Agric. Biol. Chem., 49 (5), 1257-1265, 1985 1257

Purification and Properties of a Cellulase fromAspergillus niger

Gentaro OkadaDepartment of Biology, Faculty of Education,

Shizuoka University, Shizuoka 422, JapanReceived June 20, 1984

A cellulolytic enzyme was extensively purified from a commercial crude cellulase preparationfrom Aspergillus niger by consecutive column chromatography. The purified enzyme was homo-geneous on polyacrylamide gel as well as ampholine electrophoresis. The enzymewas an acidicprotein with an isoelectric point at pH 3.67. The molecular weight of the enzyme was estimated tobe 31,000 by SDS-polyacrylamide gel electrophoresis. No carbohydrate moiety seemed to beassociated with the enzyme protein.The optimumpH was 4.0, and the optimumtemperature of the enzyme was 45~50°C. The

enzyme was completely stable over the range of pH 5.0~8.0 at 4°C for 24hr, and retained about50% of its original activity after heating at 70°C for lOmin. The enzyme was partly inactivated bylmMAg+, Hg2+, and Fe2+.

The enzyme was characterized as an endocellulase on the basis of its action on carboxy-methyl cellulose and cellooligosaccharides. The enzyme split cellopentaose, retaining the ^-configu-ration of the anomeric carbon atoms in the hydrolysis products. The Kmvalue of the enzymefor carboxymethyl cellulose was 0.086%. It was active on carboxymethyl cellulose and cellooligo-saccharides (cellotriose to cellohexaose), but not on either cellobiose or /?-nitrophenyl /?-d-glucoside under the assay conditions used.

Cellulases [l,4-(l,3;l,4)-/J-D-glucan 4-gluca-nohydrolase, EC 3.2.1.4] are essential in the

carbon cycle in nature. In most cellulolyticmicroorganisms, several cellulase componentsproduced in the cluture filtrate together con-stitute a "cellulase system," ancj insoluble cel-lulosic substrates are converted to solublesugar by their synergistic action.

Commercial crude cellulase products fromAspergillus niger have been fractionated andpartially characterized by several investi-gators.1 ~9) Ikeda et al.1Otll) reported the purifi-cation and some characteristics of a homo-geneous cellulase from A. niger, with an un-usually low optimum pH range (2.3~2.5).However,almost no detailed studies on thesubstrate specificity and mode of action of A.niger cellulases have been done.This paper describes the purification, physi-

cochemieal and enzymic properties of an en-docellulase from A. niger.

MATERIALS AND METHODS

Crude enzyme material. The ethanol-precipitated pow-der (lot No. C6600N) from a water extract ofa wheat branculture of A. niger, kindly supplied by Amano

Pharmaceutical Co., Ltd., was used as a starting materialfor the purification of cellulase.

Materials. Sodium carboxymethyl cellulose (CMC) wasCellogen BS (lot No. 1545512) supplied by DaiichiIndustrial Pharmaceutical Co., Ltd.; the degree of sub-stitution was 0.62 ~0.64. Soluble starch (lot No. DP9937)

was purchased from WakoPure Chemicals Co. p-Nitrophenyl /?-D-glucoside (/?-PNPG) was synthesized bythe method of Glaser and Wulwek,12) mp 165~166°C;[a]J>9 - 100° (c=2, H2O). Cellooligosaccharides from cel-lobiose (G2) to cellohexaose (G6) were prepared frommicrocrystalline cellulose powder (Avicel) by the method

Abbreviations: CMC, sodium carboxymethyl cellulose; G1? D-glucose; G2 to G6, cellooligosaccharides from

cellobiose to cellohexaose; /?-PNPG, /7-nitrophenyl /?-D-glucoside; SDS, sodium dodecyl sulfate; EDTA, disodiumethylenediaminetetraacetic acid.

1258 G. Okada

of Miller et al.13) All the other chemicals were commercialproducts of analytical grade.

Enzymeassays. CMC-saccharification activity was usedas the standard assay of cellulase. The reaction mixturecontained 0.5ml of 1% CMCsolution, 1.0ml of 0.05macetate buffer (pH 4.0), and 0.5ml of enzyme solution.After incubation at 30°C for an appropriate period, thereducing sugar produced per 1.0 ml of the reaction mixturewas measured by the method of Somogyi14) and Nelson.15)One unit of the enzyme activity was defined as the amountof the enzymethat catalyzed the liberation of reducingsugar equivalent to 1.0/imol of D-glucose per min understandard assay conditons.CMC-liquefaction activity was measured using viscom-

etry. The reaction mixture contained 1.5ml of 1% CMCsolution, 3.0ml of 0.05m acetate buffer (pH 4.0), and

1.5 ml of enzyme solution. The mixture was incubated inan Ostwald viscometer at 30°C and the viscosity was

measured at intervals. The cellulase activity was expressedin terms of the specific fluidity, $sp {i.e., l/rjsp).Amylaseactivity was measured under the same con-

ditions as CMC-saccharification activity, using 0.3% sol-uble starch solution as the substrate in place of CMC.Oneunit of the enzymeactivity was defined in the same wayasCMC-saccharification activity.

Aryl /?-glucosidase activity was measured using P-PNPGas the substrate. The reaction mixture contained0.2ml of0.0136m jS-PNPG, 0.4ml of0.05 M acetate buffer(pH 4.0), and 0.2ml of enzyme solution. After a suitableincubation period at 30°C, 0.4ml of the mixture was addedto 10.0ml of 0.1m sodium carbonate, and the p-nitrophenol liberated was estimated by measuring theabsorbance at 420 nm. One unit of the enzyme activity wasdefined as the amount of the enzyme that catalyzed theliberation of 1.0/miol of /?-nitrophenol per min under

standard assay conditions.

Measurementofprotein. Protein concentration was mea-sured by the method of Lowry et al.16) using crystallinebovine serum albumin (Miles Laboratories, Inc.) as astandard. The absorbance at 280nm was used formonitoring protein in column effluents.

Measurement of carbohydrate. The carbohydrate in theenzyme was measured by the phenol-sulfuric acidmethod17) using D-glucose as a standard.

Polyacrylamide gel electrophoresis. Disc gel electro-phoresis was done in 7.5% polyacrylamide with a Tris-diethylbarbituric acid buffer (pH 7.0) by the method ofDavis.18) SDS-polyacrylamide gel electrophoresis was

done in 10% polyacrylamide containing 0.1% SDS by thetechnique of Weber and Osborn.19) In both cases, the gelswere stained overnight in 0.05% Coomassie Brilliant BlueR-250-50% methanol-10% acetic acid.

Isoelectric focusing. The isoelectric point of the enzymewas measured using LKB Ampholine electrofocusingequipment (110ml) by the method of Vesterberg andSvensson.20)

Polarimetric procedure. Cellopentaose (22.5 mg) wasdissolved in 1.5 ml of water (1.5%, w/v), and centrifuged toremove.undissolved materials. The supernatant was left atroomtemperature overnight to ensure mutarotation. Analiquot of 0.5ml of buffered enzyme solution at pH 4.0(ll.0 units) was added to 1.0ml of the G5 solution, andthoroughly mixed. Then a part of this mixture was quicklytransferred to a 1-dm polarimeter tube, and changes in therotation were measured at 25°C at intervals using a

polarized sodium d lamp as a light source. After in-cubation for 23 min, one drop of concentrated ammoniumhydroxide was added to the polarimeter tube, and therotation was again measured after thorough mixing.

Measurement ofKmfor CMC.A mixture of 1.0ml ofeach 0.1 - 1.0% CMCsolution and 2.0ml of0.05m acetatebuffer (pH 4.0) was equilibrated at 30°C. The reaction wasstarted by the addition of 1.0ml of the purified enzyme(0.095 unit) warmed to the same temperature. Afterincubation for 10min, 1.0ml aliquots were removed, andthe reducing sugar liberated was measured by the col-orimetric method of Somogyi and Nelson. The Kmvaluewas calculated from Lineweaver-Burk plots.21}

Paper chromatography. Hydrolysis products of the en-zyme were detected by paper chromatography. After anappropriate period of incubation, 20fA aliquots of theenzymatic hydrolysates of £-PNPG, G2~G6, CMC,Avicel, and solutions of authentic sugars used as standardswere spotted on Whatman No. 1 filter paper.Chromatograms were developed by the descending tech-nique with «-butanol-pyridine-water (6 : 4 : 3, v/v) at roomtemperature for 20hr. Products were detected by the

dipping procedure using alkaline silver nitrate reagent.22)

RESULTS

Purification of the enzymeUnless otherwise stated, all fractionations of

the enzyme were done in a cold room (4°C).The enzymepreparations at various stages ofpurification were concentrated by ultrafll-tration with a Diaflo UM-10 membrane(Amicon Corp.).Step 1: Amberlite CG-50 column chromatog-

raphy. The crude cellulase powder (12g) wasdissolved in 400ml water, and fractionated

with ammoniumsulfate. The precipitate sedi-menting at 60 ~ 80% saturation of ammonium

A Cellulase from A. niger 1259

c E50-

I §f I""

g0.8--a20- \ \ pi g

I I \ l\ !\ 0.3-<^Q4-|fO- / \ f\\ \ 0.2-f

0L pi ^ å à"---.-..å  '^-K^/^j^^.. //à"å ^k, 10 20 40 60 80 100 120 U0 160

Tube number (1tube=60ml)Acetate buffer

h-0.2M, pH3.5-+ Q3M, pH45-^-<~0AM,pH5.5 i

Fig. 1. Elution Patterns of the AmmoniumSulfate Precipitate of Crude EnzymePreparation on AmberliteCG-50 Column Chromatography.#. protein concentration 0428Onm)' O> CMC-saccharification activity; A, amylase activity; x , aryl /?-

glucosidase activity. Active fractions (tubes No. 70 ~91) were combined.

f .3 =8(-rA8. . 1.6-

I a 2 hS 5e-536- ;/| i.2--

I rl A //\ \?8rgA-C2A- / \ //' h\ 0.8-g8 o o / .o'/ à"\ S

* à" * r vk \\ iiA-82-212- J\ I / \ V\ 0.6-f

0 ° ° ^20 30 4<P 50^ 60Tube number (Hube=4ml )

Fig. 2. Gel Filtration Patterns of the Cellulase Fractions on Bio-Gel P-150 after amberlite CG-50 ColumnChromatography.All symbols are the same as in Fig. 1. Active fractions (tubes No. 40~52) were combined.

sulfate was dissolved in 0.2m acetate buffer(pH 3.5) and dialyzed against the same buffer.The dialyzed solution was put on a column ofAmberlite CG-50 (4.4 x 70 cm) preequilibratedwith 0.2m acetate buffer (pH 3.5), and elutedstepwise with the results shown in Fig. 1.Fractions No. 70 to 91, which contained mostof the cellulase activity, were pooled and con-centrated to about 4.7ml.Step 2: Gel filtration on 1st Bio-Gel P-150.Theconcentrated samplewasput on a columnof Bio-Gel P-150 (2.2 x 67cm) preequilibratedwith 0.05 m acetate buffer (pH 5.0), and elutedwith the same buffer. The fractions containingcellulase activity (tubes No. 40~52 in Fig. 2)

were pooled and concentrated to about 3.3 ml.Step 3: Gel filtration on 2nd Bio-Gel P-150.The same chromatography as in step 2 wasdone again. The fractions containing cellulaseactivity were pooled and concentrated toabout 3.4ml.

Step 4: Gel filtration on 1st Sephadex G-50.The concentrated sample was passed through aSephadex G-50 column (2.2x62cm) pre-equilibrated with 0.05m acetate buffer (pH5.0). The active fractions of cellulase werepooled and concentrated to about 3.7ml.Step 5: Gel filtration on 2nd Sephadex G-50.The same chromatography as in step 4 wasdone again. The fractions containing cellulase

1260 G. Okada

Table I. Summaryof Purification of a Cellulase from A. niger

A c tiv ity

S tep of puri fic ati onV o lu m e

(m l)

P r o te in

(n ig)U n its

Y ield

( % )

S p e cifi c a c tiv ity

(u n its/m g )

1. Am b e rl i te C G- 5 0 1 32 7 7 24 .4 20 6 57 .9 1 0 0 2 8. 52

2. 1 st B io -G el P - 15 0 4 8 5 6 6.8 19 95 0. 6 96.6 35. 20

3 . 2n d B io - Ge l P -1 50 4 7 4 7 3.8 1 7842.0 86. 4 37. 66

4. 1 s t S ep ha d ex G- 5 0 4 3 17 9.7 14 18 1. 9 6 8.7 78. 92

5 . 2 n d S e p ha d e x G - 5 0 4 4 14 8.4 13 98 4. 7 6 7.7 9 4. 24

6. 3 r d Bi o- Ge l P -1 50 32 10 8.6 12 68 7. 4 6 1. 4 1 16. 83

activity were pooled and concentrated toabout 3 ml.Step 6: Gel filtration on 3rd Bio-Gel P-150.The concentrated sample was passed through aBio-Gel P-150 column (2.2x40cm) pre-equilibrated with 0.05m acetate buffer (pH

5.0). A protein peak containing only cellulaseactivity was obtained. The active fractionswere then combined.

Table I summarizes the purification pro-cedure. The final purified enzyme had a spe-

cific activity of 116.8 units per mg of proteinin a final yield of about 60%, and was usedfor the subsequent characterization.

Purity and molecular weight of the purifiedenzyme

The purified enzyme showed a single proteinband on polyacrylamide gel electrophoresis(Fig. 3). No carbohydrate moiety associatedwith the enzyme protein was detected by thephenol-sulfuric acid method.The molecular weight of the purified enzymewas estimated to be 31,000 by comparison ofits relative mobility on SDS-polyacrylamide

gel electrophoresis with those of standard pro-teins (Fig. 4).

Isoelectric pointThe purified enzyme was subjected to

isoelectric focusing to find its isoelectric pointand also to check on its homogeneity. A sharp,single peak was obtained as can be seen in Fig.5. The enzyme was found to be an acidic

protein, homogeneousand with an isoelectricpoint at pH 3.67.

Fig. 3. Polyacrylamide Gel Electrophoresis of thePurified Cellulase.A sample of the purified enzyme (9fig) was put on a gelcolumn of 7.5% polyacrylamide and run at 4°C under aconstant current of 2mAper gel.

Effects of pH on the activity of the enzymeThe effects of pH on the activity of the

purified enzyme were studied under standardassay conditions at 30°C for lOmin using ace-tate buffers (0.05m) at pH 3.5-6.0 and

Mcllvaine buffers (0.05m) at pH 3.5-8.0. Theenzyme exhibited a single maximumat pH 4.0in each buffer series.

Stabilities of the enzyme toward pH andtempera ture

Two series of enzyme solutions were pre-

A Cellulase from A. niger 1261

9 -

^-> \ Bovine serum albumin

'o \x5" \

*~> *\ Ovalbumi n£ \.91 \

|3 - \ Cellulase

| \o \

r å \-*å  Myoglobin >

Cytochrome c \

10 0.2 0.A 0.6 0.8 1.0Mobility

Fig. 4. Estimation of the Molecular Weight of thePurified Cellulase by SDS-Polyacrylamide Gel Electro-phoresis.

The purified enzyme (20^g) was put on a gel column of10% polyacrylamide containing 0.1% SDS. Electro-phoresis was done at room temperature at a constantcurrent of 8mA per gel. Cytochrome c (MW 12,400),myoglobin (MW 17,800), ovalbumin (MW 45,000), and

bovine serum albumin (MW67.000) were used as themolecular weight markers.

1.0- -10

ob .à"à"å 9

å  8

|a5" As ::...à"à"à"à"å  M -3

A %....

..../å  7 X - å '

0 I , x , <£-> ?'°-°-n . 10 10 20 30 40 50 60

Tube number (1 tube=2.0ml)

Fig. 5. Isoelectric Focusing of the Purified Cellulase.The purified enzyme containing 1.8mg of protein and116units of enzyme activity was put into a 110ml LKBcolumn. After electrophresis at 4°C for 63hr under aconstant voltage of 900V, each 2.0ml fraction was col-lected, and its enzyme activity, protein concentration andpH were assayed. -#-, protein concentration (A280 nm);-O-, CMC-saccharification activity; #, pH.

,0c ^aa-ooo

t. 80- // \* / \

Z60å o/ \« x \

20-

° 3.5 4.0 A.5 5.0 5.5 6.06.5 7.0 7.5 8.0

PH

Fig. 6. Stability of the Purified Cellulase with Respect topHand Temperature.O, held at 4°C for 24hr before assay for enzyme activity;x , held at 45°C for 2hr before assay for enzyme activity.

Experimental details are described in the text.

100 - à"-à"-à"-à"

S80- \

i \

K \aI 1 ' 1 ' 1 à"-à" à"

30 40 50 60 70 80 90100

Temperature (°C )

Fig. 7. Thermal Stability of the Purified Cellulase.Experimental details are described in the text.

pared, each solution containing an equalamount (0.1 ml) of the purified enzyme (0.540unit) individually adjusted to a pH value from

3.5 to 8.0 by adding.4.9ml of0.02m Mcllvainebuffer. After one set of solutions of eachenzyme had been kept at 4°C for 24hr, theother set at 45°C for 2hr, both series of

buffered enzymesolutions were diluted to anappropriate concentration with 0.05 m acetatebuffer (pH 4.0). Samples were then examinedfor remaining cellulase activity by the standardassay at 30°C for lOmin. The results are

shown in Fig. 6. The enzyme was completelystable over the range of pH 5.0-8.0 at 4°Cand pH 5.5 -6.5 at 45°C, under the conditionsused.

The purified enzyme solutions (0.054 unit) in0.5ml of lniM acetate buffer (pH 5.0) were

1262 G. Okada

io° r7\t -7 \f \

q I " 1 " 1 1 1 1 1 1

30 405060 7080 90100

Temperature ( °C )Fig. 8. Effects of Temperatureon the Activity of thePurified Cellulase.Experimental details are described in the text.

5 10 15

Reducing power as glucose ( ug/ml)

Fig. 9. Relationship between Increases in Fluidity andReducing Power,during the Hydrolysis of CMCby thePurified Cellulase.

Reaction conditions: 1.5 ml of 1% CMCsolution, 3.0ml of0.05m acetate buffer (pH 4.0), 1.5ml of 0.000014%purified enzyme, at 30°C for every 5 min of incubation.

heated at various temperatures for lOmin,then cooled in running tap water. The remain-ing cellulase activity was then determined with0.25ml of each enzyme solution by the stan-dard assay at 30°C for 10min. The results (Fig.7) indicate that the enzyme is completely stableat temperatures below 60°C. The enzymere-tained about 50% of its original activity onheating at 70°C, but was completely inacti-vated by heating at 80°C, under the conditionsused.

Effects of temperature on the activity of theenzyme

Enzyme solutions (0.027 unit) in 1.5 ml con-taining 0.05m acetate buffer (pH 4.0) wereincubated with 0.5ml of 1% CMCsolution atdifferent reaction temperatures for 10 min. Thecellulase activity per 1.0ml of the reactionmixture was assayed. The optimum tempera-ture for the activity of the enzyme was45~50°C, as shown in Fig. 8.

Effects of various metal ions and several enzymeinhibitors on the activity of the enzyme

Mixtures containing 0.5ml of the enzymesolution (0.135 unit) plus 1.0ml of bufferedmetal or inhibitor solution at pH 4.0 (or bufferalone) were held at 30°C for 10min. Then

0.5 ml of 1% CMCsolution was added quicklyand after incubation at 30°C for 10min, theremaining cellulase activity per 1.0 ml of each

reaction mixture was assayed. The final con-centrations of metal ions and inhibitors in thereaction mixtures were lmMand 0.1 mM,re-spectively. Inactivation of the enzyme by themetal ions tested was found to be partial with1 mMAg+, Hg2+ and Fe2+, corresponding toabout 75, 67, and 55% inhibition, respectively.EDTAand the sulfhydryl reagents tested hadno effect on the activity of the enzyme.

Degree of randomness of CMC-saccharificationactivity in the enzyme

The degree of randomness of the enzymeinthe hydrolysis of CMCwas examined. Theenzyme was incubated with CMCunder stan-dard assay conditions, and the decrease inviscosity of the incubation mixture and theformation of reducing sugars were measuredevery 5min. Then the increases in specificfluidity (</>sp) were plotted against the increasesin reducing power. A straight line with theslope of an angle of about 30° was obtained forthe enzyme (Fig. 9). This result indicates thatthe purified enzyme is a typical endo-ty\>Qcellulase with a medium randomness on hy-drolysis of CMC.

Anomeric configuration of productsThe anomeric configurations of the reactionproducts formed by the enzyme were exam-ined. The results are shown in Fig. 10. The

A Cellulase from A. niger 1263

Table II. A Summaryof the Values of Kmand Specific Activity forCMCof Endocellulases from Different Microorganisms

E n zy m e so u rce K m ( % ) Specific activity(u n its/m g )

Asp ergil lus n iger cell ulase 0. 08 6 11 6. 83

T r ich o d e rm a virid e c ellu o la ses II-A 23) 0.0 81 2 9 .8 3

II -B 2 3) 0 .09 6 4.95

TTT24 ) 0 .05 4 20 .0 0

H yp oc r ea n ig ri ca ns c el l ul as es O II -C 0. 06 4 1 2.5 7

tu b 0 .06 6 1 7.4 3

G. Okada and K. Kitagawa, unpublished results.

+0.34 å  y.

+0.30 å  /

o /

|+0.26å  ^

t +0.22 å  /

+0.18 " /

+°'140 10 20 30 40

Time (min)

Fig. 10. Changes in Optical Rotation during the Hy-drolysis of Cellopentaose by the Purified Cellulase andafter Base-catalyzed Mutarotation.A drop of concentrated ammoniumhydroxide was addedto the digest at the time indicated by an arrow, and theoptical rotation was followed until 35 min of incubation.Experimental details are described in the text.

optical rotation of G5 increased gradually in apositive direction after addition of the enzyme.However, an immediate increase in the opticalrotation was observed upon addition of am-moniumhydroxide to the reaction mixture.This indicates that the j8-glycosidic linkage of

the substrate was retained.

Kmvalue for CMCThe Kmvalue of the enztyme for CMCwasestimated from Lineweaver-Burk plots. TableII summarizesthe Kmvalues of this enzymeand some purified endocellulases from othermicrobial sources, with the data of the specificactivity values for the individual enzyme.

Substrate specificityHydrolysis products formed from varioussubstrates by the purified enzyme were iden-tified by paper chromatography. The standardreaction mixture (1.0ml) contained 5mMofeach substrate (except for CMC,which wasused at the final concentration of 0.25 percent), 0.05m acetate buffer (pH 4.0) and thepurified enzyme (2.67 units). The reaction mix-tures were incubated at 30°C for 1/6, 1/3, 2/3, 1,2, 4, 24, and 48hr. After incubation, 20^1

aliquots of the hydrolysates were removedatthe intervals indicated above and identified bypaper chromatography. The relative amountsof hydrolysis products on the chromatogramswere estimated from the color intensity and thesize of the spots.The enzyme could not attack either G2 or /?-PNPG even after a prolonged incubation(48hr). When 10 times more enzyme (26.7units) was used, G3 was only slightly hy-

drolyzed to give Gx and G2 after prolongedincubation (24hr). G4 was not attacked com-pletely even after 24 hr of incubation, while G5was hydrolyzed thoroughly after 4hr incu-bation to give products of G1 to G3(G2^G3|>G1). On the other hand, G6 washydrolyzed completely within lOmin of in-

cubation to give the products of Gt to G4(G2>G4>G3|>G1). The enzyme easily hy-

drolyzed CMCin random fashion, howeverGl5 cellooligosaccharides (G2 ~G6) and sub-stituted cellooligosaccharides were practicallynot detected on the chromatograms, under the

1264 G. Okada

conditions used.

DISCUSSION

A highly purified cellulase was separatedfrom a commercialcrude cellulase preparationof A. niger by ion-exchange chromatographywith Amberlite CG-50, followed by the re-petition of gel filtration on Bio-Gel P-150 andSephadex G-50. The purified cellulase prepara-tion was homogeneous on polyacrylamide gelas well as ampholine electrophresis, and wascompletely free from aryl jS-glucosidase.

The p/ value of the purified cellulase (pi3.67) fairly closely resembles to that of an acid-cellulase of A. niger (p/ 3.3).1O) The molecularweight of the purified enzyme (MW31,000) isvery close to that of cellulase II-A (MW30,000) from Trichoderma viride23) whileIkeda et al.1Otll) reported that an acid-cellulasefrom A. niger has a molecular weight of46,000, containing both glucosamine andarabinose. Nocarbohydrate moiety seemedtobe associated with the purified enzyme protein.

No inhibition of the purified enzyme wasobserved with EDTA and the sulfhydryl re-agents tested. This suggests that metals andsulfhydryl groups are not essential for thecellulolytic action of the enzyme, as is the casewith highly purified cellulases from T.viride.23 >24)

During the hydrolysis of CMCby cellulasecomponents, there is usually a characteristicpattern in the relationship between loss ofviscosity and increase in reducing power.Consequently, when the increases in specificfluidity (</>sp) in the enzymic reaction are plot-ted against the increases in reducing power, theslopes of the resulting curves are different. Ithas been reported that the highly purifiedcellulase components from T. viride23a4) andIrpex lacteus25) have different degrees of ran-domness during the hydrolysis of CMC. In

comparison with these data, this purified en-zyme was characterized as a medium-randomtype cellulase.No study has been madeon the anomericconfigurations of the reaction products formed

by A. niger cellulases. On the basis of the

observed mutarotation upon the hydrolysis ofG5 by the purified enzyme, we concluded thatthe anomeric carbon atoms of the reactionproducts retain the /^-configuration. In thisrespect, the enzyme resembles T. viride cel-lulases (II-A, II-B and III),24'26) /. lacteus

cellulases (Ex-1, S-l and F-l)25) and a cellulasepurified from Myrothecium verrucaria.21)

However, it is quite different from the cellulasepreparation fractionated from Cellvibriogilvus.28)

The values of both apparent Kmand specificactivity of the purified enzyme for CMCwerecompared with those obtained with purifiedendocellulases from other microbial sources(Table II). The Kmvalue of this enzyme isclose to that of T. viride cellulase II-A, asshown in Table II. However, the specific ac-tivity of the enzymeis about 4 times higherthan that of T. viride cellulase II-A.Consequently, on the basis of both molecularweight and specific activity for CMCof thesetwo enzymes, it seems probably that the turn-over number of the purified enzyme is about4 times larger than that of T. viride cellulase II-A.23)

The enzyme was incapable of attacking ei-ther G2 or jS-PNPG, but hydrolyzed variousother substrates such as CMCand cellooligo-saccharides (G3~G6) to various extents. G3

was only slightly attacked using a fairly largeamount of enzyme. G4 was hydrolyzed muchmore slowly by the enzyme than G5 or G6. Themodeof action of the enzymeon cellooligosac-charides was similar to that of an endocellulasefrom A. niger reported by Clarke and Stone,7)and Hurst et al.29) In case ofCMC, though theenzymefavored this substrate, Gl5 cellooligo-saccharides (G2~G6) and substituted cel-looligosaccharides migrating faster than G6were not detected on the chromatograms ashydrolysis products even after prolonged in-cubation. This suggests that the enzyme ran-domly attacks CMCto form products havinglonger chain lengths than G6.The mode of actions of the purified enzyme

on CMC and cellooligosaccharides indicates

A Cellulase from A. niger 1265

that this A. niger cellulase is an endoeellulaserCMCaseV

(CMCase)Acknowledgments. The author wishes to express his

thanks to Miss C. Yamada and Mr. T. Tanaka for theirtechnical assistance during this work, and also to AmanoPharmaceutical Co., Ltd. for supplying the starting en-zyme material.

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