion that the temperature optimum for the FPdegrading enzyme decreased from 65° C to 60° Cwhen the incubation period was increased from2 h to 24 h.

Although there are many reports describingfungal extracts capable of degrading solublecellulosic derivatives only a small num ber of theseappear capable of extensive degradation of highlyordered (crystalline, insoluble) celluloses tosoluble sugars (Rosenberg, 1978). Culture filtratesof T. auran tiacus exhibited (3-glucosidase activityas well as activity on both soluble and insolubleforms of cellulose.

The assay procedure using filter paper as acellulose source has proved to be the most satis­factory for routine use in the estimation of hydro­lysis of native cellulose (Folan and Coughlan,1978; Griffin, 1973; Mandels and Weber, 1969).Filter paper, though partly degraded and moresusceptible to hydrolysis by cellulase than cotton,is considered as highly crystalline and difficult tohydrolyse.

9

( b)

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0.5

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pHEffect of pH changes on the Stability of{3-g1ucosidase and cellulase activities. Cul­ture filtrates (0.2 ml) were mixed with theappropriate buffer (l.8 ml) and the resul­tant pH measured before incubation at30° C for 3 h. The buffers used were asdescribed for Fig. 4. After 3 h incubation0.5 ml was withdrawn and assayed forenzyme activity. (3-glucosidase and CM­cellulase was assayed at 70° C in citrate-

600 (e)

300

450

Fig. 6.

261

C.C. TONG AND A.L.J. COLE

The optimum pH for activity was pH 5.0 for(3-glucosidase and FP degrading enzyme and pH4.3 for eM-cellulase activity. This compares withthe high pH optimum of 5.5 for cellulase fromPyricuiaria oryzae (Hirayama et ai. 1978) and alow pH optimum at 2.5 for Aspergillus nigercellulase (Ikeda et ai. 1973). At neutral pHvirtually no activity was detected. The optimumpH for the activity of the FP degrading enzymecould be lower than pH 5.0. When the incubationtime of the assay was decreased from 24 h to2 h (Fig. 5) the pH activity optimum moved topH 4.3. This shift is probably related to thestability of the enzyme.

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(Received 25 June 19821

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