The Action Cellulolytic Enzymes from Myrothecium verrucaria · Biochem. J. (1961) 79, 185 TheActionofCellulolytic EnzymesfromMyrotheciumverrucaria BY G. HALLIWELL DepartmentofEnzymology,

Biochem. J. (1961) 79, 185

The Action of Cellulolytic Enzymes from Myrothecium verrucaria

BY G. HALLIWELLDepartment of Enzymology, Rowett Research Institute, Buck8burn, Aberdeen

(Received 27 July 1960)

Although an extensive literature is availabledescribing cell-free cellulolytic preparations whichattack soluble cellulose derivatives, there is adearth of information on the enzymic breakdownof insoluble cellulose, and virtually nothing isknown about the breakdown of undegradedcellulose of the type associated with native cottonfibres [For reviews see Siu (1951), Halliwell (1959)].Despite the failure of cell-free preparations madefrom them to attack undegraded cellulose, trulycellulolytic organisms can quantitatively transformall types of cellulose, undegraded or otherwise, tosoluble products.The present report concerns an examination of

the breakdown of an acid-swollen cellulose byculture filtrates from Myrothecium verrucaria. Thissubstrate is an insoluble although degradedcellulose and was chosen firstly because the rela-tively mild method of preparation involves onlysmall changes in the degree of polymerization(Walseth, 1952), and secondly only after havingshown that our enzyme preparations produoedsignificant solubilization of this substrate. Condi-tions governing the enzymic breakdown of in-soluble cellulose are described below and comparedwith the enzymic hydrolysis of the soluble cellulosederivative, carboxymethylcellulose.

MATERIATLS AND METHODS

Preparation of the culture filtrateCultures of M. vemrcaria (I.M.I. 45541, Commonwealth

Mycological Institute, Kew, Surrey), grown on the saltmedium of Saunders, Siu & Genest (1948) with cellulosepowder (Whatman) or de-waxed cotton fibres as substrate,were aerated at 280 by incubation in a reciprocal shakingmachine operating at 60-90 strokes of 38 mm. horizontalmovement per minute. After fermentation for 1-3 weeksmost of the organisms and all of the residual cellulose wereseparated off by filtration through a sintered-glass filter(porosity 3). The filtrate was centrifuged at 54 000g for30 min. at 10 and subsequently passed through a sintered-glass bacteriological filter (porosity 5/3). In the later workthe final filtration was found to be unnecessary by inocula-tion on cellulose-agar slopes. The cell-free culture super-natant or filtrate fraction was used as the source of cellulo-lytic enzymes. For enzyme assays the following averagevolumes were used: for method (a) 0-2 ml. of undilutedfiltrate; for method (b) 0.1 ml. of filtrate diluted 50 times.

The solubilization of cellulose by the organism wasdetermined by washing the mixture on the sinter withhydrochloric acid, aq. ammonia solution, Teepol andethanol, followed by drying and weighing as describedelsewhere (Halliwell, 1957a).

Sub8tratesNative cotton fibre8. Texas cotton (kindly supplied by the

British Cotton Industry Research Association) was de-waxed by treatment with ethanol, ether and finally withsodium hydroxide under nitrogen (Halliwell, 1957a).

Cellulose powder (Whatman). This was standard-gradeashless powder for chromatography.

Swollen cellulose. This insoluble preparation was madefrom Texas cotton fibres and also from cellulose powder(Whatman) by soaking in phosphoric acid for 2 hr. at 10(Halliwell, 1957a).Sodium carboxymethykellulo8e. A soluble cellulose

derivative, with a degree of substitution of about 0-5, wasgenerously supplied as Cellofas B by Imperial ChemicalIndustries Ltd.

Other materiale. These were: cellobiose and glucono-1 --4-lactone (L. Light and Co. Ltd.); glucose (A.R.; Hopkin andWilliams Ltd.).

Analytical procedureDetermination of the degree of hydrolysis of the soluble

cellulose derivative carboxymethylceUulose. Hydrolysis ofcarboxymethylcellulose to 'reducing' sugars was followedby a modification of the colorimetric procedure of Park &Johnson (1949). The latter method was satisfactory withpure aqueous solutions of glucose, but not under assayconditions in the presence ofphosphate buffer, owingto pre-cipitation of reagents. This was overcome by substitutingphosphoric acid for sulphuric acid in the ferric alum solu-tion as follows: (a) 0-1 g. of ferric ammonium sulphate(A.R.) was dissolved in 1-5 ml. of conc. phosphoric acid(A.R., 90%) and 48-5 ml. of water; (b) to solution (a) wasadded a mixture containing 0.15 g. of Duponol (DuponolME, dry, kindly given by E. I. du Pont de Nemours and Co.Inc., Wilmington, Del., U.S.A.) in 1-0 ml. of conc. phos-phoric acid and 49 ml. of water.

Modified procedure for determination of 'reducing' sugar.The sample, containing 1-14,ug. of sugar, was neutralized(see below under enzyme assay) and made up to 3 ml. withwater. Deproteinizing was unnecessary. After addition of1 ml. of cyanide-carbonate solution (0-064% of potassiumcyanide + 0-52% of sodium carbonate anhydrous) followedby 1 ml. of 0-05% potassium ferricyanide, the mixture washeated in a boiling-water bath for 15 min., transferred to acold-water bath for 5 min. and treated with 2 ml. of ferricalum-Duponol-phosphoric acid reagent. Colours were

185

G. HALLIWELLdeveloped at room temperature for 15 min. before beingread on a Spekker absorptiometer (Hilger) in a 1 cm. light-path with Ilford orange filters (no. 607, peak transmission600 m).

Glucose and cellobiose gave straight-line calibrationcurves up to 14,ug. of sugar when analysed by the pro-cedure above. Absorptiometer readings obtained after15 min. of colour development increased slightly on furtherstanding, but for periods of colour development up to75 min. this error was eliminated by including a reagentcontrol whose colour value increased by the sameamount.

Determination of protein. The method of Lowry, Rose-brough, Farr & Randall (1951) was used on a Spekkerabsorptiometer in a 1 cm. light-path with Ilford red filters(no. 608, peak transmission 700 m,u). As the copper sul-phate-tartrate reagent tends to settle out on standing,tartrate was replaced by trisodium citrate dihydrate(A.R.). Crystalline bovine plasma albumin (ArmourLaboratories) provided the standard reference protein. Theprocedure was used to follow protein formation in culturefiltrates and to determine enzyme protein before and afterdialysis.

Method of enzyme assay (a) with insoluble substrate,cellulose. As a result of preliminary experiments the follow-ing procedure was adopted. To about 26 mg. (or 1 mg. inearlier experiments) of cellulose [cotton fibres, cellulosepowder (Whatman) or swollen forms of these celluloses]was added 1-3 ml. of acetate buffer, pH 5-5, (0.2N-aceticacid and 0'2N-sodium acetate), water and cell-free culturefiltrate in a final volume of 4 ml., contained in 15-ml. R.B.heat-resistant, centrifuge tubes (110 mm. x 16 mm.;Measuring and Scientific Equipment Ltd.), or 25-ml. testtubes (125 mm. x 19mm.; Griffin and George Ltd.). Testtubes were used when the residual cellulose was estimatedafter separation from the aqueous phase by means of aifiter-stick. Centrifuge tubes were employed when cellulo-lysis was measured by increase in soluble carbohydratesformed from insoluble cellulose (see below). Cell-freeculture filtrate-substrate mixtures were incubated for1 hr. at 370, and the enzyme was inactivated by addition ofsufficient 0-6N-sulphuric acid (about 0-5 ml.) to lower thepH to 1-90-1-95. Enzyme and substrate controls wereincluded.

Cellulose in the acidified suspensions was determinedcolorimetrically by two methods: (a) directly, the cellulosein its original tube being filtered, washed on the filter-stick and finally estimated with dichromate (Halliwell,1958); (b) indirectly, from the amount of soluble carbo-hydrates present in the aqueous phase: the acidified assaymedium containing cellulose in centrifuge tubes was centri-fuged at 1300-1800g for 3 min. at room temperature, andthe supernatant passed through a sintered-glass crucible(porosity 3). The carbohydrates in the filtrate were quanti-tatively determined with dichromate, the values beingcalculated from a standard calibration curve prepared withglucose (Halliwell, 1960). The dichromate method is pre-ferable to reducing-sugar methods for this purpose becauseit determines both 'reducing' and 'non-reducing' carbo-hydrates and thus gives a value much closer to the actualdegree of solubilization (cf. Fig. 3).The preliminary centrifuging was used to deposit the

insoluble cellulose firmly, thus facilitating separation ofmost ofthe liquid phase. Filtration was essential to remove

fine floating particles of insoluble cellulose, which failed toprecipitate even on prolonged centrifuging.

Acidification of cellulose to pH 1-9 produced no solubili-zation of this substrate during the period required forcentrifuging and filtration. Any solubilization that mightoccur under experimental conditions would be recognizedin the substrate controls.

In some enzymic digests of cellulose the buffer solutionsand volumes used were different from those given above forthe standard assay with acetate. However, when 0-8-1-3 ml. of acetate buffer, pH 5 5, or 1-6 ml. of phosphatebuffer (0-067M-disodium hydrogen phosphate +0-067M-potassium dihydrogen phosphate, pH 5.5) or citrate buffer(0.IM-citric acid+0-2M-disodium hydrogen phosphate,pH 5.5) were employed, enzymic activities on swollencellulose powder were identical.

Filtrates obtained from enzymic incubations withcellulose in presence of phosphate or acetate buffers wereexamined by the indirect dichromate procedure (b) outlinedabove. As citrate buffer reduces dichromate, cellulolysis insuch a medium was determined by direct procedure (a)above. Citrate was used only in measuring thepH optimumof the enzyme on swollen cellulose powder.Method of enzyme assay (b) with soluble substrate, carboxy-

methylcellulose. To 0-3 ml. of 1% (w/v) carboxymethyl.cellulose was added 0-15 ml. of acetate buffer, dilutedcell-free culture filtrate and water to 0-6 ml., final pH 5-5.The mixture was incubated at 370 for 1 hr. and the reactionterminated by addition of 0-25 ml. of 0.13% anhydroussodium carbonate and 1 ml. of cyanide-carbonate (0.064%of potassium cyanide + 0-52% of sodium carbonate, seeabove). After addition of 2-15 ml. of water (total volumenow 4 ml.) subsequent stages, commencing with theaddition of 1 ml. of potassium ferricyanide, are identicalwith those described above (see 'Modified procedure fordetermination of reducing sugar'). The value, 0-25 ml. of0.13% sodium carbonate, is experimentally determined asnecessary to neutralize the enzyme reaction mixture toabout pH 7-0, whereas the cyanide-carbonate adjusts thereaction of the neutralized mixture to about pH 10-7, thusattaining the required alkalinity of the standard glucosecurve and simultaneously terminating enzymic activity.The diluted cell-free culture filtrate itself gave a negligiblevalue when oxidized with ferricyanide, thus avoiding thenecessity for deproteinization before sugar determination.

RESULTS

In the work described below the term cellulase isused to describe the activity in cell-free prepara-tions attacking insoluble cellulose (swollen cellulosepowder, swollen de-waxed fibres, cellulose powderand de-waxed fibres), with the proviso that a truecellulase (as yet not isolated) should produceextensive solubilization of undegraded cellulose(e.g. untreated fibres) in the manner of the parentmicro-organism. Enzymic activity on the solublecellulose derivative, carboxymethylcellulose, isreferred to as carboxymethylcellulase.

Decomposition of de-waxed cotton fibres and ofcellulose powder by cultures of Myrothecium verru-caria. Fig. 1 illustrates the capacity ofthe organism

186 1961

VCELLULASES FROM MYROTHECIUM VERRUCARIA

to solubilize these less-degraded forms of cellulose.Continuous shaking was beneficial to extensivecellulolysis of both forms of cellulose and was alsoessential when profitable volumes of medium wererequired. In stationary cultures growth was muchslower, and it was necessary to use smaller quanti-ties of medium to avoid submerging the fungus.

Decomposition of de-waxed cotton fibres and ofceUulose powder by enzyme filtrates of Myrotheciumverrucaria. The degree of solubilization of thesesubstrates by the cell-free culture filtrate (Table 1)is only a small fraction of that shown on the samesubstrates by the whole culture (Fig. 1). The initialrate of solubilization of undegraded fibres by thecell-free culture filtrate is comparable with thatshown by whole organisms during growth, but the

90

0

C

PtI

Q

8

0

wd-

80

70

60

50

40

30

20

10

2 4 6 8 10 12 14 16Duration of incubation (days)

Fig. 1. Solubilization of cellulose by cultures of Myro-thecium vemrcaria. 50-ml. conical flasks containing (a)50 mg. of cellulose powder (Whatman) in 10 ml. of saltsmedium, pH 6-6, or (b) 50 mg. of de-waxed Texas cottonfibres in 4-5 ml. of salts medium for shaken cultures (or in1 ml. for stationary cultures)were inoculated with 1 ml. of aheavy spore suspension of the organism prepared fromcultures of M. verrucaria grown on filter-paper strips onagar slopes in test tubes. The flasks were aerated byreciprocal shaking at 120 strokes of 50 mm.' horizontalmovement per minute for cellulose powder (or 60 strokes of38 mm. for cotton fibres) and at 280 for the periods shown.Flasks were removed at intervals, the mycelium-celluloseresidue was filtered on sintered glass, and washed and driedas described under Materials and Methods. Cellulosepowder (Whatman) in shaken culture (0), in stationaryculture ( x ); de-waxed cotton fibres in shaken culture (0),in stationary culture (M). Control flasks containing celluloseand medium (no inoculum) at 280 in shaken or stationaryincubations showed no loss in weight of cellulose.

extent of solubilization by the enzyme preparationbecomes constant after 22 hr., whereas with wholeorganisms, where growth is in progress, it in-creases to 80% in 16 days. It is first necessary tosubject the cellulose to chemical treatment, suchas swelling in acid, before cellulolysis by the culturefiltrate becomes significant.

Activity of cellula8e on 8wollen cellulose powder atdifferent pH values. Citrate-phosphate buffer,covering the range pH 2-8 (Fig. 2) was used inmixtures, containing cell-free culture ffitrate and

Table 1. Solubilization of different form8 of cellulo8eby cell-free filtrates from Myrotheciun verrucariaSwollen cellulose powder (26 mg.) or swollen de-waxed

Texas fibres (26mg.) was incubated at 370 with 0-2 ml. of cell-free filtrate in acetate buffer, pH 5-5, for 1 hr. and 22 hr.Cellulose powder or de-waxed Texas fibres (5 mg.) wasexamined under the same conditions with 0-5 ml. of cell-free filtrate. Enzymic activity was estimated from thesoluble carbohydrates formed. In the determination ofcarboxymethylcellulase activity by the standard methodthe cell-free ifitrate was used at about one-twentieth of thefinal concentration of thatpowder.

Type of celluloseCellulose powderDe-waxed fibresSwollen cellulose powderSwollen de-waxed fibresCarboxymethylcellulose(degree of hydrolysis)

7-

tD

0

2

I1

with the swollen cellulose

Degree of solubilization(% of initial wt.) in

A

1 hr. 22 hr.34

11 568 450-4

2 3 4 5 6 7 8pH

Fig. 2. Effect of pH on the activity of cellulase on swollencellulose powder. Enzymic activity, measured by thestandard method but in citric acid-phosphate buffer, wasterminated by addition of sulphuric acid followed byfiltration and determination of the residual cellulose withdichromate.

187Vol. 79

G. HALLIWELL

substrate, which were checked for constancy of pHduring incubation. Optimum activity for theenzyme occurs about pH 5, with some hydrolysisstill evident at pH 2-5. As enzymic activity atpH 5-5 is very similar to that at pH 5-0 the formerpH was used in the standard assay medium in orderto facilitate comparison with results of otherworkers in the field.

Effect of 8ub8trate concentration on enzymricactivity. In the original cell-free culture filtratesthe relationship between cellulose made solubleand initial weight of substrate indicated that satur-ation of the enzyme occurred at about 1 mg. ofcellulose/4 ml. incubated. Activities were approxi-mately proportional to the concentrations ofenzyme used. In later experiments cell-free cultureifitrates obtained from subcultures of the originalorganism provided different curves with saturationoccurring at 27 mg. of cellulose/4 ml. of incubationmixture (Fig. 3).The Figure also illustrates agreement between

the two methods used to measure enzymic cellulo-

6

51-:bD0

I- 4

0

0

::2

0

0 0U -

0

A3 9 15 21 27 33

SFig. 3. Effect of weight of swollen cellulose powder on theamount solubilized by cellulase. Standard conditions ofassay except that incubated mixtures, after inactivationwith sulphuric acid, were filtered through sintered cruciblesto obtain both the filtrate and residual cellulose. The latterwas washed and determined gravimetrically. The filtratewas examined for the amount of soluble carbohydrates(see Methods section). Cellulose solubilized as determinedfrom residual weight of cellulose, with culture filtrate A(0-5 ml.) (U), (1 ml.) (E); with another filtrate (0-2 ml.)(0). Cellulose solubilized as determined from solublecarbohydrates in the filtrate, with culture filtrate A (1 ml.)(0). S, Conen. of substrate (mg./4 ml.).

lYsis: (1) direct loss in weight of cellulose, and(2) production of soluble saccharides.

Carboxymethylcellulase activity in the cultureifitrate was examined as in Table 2 with saturationbeing obtained at about 3 mg. of carboxymethyl-cellulose in the 0-6 ml. of assay volume.The K. values of the culture filtrate on swollen

cellulose powder and on carboxymethylcelluloseare calculated by the graphical method of Line-weaver & Burk (1934) (Fig. 4), and give values of

Table 2. Effect of 8ub8trate concentration on theproduction of reducing sugar (a8 glucose) fromcarboxymethylcelluloaeAssay of the enzyme was made by the standard pro-

cedure. The cell-free culture filtrate was used at about one-fortieth of the concentration of that in Fig. 3 (-).

Carboxymethyl-cellulose in a Reducingtotal volume sugar producedof 0-6 ml. (as glucose)

(mg.) (.g.)0-11 2-60-23 4-10-46 5-51-17 7-32-0 8-33-0 8-93-5 8-84-0 8-1

- 3-5 -0-7 0-7 2-1 3-5 4-9 6-3 7-7 9-1 (CMC)- 0-28 0 0-28 0-56 0-84 (C)

l/SFig. 4. Determination of the dissociation constant (K.) ofthe enzyme-substrate complex for cellulase and carboxy-methylcellulase by plotting the reciprocal of substrateconcentration (S) against the reciprocal of rate of solubili-zation (V) of cellulose (for cellulase) or rate of formation ofreducing sugar (for carboxymethylcellulase) from data ofFig. 3 (cellulase) and Table 2 (carboxymethylcellulase).0, Cellulase (C); El, carboxymethylcellulase (CMC).

1961188

CELLULASES FROM MYROTHECIUM VERRUCARIA

1 g. of cellulose/l. and 0-5 g. of carboxymethyl-cellulose/l. for the dissociation constant of theenzyme-substrate complex.

Effect of concentration of cell-free culture filtrateon the solubilization of swollen cellulose powder. Athigh enzyme concentrations the reaction velocity isnot proportional to the amount of culture filtratepresent (Fig. 5) probably owing in part topreferentialenzymic attack on the more susceptible amorphousregions of the swollen cellulose. The effect was notdue to the presence of a dialysable inhibitor as thedialysed culture filtrate possessed slightly lessactivity than the undialysed preparation whenequal amounts of filtrate were used, based on

protein content. Likewise, possible end productssuch as glucose or cellobiose produced no markeddecrease in cellulolysis (see below). The effect couldalso indicate the presence of non-dialysable in-hibitor in the cell-free culture filtrate.

Solubilization of cellulose as affected by the dura-tion of enzymic activity. The rate of breakdown ofswollen cellulose powder is almost linear for the first45 min. but thereafter decreases progressively

16F

14F

121F

101

8

6

4

2

v

Ev0

-'-a

1 2 3 4 5 6 7Concn. of enzyme

4 8 12 16 20 24Period of incubation (hr.)

Fig. 5. Effect of enzyme concentration and of duration ofincubation on the solubilization of swollen cellulosepowder. Cellulase activity was measured and terminatedby the standard method. Enzyme concentration was

expressed as units in 4 ml., each unit corresponding to0-05 ml. of cell-free culture filtrate. Soluble carbohydratesin the ifitrate were determined with dichromate. Cellulosemade soluble (0-3 mg.) as affected by concentration ofenzyme (0). Cellulose made soluble (0-16 mg.) as in-fluenced by period of incubation (0).

(Fig. 5). This presumably results from the earlybreakdown of the less refractory amorphous areasin cellulose and from the instability of the enzymeat 370 (see below) together with its increasing degreeof unsaturation as the substrate is solubilized.

Effect of agitation on enzymic activity on cellu10seand carboxymethylcellulo8e. Previous paragraphshave indicated that approximately 30-fold morecellulose was required to saturate later culturefiltrates than was the case in earlier experiments.In an unsuccessful attempt to explain this differ-ence, the action of cellulase (cell-free preparation)was examined with and without agitation on aWarburg apparatus operating at 107 completestrokes of 50 mm. horizontal movement per minute,when activity was found to be unaffected or in-creased only slightly by shaking. The same enzymepreparation examined under similar conditionswith carboxymethylcellulose as substrate lost 72%of its activity compared with an unshaken incuba-tion (Table 3).

Activity of cellulase after heat treatment. Cellulaseactivity in the preliminary experiments (in whichthe culture filtrate was saturated with 1 mg. ofcellulose) was completely terminated by immersingthe reaction tubes containing filtrate and substratein hot water (970) for 20 min. In presence of thelarger amounts of cellulose (27 mg.) required tosaturate subsequent ifitrates, the same heat treat-ment was less effective and allowed variableamounts of activity to continue. The effect wasfurther examined by using shorter and longerperiods of heating as follows: cell-free culturefiltrates-cellulose mixtures incubated for 1 hr.were subjected to the following treatments: (a)immediate filtration (control), (b) immersion at 970for 10 mm. followed by incubation at room temper-ature (200) for 30 min., (c) as in (b) but with a30 min. heating period. In each case the cellulosewas finally filtered, washed with hydrochloric acid,water, ethanol and dried at 1050 before beingweighed, when the degree of solubilization in (b)and (c) were 116 and 97% respectively ofthatin (a).

It appears that one or more of the components ofthe enzymic preparation, possibly one concernedwith the shorter but still insoluble chains, is notreadily inactivated by brief periods of heat-treatment. Assays were more conveniently termin-ated by addition of dilute acid as described in theexperimental section.The filtrate was also examined for cellulase and

carboxymethylcellulase activity by the standardprocedures, after a preliminary incubation underassay conditions in the absence of substrate for1 hr. and at different temperatures (Fig. 6). Theresults suggest that carboxymethylcellulase is lessstable than cellulase, but in effect this is due to theexperimental conditions of the respective assays

Vol. 79 189

Table 3. Effect of 8haking on the activty of ceUulluse and carboxYmethyksellulas8eCellulase and carboxymethylcellulase activities were determined by the standard methods on swollen cellulose

powder and on carboxymethylcellulose. Procedures for terminating and for estimating enzymic activity aredescribed in the table. Tubes were shaken in a Warburg bath at 107 complete strokes of 50 mm. horizontal move-ment per minute and held in suitable positions (about 350 to the horizontal for cellulose) to obtain similardegrees of agitation with both enzymes. Unshaken tubes: Shaken tubes:

Cell-free Method of terminatingfiltrate enzymic activity

1, 2 Immediate filtration andsolvent washing (hydro-chloric acid, water, ethanoland oven-drying)

2 Addition of sulphuric acidto pH 1-95, rapid filtrationunder pressure and solventwashing (as above)

3 Addition of sulphuric acidto pH 1-95, rapid filtrationunder pressure and solventwashing. Filtrate retainedfor next experiment

3 Filtrate from previousexperiment

4 Addition of sulphuric acidto pH 1-95, filtrationunder gravity, filtrateanalysed

3 Addition of carbonate-cyanide to pH 10-7

Method of estimatingenzymic activityCeUulase activity

Gravimetric, on residualcellulose

As above

As above

Dichromate-sulphuric acidoxidation of soluble carbo-hydrates in the filtrateAs above

CarbozymethylceUulase activity

Ferricyanide determinationof reducing sugars formed

cellulose solubilized(% of initial

weight)

23

21

15

15

15

Unshaken tubes:reducing sugarformed (% ofsubstrate)

0-4

cellulose solubilized(% of that solubilizedin unshaken tubes)

105

98

106

108

98

Shaken tubes:reducing sugars

formed (% of thatin haken tubes)

28

rather than to inherent instability of that enzyme.Thus when the cell-free preparation itself was in-cubated in absence of substrate for 1 hr. (ratherthan under the respective conditions for the twoenzymes) before removal ofsamples for the standardcellulase and carboxymethylcellulase assays, thelatter enzyme appeared to be slightly more stablethan cellulase (Fig. 6).

Effect of possible inhibitor non cellulase activity.Glucono-1 -+ 4-lactone or glucose at a final concen-tration of 2-5 mm had no effect on the rate ofsolubilization of cellulose by the cell-free culturefiltrate, whereas the same molarity of cellobiosealways produced slight inhibition, but never morethan 10%. At the high concentration of 25 mM(18 mg. of glucose or 34 mg. of cellobiose in thepresence of 26 mg. of cellulose) 15, 10 and 30%inhibition was obtained with gluconolactone,glucose and cellobiose respectively. The reactionmedium containing carbohydrates equivalent to1 mg. of glucose (dichromate method) obtainedafter incubation of cellulose and enzyme for 1 hr.was transferred to a fresh assay system and pro-

duced only 8% inhibition of cellulolysis, indicatingthat end products have negligible effect oncellulolysis.

Adsorptim of cellulase by celluloseImmediately after mixing cellulase and its sub-

strate (swollen cellulose powder) the aqueous phaseis relatively free from enzyme owing to the latter'sadsorption, the extent of which depends on theinitial quantity of cellulose present and on sub-sequent incubation of the mixture (Table 4).

In Table 4 separate samples were used for the 0and 1 hr. experiments in order to avoid delays infiltration, but substantially similar results wereachieved when the original sample of cell-freeculture filtrate and cellulose (26 mg.) was suc-cessively centrifuged, the supernatant filtered offand the cellulose reincubated with fresh buffersolution. Thus after incubation for 0 and 1 hr.,supernatant fractions containing 17 and 20%respectively of the original enzymic activity wereobtained by successive centrifugings. The de-posited cellulose was then washed once (or three

1961190 G. HALLIWELL

Vol. 79

times) witof the cellenzyme oIfor some a

and for th(enzyme ccof the cellsubstrate.

M. verrbilization 4

cell-free cl

100r

90g

80 F

701-

601-

50 F40F

30 F

201-

Fig. 6. ReEcell-free pri+AmT%Arsa.+.lrr

CELLULASES FROM MYROTHECIUM VERRUCARIA,h dilute acetate buffer. On reincubation (4% solubilization) on this substrate. For celluloselulose it was found to retain 49% of the powder prolonged incubation, with addition ofriginally added. All values are corrected further quantities of cell-free filtrate, provides aidsorption of enzyme by the sinter itself constant maximum of 26% solubilization achievede non-linearity of enzyme activity at high in 72 hr., comparable with the values given by ourmncentrations. It is noteworthy that 86% earliest preparations (Halliwell, 1957b). The latter,lulase has survived 2 hr. incubation with however, contrasted with present cell-free culture

filtrates in being more active (requiring lessDISCUSSION enzymic material to produce this degree of solubili-

~ucaria produces almost complete solu- zation) on cellulose powder, but less active onof de-waxed cotton fibres (Fig. 1), whereas swollen cellulose powder. This activity was asso-alture filtrates have an insignificant effect ciated with a lemon-yellow colour present in the

early culture media, but less evident in morerecent metabolism fluids. Such apparent variancein chemical properties between cell-free prepara-tions may also account for the different amounts ofcellulose required to saturate the various cell-freeculture filtrates described above.

After conversion of cotton fibres or cellulose0 \\ powder into the swollen form, both the rate and

extent ofbreakdown are increased (Table 1) so thatalmost complete solubilization (90%) can beachieved in 22 hr. by addition of larger amounts ofenzyme.

U\\ The results show that the procedure describedfor the determination of activity on swollen, butinsoluble, forms of cellulose powder and fibres is as

0 rapid as it is on the soluble carboxymethylcellulose(Fig. 3, Table 2) under normal conditions ofmeasurement. It thus seems preferable and

\\v possible to employ an insoluble cellulosic substratein studies involving cellulase rather than to sub-

c * stitute carboxymethylcellulose.20 40 60 80 100° Basu & Pal (1956) observed that carboxymethyl-20 40 60 80 1000 cellulase preparations from several fungi, includingTemp. of incubation M. verucaria, were denatured (40%) if agitated

3idual enzymic activity after incubation of the during a 3 hr. assay procedure, whereas Myers &eparation in absence of substrates at different Northcote (1959) failed to obtain any inhibition of

Y0 n r.I.maa onf .AX LLUar V n.% U [1UIUyl-mprzure. xrea-menulz : oceuue tu), carooxymetnyi-cellulase (-); the enzyme preparation was incubated inacetate buffer, pH 5-5, under the appropriate assay condi-tions for cellulase and carboxymethylcellulase (see Materialsand Methods), but in the absence of substrate (cellulose orcarboxymethylcellulose), at 200, 370 or 540 for 1 hr. and thentransferred to a bath at 370 followed immediately byaddition of the respective substrates (cellulose or carboxy-methylcellulose) and a further incubation of 1 hr. at 370.Controls, assayed in the normal manner for 1 hr. at 370,were taken to represent 100% enzymic activity (c onabscissa). Treatment B: cellulase (0), carboxymethyl-cellulase (E); the cell-free preparation was diluted fourfoldin acetate buffer, pH 5-5, followed by (a) immediate assay(controls) on swollen cellulose powder or on carboxymethyl-cellulose, these activities being taken as 100% (c onabscissa), or (b) incubation at 200, 370 or 540 for 1 hr. or970 for 10 min. when samples were removed and assayed onswollen cellulose powder or, after dilution, on carboxy-methylcellulose under the usual conditions (see Methods)for 1 hr. at 37. Cellulase activities were determined fromthe amount of soluble carbohydrates formed.

Table 4. Ad8orption of cellulase by differentamount8 of cellulo8e

ES (0) and ES (1) represent enzyme-swollen cellulosepowder-buffer mixtures prepared in the standard manner(see Materials and Methods section) with mixing, followedby (a) immediate filtration (no centrifuging), ES (0), or (b)incubation for 1 hr. at 370, ES (1), before filtration througha sintered-glass crucible. The enzyme in each filtrate wasdetermined by assay on fresh cellulose-buffer as inMaterials and Methods and the assay value (solubilizationof cellulose) is expressed as a percentage of the enzymicactivity of a control ifitrate obtained in a similar manner toES (0), but in absence of cellulose.

Cellulose(mg.)24127

Enzymic activity in filtrate(% of initial)

ES (0) ES (1)2 276 28

16 36

191

192 G. HALLIWELL 1961

the same enzymic activity during a 20 mi.shaking period with a preparation from snails. Inour own work, a 1 hr. period of agitation producedsevere loss (72%) of carboxymethylcellulase, butallowed cellulase activity to continue unimpaired(Table 3). The results achieved by Myers &Northcote (1959) are possibly due to the slowerrate of shaking.

Fig. 6 indicates that carboxymethylcellulase losesactivity more rapidly under its own particular assayconditions when devoid of substrate than doescellulase. On the other hand cellulase appears tobe slightly less stable than carboxymethylcellulasewhen the cell-free culture filtrate, without additions,other than fourfold dilution in buffer, is incubatedat different temperatures (in absence of substrate)before transfer to the two standard assay systems.Under their respective assay conditions but with-out added substrate the cell-free culture filtrateloses 50% of carboxymethylcellulase and 10% ofcellulase activity during a 1 hr. incubation at 37°.The possibility that end products resulting from

enzymic activity on cellulose might inhibit furtherbreakdown of this substrate does not exist underthe present conditions. Only when glucose orcellobiose was added in very large amounts (18and 34 mg. respectively in presence of 26 mg. ofcellulose) did significant (10-30%) reduction inactivity occur in 1 hr. End products of cellulolyticactivity likewise failed to inhibit cellulase whentransferred to a fresh enzymic assay on cellulose.Further, Fig. 5 also indicates that the accumulationof end products on prolonged incubation of enzymewith substrate for 22 hr. does not prevent solubili-zation from rising to at least 60% breakdown.Larger amounts of cell-free culture filtrate per-mitted 90% solubilization of cellulose in the sameperiod. Gilligan & Reese (1954) have reported thatrelatively high concentrations of cellobiose wererequired to produce appreciable inhibition of thehydrolysis of hydrochloric acid- and phosphoricacid-treated, ground cotton by filtrates fromTrichoderma viride.

Table 4 illustrates the high affinity of cellulosefor its enzyme and probably accounts for the lackof any beneficial effect of agitation on cellulolysis.Cellulase is strongly adsorbed by its insoluble sub-strate and is slowly liberated during solubilizationof the cellulose.

Cellulase (as examined on acid-swollen cellulosepowder) is in fact a relatively stable enzyme,strongly adsorbed by its substrate and thusdifficult to recover.

SU1WMARY1. The action of cell-free filtrates from Myro-

thecium verrucaria was examined on various forms

of insoluble cellulose and on the soluble cellulosederivative, carboxymethylcellulose.

2. Unlike the parent organism which completelysolubilizes undegraded forms of cellulose (de-waxedcotton fibres), cell-free culture medium has anegligible effect (4% solubilization) on the samesubstrate unless this is first swollen with phosphoricacid. After such treatment of cellulose, cellulolyticfiltrates produce as much as 90% solubilization ofswollen cellulose powder or of swollen de-waxedcotton fibres. Conditions governing the activity ofthe cell-free filtrates on swollen cellulose powderare described.

3. The preparation has an optimum activity onswollen cellulose powder at about pH 5.

4. Values of 1 g. of swollen cellulose powder/l.and of 0 5 g. of carboxymethylcellulose/l. wereobtained for the Michaelis constants.

5. At pH 5-5 and temperatures between 20° and540 the cell-free filtrate loses cellulase slightly morerapidly than carboxymethylcellulase activity. Incontrast, under the appropriate assay conditionsfor the two activities, carboxymethylcellulase isdenatured more rapidly than cellulase.

6. Agitation of cell-free filtrates in presence ofcellulose has no marked effect on the degree ofcellulolysis. With carboxymethylcellulose as sub-strate, however, agitation produces 70% inhibition.

7. Glucose, cellobiose or reaction products ofenzymic cellulolysis fail to retard enzymic solu-bilization of cellulose unless present in amountscomparable with the initial weight of cellulose.

8. Cellulase in cell-free filtrates is stronglyadsorbed on its insoluble substrate and is onlyslowly released as the cellulose itself is solubilized.

I sincerely thank Dr G. A. Levvy for his advice andencouragement and Mr G. Pratt for valuable technicalassistance.

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56, 658.Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall,R. J. (1951). J. biol. Chem. 193, 265.

Myers,F. L. & Northcote, D. H. (1959). Biochem. J. 71, 749.Park, J. T. & Johnson, M. J. (1949). J. biol. Chem. 181, 149.Saunders, P. R., Siu, R. G. H. & Genest, R. N. (1948).

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