APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1986, p. 1041-1046 Vol. 51, No. 50099-2240/86/051041-06$02.00/0Copyright © 1986, American Society for Microbiology

Production and Characteristics of Avicel-DisintegratingEndoglucanase from a Protease-Negative Humicola grisea var.

thermoidea MutantSHINSAKU HAYASHIDA* AND KAIGUO MO

Department ofAgricultural Chemistry, Kyushu University, Fukuoka 812, Japan

Received 7 October 1985/Accepted 21 February 1986

Mutational experiments were performed to decrease the protease productivity of Humicola grisea var.thermoidea YH-78 using UV light and N-methyl-N'-nitro-N-nitrosoguanidine. A protease-negative mutant, no.140, exhibited higher endoglucanase activity than the parent strain in mold bran culture at 50°C for 4 days. Theculture extract rapidly disintegrated filter paper but produced a small amount of reducing sugar. About 30%of total endoglucanase activity in the extract was adsorbed onto Avicel. The electrophoretically homogeneouspreparation of Avicel-adsorbable endoglucanase (molecular weight, 128,000) showed intensive filter-paper-disintegrating activity but did not release reducing sugar. The preparation also exhibited a highly synergisticeffect with the cellulase preparation from Trichoderma reesei in the hydrolysis of microcrystalline cellulose.This endoglucanase was observed via scanning electron microscopy to disintegrate Avicel fibrils layer by layerfrom the surface, yielding thin sections with exposed chain ends. A mutant, no. 191, producing higher proteaseactivity and an Avicel-unadsorbable, Avicel-nondisintegrating endoglucanase was isolated. The purifiedenzyme (molecular weight, 63,000) showed no disintegrating activity on filter paper and Avicel and a less syner-gistic effect with the T. reesei cellulase in hydrolyzing microcrystalline cellulose than did the former enzyme.Endoglucanase was therefore divided into two types, Avicel disintegrating and Avicel nondisintegrating.

There are three types of cellulolytic enzymes: cellobiohy-drolase (Avicelase), endoglucanase (carboxymethyl cel-lulase), and 3-glucosidase. However, the mode of action ofthese enzymes on native cellulose has not yet been com-pletely clarified. Generally, endoglucanase does not exten-sively degrade crystalline cellulose but does effectivelydegrade substituted soluble substrates. The role ofendoglucanase in the degradation of native cellulose remainsobscure (2).As reported previously, Humicola grisea var. thermoidea

YH-78 produces a novel type of endoglucanase (11). Thisenzyme adsorbed onto Avicel and showed a highly syner-gistic effect with the cellulase from H. insolens YH-8 inhydrolyzing microcrystalline cellulose. To certify the pres-ence of an endoglucanase that could degrade native cellu-lose, we tried to isolate a mutant that could produce lessprotease and higher amounts of Avicel-adsorbable endo-glucanase than the parent strain by the procedure describedin a previous paper (4) on the high productivity of raw-starch-digesting glucoamylase in a protease-negative,glucosidase-negative mutant of Aspergillus awamori var.Kawachi.

This paper describes the isolation of a protease-negativemutant from H. grisea var. thermoidea YH-78 and theproduction, purification, and properties of Avicel-disintegrat-ing endoglucanase from it. For comparison, results with ahigh-protease mutant are presented.

MATERIALS AND METHODSInduction and isolation of mutants. H. grisea var.

thermoidea YH-78 was used as the parent material. Inductionand isolation of mutants were carried out by the methoddescribed previously (4). The parent culture was grown on aslant of carrot medium (carrot, 2,000 g; chloroamphenicol, 50

* Corresponding author.

mg; agar, 20 g; tap water, 1,000 ml) for 7 days at 50°C. Thespores were collected and suspended in sterile water toproduce a spore suspension of not less than 108 spores per ml.Spore suspension (10 ml) was treated with 10 ml of freshlyprepared N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) ata concentration of 4 mg/ml in 0.1 M acetate buffer, pH 4.5, for30 min in sterilized tubes. The suspension was diluted withsterile water and then centrifuged for 5 min to remove thesupernatant. The precipitated spores were washed twice withsterile water and then inoculated into 100 ml of sterile minimalmedium (glucose, 50 g; sodium nitrate, 3 g; potassiumdihydrogen phosphate, 1 g; iron sulfate, 0.01 g; potassiumchloride, 0.5 g; magnesium sulfate, 0.2 g; deionized water,1,000 ml) and shaken at 50°C for 40 h. The mycelia wereseparated by filtration through sterile glass wool, and thefiltrate was plated onto casein medium for primary screening.Those colonies with larger and smaller halos (clear zones) oncasein medium were transferred to carrot medium slants andfurther incubated at 50°C for 7 days. Several transfers weredone to test the stability of the mutants, and then they wereplated individually onto casein medium for a secondscreening for protease productivity. The colonies withsignificantly large and small halos compared with the parentstrain were selected and cultured on carrot medium slants for7 days at 50°C. Spores of that strain were inoculated into solidwheat bran medium and incubated at 50°C for 4 days. Culturefiltrates were collected and tested for enzyme activity in athird screening. The desired mutants were selected. Theselected mutant was subjected to the combined action ofMNNG-UV light. Spores of the selected mutant weresuspended in sterile water, and 10 ml of spore suspension wasadded to 10 ml of previously prepared MNNG (2 mg/ml)solution, dispensed into sterile petri plates, and thenimmediately exposed to a UV lamp (wavelength, 253.6 nm;distance, 42 cm) at a distance of around 36 cm for 30 min withcontinuous stirring with a sterile magnetic stirrer. The

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1042 HAYASHIDA AND MO

suspension was serially diluted with sterile water and platedonto casein medium for screening. Further screening of theselected mutants was done by the procedure described above.

Culture of mutants. The selected mutants were grown onslants of carrot medium for 7 days at 50°C. The spores werecollected, inoculated into wheat bran medium (wheat bran,25 g; Avicel, 2.5 g; tap water, 25 ml) in 500-ml Erlenmeyerflasks, and incubated at 50°C for 4 days. Enzymes wereextracted by addition of 150 ml of tap water and left to standat 4°C for 5 h. The filtrate was collected and tested forenzyme activity.

Chemicals. CM (carboxymethyl)-cellulose (DS, 0.41) waspurchased from Wako Pure Chemical Industries Pharmaceu-tical Co., Osaka, Japan. Avicel was obtained from AsahiKasei Co., Tokyo, Japan, and used as microcrystallinecellulose. Pulp Floc and filter paper were supplied by NihonMining Industry Co., Tokyo, Japan, and Toyo-roshi Co.,Tokyo, Japan. Filter paper (no. 1) was used as squares (1 by1 cm). Sumyzyme C (cellulase from Trichodermna lreesei) waskindly supplied by Shin-Nihon Kagaku Kogyo Co., Anjyo,Japan.Enzyme assay. (a) Endoglucanase activity. The reaction

mixture, containing 1% CM-cellulose in 5 ml of 0.1 M acetatebuffer, pH 5.0, and 5 ml of appropriately diluted enzymesolution, was shaken on a Monod shaker at 90 strokes permin at 50°C for 30 min. Reducing sugar formed was deter-mined by the micro-Bertrand method (6). One unit ofendoglucanase activity was defined as the amount of enzymereleasing 1 pLmol of reducing sugar from the substrate permin.

(b) Avicelase activity. The reaction mixture, containing 100mg of Avicel in 5 ml of 0.1 M acetate buffer (pH 5.0) and 5 mlof enzyme solution, was shaken on a Monod shaker at 90strokes per min at 50°C for 24 h. One unit of Avicelaseactivity was defined as the amount of enzyme releasing 1pmol of reducing sugar from the substrate per min.

(c) Protease activity. Protease activity was determined byincubating 1 ml of enzyme solution with 5 ml of 1.2%Hammarsten casein in phosphate buffer (pH 7.0) for 10 min.One unit of protease activity was defined as that whichproduced 1 pg of tyrosine per min at 50°C, measured by theFolin procedure (7).

(d) Filter-paper-disintegrating activity. The reaction mix-ture, containing 10 strips of filter paper (1 by 1 cm) in 5 ml of0.1 M acetate buffer (pH 5.0) and 5 ml of enzyme solutionwas shaken on a Monod shaker at 90 strokes per min at 50°C.The time for complete disintegration of filter paper wasobserved.

Adsorbability of endoglucanase activity to Avicel. Enzymesolution (2.0 U/ml) in 5 ml of 0.1 M acetate buffer (pH 5.0)was applied to 1 g of Avicel, followed by standing at 4°C for10 min. After centrifugation, endoglucanase activity in thesupernatant fluid was assayed, and the adsorption rate wascalculated.

Purification of enzymes. Based on the procedures de-scribed above, mutants no. 140 and 191 were cultivated for 4days for enzyme production and the resulting culture ex-

tracts were collected for purification.(i) Step 1. The culture extract was concentrated by pre-

cipitation with ammonium sulfate at a 60% (wt/vol) concen-

tration and kept overnight. The resulting precipitate was

collected by filtration and dissolved in a small volume ofdeionized water. The supernatant was dialyzed by PVA-Hollow Fiber (Kuraray Co., Osaka, Japan). The enzyme

solution was then adjusted to pH 3.0 with 1 N hydrochloricacid and allowed to stand for 48 h. The precipitate was

removed by centrifugation at 10,000 rpm. for 20 min. Thesupernatant was collected, concentrated, and desalted byfiltration through a Sephadex G-50 column (4.5 by 103 cm).The lyophilized preparations were used as crude enzyme.

(ii) Step 2. The concentrated sample from step 1 wasapplied to a DEAE-Sephadex A-50 column (2.5 by 57 cm)previously buffered (pH 5.5) and eluted with a linear gradientfrom 0.05 to 1 M phosphate buffer (pH 5.5) in 500 ml at 4°C.The flow rate was 5 ml/20 min. The endoglucanase activity ofeach fraction (5 ml) was measured before and after adsorp-tion onto Avicel. Fractions which contained endoglucanaseactivity adsorbed onto Avicel were combined, concentrated,desalted by filtration through a Sephadex G-50 column, andfinally lyophilized.

(iii) Step 3. The concentrated sample from step 2 wasapplied to a Sephadex G-100 column (2.7 by 100 cm).Filtration (each fraction, 5 ml) was carried out with deion-ized water at 4°C at a rate of 5 ml/20 min. Those fractionswhich showed the endoglucanase activity adsorbed ontoAvicel were combined and lyophilized.

(iv) Step 4. The concentrated sample from step 3 wasfurther applied to a DEAE-Sephadex A-50 column by theprocedure described above.

(v) Step 5. The concentrated sample from step 4 wasfinally applied to a Sephadex G-200 column (2.7 by 100 cm).Filtration was carried out with deionized water at 4°C at arate of 5 ml/20 min. Endoglucanase fractions which showedadsorbability to Avicel were combined and lyophilized. Thelyophilized sample was designated as purified Avicel-adsorbable endoglucanase.

Hydrolysis of cellulosic substrates. The hydrolysis rate wascalculated from the amount of glucose formed with enzymeas a percentage of that formed by acid hydrolysis (9).

Scanning electron microscopy. Specimens were dehydratedby immersion for 5 min in various concentrations of ethanol,added to 100% isoamyl acetate, and allowed to stand at roomtemperature for 10 min. Then the specimens were trans-ferred to liquid carbon dioxide and dried to the critical pointin a JCPD-5 critical-point drier (Japan Electron OpticusLaboratories Ltd.). A conductive coating of gold-palladiumwas applied in an enscope sputter coater (JEOL Ltd.), andthe specimens were examined in a JSM-25S scanning elec-tron microscope (JEOL) at an accelerating voltage of 25 kV.

General analytical procedures. Protein was determined bythe method of Lowry et al. (7) with crystalline serumalbumin as the standard. Disc electrophoresis in 7.5% poly-acrylamide gel at pH 8.3 in Tris-glycine buffer was carriedout by the method of Davis (1). Molecular weights wereestimated by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (10). Viscometric assay was performed insize 150 Cannon-Fenske viscometers in a water bath at 50°C.

RESULTSSelection of mutants. At first, 287 colonies with larger and

smaller halos on casein medium were primarily selectedfrom about 3,000 colonies and transferred to carrot mediumslants. Several transfers were repeated and then platedindividually onto casein medium. Thirty isolates were sec-ondarily selected on the basis of higher and lower ratios ofthe diameters of the halos to that of the colony on the caseinmedium. These isolates were further tested for their abilitiesto produce protease and cellulase in solid wheat bran me-dium. A mutant, no. 101, exhibiting the lowest protease, thestrongest filter-paper-disintegrating, and the highest Avicel-adsorbable endoglucanase activities was obtained. Mutantno. 191, which increased in protease productivity and

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TABLE 1. Comparison of enzyme activities of culture filtratesfrom mutants and the parent strain"

Mutant Disintegrating Enzyme ativity (U/ml) Adsorbabilityno. activity on Endo- Avicelase on Avicel

filter paper Proteaseguaae (03 %

140 + + + + + 6.4 1.9 3.5 30113 + + 6.4 2.0 3.1 7139 + 10.0 0.8 8.8 5125 + + + 11.0 1.8 2.4 21142 + + + 11.6 1.5 1.5 13Parent + + 18.2 1.2 1.2 11191 - 39.0 0.7 5.3 0

" Mutants were incubated in wheat bran medium at 50°C for 4 days.

showed little filter-paper-disintegrating activity and no

adsorbability of endoglucanase activity to Avicel, was alsoselected. Selected mutant no. 101 was exposed again tocombined MNNG and UV light treatment and then platedonto casein medium. As a result, of about 900 colonies, 85with very small halos on casein medium were isolated andfurther screened. Eleven mutant colonies were selected by a

second screening. At a third screening, mutant no. 140exhibited the highest Avicel-adsorbable endoglucanase, thestrongest filter-paper-disintegrating, and the lowest proteaseactivities. Mutants no. 140 and 191 were maintained in carrotmedium as desired mutants and stored at 4°C. A comparisonof the enzyme productivities of some mutants is summarizedin Table 1. The protease activity of mutant no. 140 was 65%lower, whereas its endoglucanase activity was 58% higherthan that of the parent strain. The culture filtrate of mutantno. 140 exhibited the highest filter-paper-disintegrating ac-

tivity and the highest adsorbability to Avicel among mutants.Enzyme production. Mutants no. 140 and 191 were inocu-

lated onto wheat bran medium and incubated at 50°C.Maximum amounts of endoglucanase and Avicelase were

observed after 4 days. The pHs of culture filtrates increasedslowly from 5.8 to 7.9.

Purification of Avicel-adsorbable endoglucanase. Lyophi-

1.0

, X2c

0.5 8C=

CL3

CO.

JO.050 20 40 60 80 100

FRACTION NUMBER t5mlIFIG. 1. DEAE-Sephadex A-50 column chromatography of the

endoglucanase from mutant no. 140. Symbols: 0 O, A.80;* *, endoglucanase activity before adsorption onto Avicel;0---0, endoglucanase activity after adsorption onto Avicel; -- -,

phosphate concentration. Experimental details are described in thetext.

E

0 20 40 60 80FRACTION NUMBER 15m11

FIG. 2. Sephadex G-200 column chromatography of the endo-glucanase from mutant no. 140. Symbols: 0 O. A2,,8: * *,endoglucanase activity before adsorption onto Avicel; 0---0.

endoglucanase activity after adsorption onto Avicel. Experimentaldetails are described in the text.

lized crude enzyme was dissolved in 0.05 M phosphatebuffer (pH 5.5) and applied to a DEAE-Sephadex A-50column. The chromatographic pattern is shown in Fig. 1.The endoglucanase activity of fractions no. 29 to 60 de-creased significantly after adsorption to Avicel. These frac-tions were combined, concentrated, desalted by filtrationthrough a Sephadex G-50 column, and then applied toSephadex G-100, DEAE-Sephadex A-50, and SephadexG-200 columns. The chromatographic pattern showed one

peak between fractions no. 21 and 38 (Fig. 2). Theendoglucanase activity of these fractions was nearly lostafter adsorption to Avicel. These fractions were combinedand lyophilized. The lyophilized sample was designated as

purified Avicel-adsorbable endoglucanase and kept in an

evacuated desiccator at 4°C. Recovery and specific activityare summarized in Table 2.

For comparison, the Avicel-unadsorbable endoglucanasefrom mutant no. 191 was also purified by the proceduredescribed above. Recovery and specific activity are summa-

rized in Table 3.Homogeneity of purified endoglucanases. The pu ified,

Avicel-adsorbable endoglucanase was homogeneous o discelectrophoresis, and the Avicel-unadsorbable endoglucanasealso showed a single protein band on disc electrophoresis(Fig. 3). Both enzyme preparations showed intensiveendoglucanase activity but no Avicelase activity.

Properties of mutant endoglucanases. (i) Molecular weight.

TABLE 2. Purification of Avicel-adsorbable endoglucanase fiomprotease-negative mutant no. 140

Step Total protein Total activity Sp act YieldStep (mg) (U) (u/mg) (%)

Crude enzyme 2,100 7,245 3.5 100DEAE-Sephadex A-50 540 5,670 10.5 78.3Sephadex G-100 310 3,798 12.3 52.4DEAE-Sephadex A-50 89 1,925 21.8 26.6Sephadex G-200 48 1,369 28.7 18.9

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TABLE 3. Purification of Avicel-unadsorbable endoglucanasefrom mutant no. 191

Step Total protein Total activity Sp act YieldStep (mg) (U) (U/mg) (%)

Crude enzyme 2,400 4,039 1.7 100DEAE-Sephadex A-50 501 2,430 4.9 60.2DEAE-Sephadex A-50 97 981 10.1 24.3Sephadex G-200 28 408 14.4 10.1

Logarithmic plots of reference proteins versus their relativemobilities are shown in Fig. 4. The molecular weight ofAvicel-adsorbable endoglucanase from mutant no. 140 wasestimated to be 128,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and that of Avicel-unadsorbable endoglucanase from mutant no. 191 was esti-mated to be 63,000.

(ii) Thermostability and pH stability. Portions of enzymesolutions (2.0 U/ml) prepared in 0.1 M acetate buffer (pH 5.0)were kept for 10 min at various temperatures. Avicel-adsorbable endoglucanase was stable at 65°C, and Avicel-unadsorbable endoglucanase was stable at 70°C. Both werestable at pH 3.0 to 11.0.

(iii) Effect of pH on adsorption to Avicel. The adsorbabilityof endoglucanase activity to Avicel was tested at variouspHs. The pH for adsorption to Avicel ranged from 4.0 to 8.0.

(iv) Adsorbability of endoglucanase activity to cellulosicsubstrates. Prepared endoglucanase solution with endoglu-canase activity of 2.0 U/ml from mutant no. 140 was 72%adsorbed to Avicel and 70% adsorbed to cellulose powder.

(v) Decreases in the viscosity of CM-cellulose. Bothendoglucanases could rapidly decrease the viscosity of a0.5% solution of CM-cellulose in 0.1 M acetate buffer (pH5.0). Avicel-adsorbable endoglucanase (2.0 IU/ml) caused a76.7% reduction in viscosity within 2 min, and Avicel-unadsorbable endoglucanase (2.1 IU/ml) decreased viscosityby 75.7% within 2 min (Fig. 5).

(vi) Actions on filter paper and Avicel. Avicel-adsorbableendoglucanase completely disintegrated filter paper andAvicel at 50°C within 30 min. However, Avicel-unadsorb-able endoglucanase showed little disintegrating activity on

201-

0 10-.8-.ase 6

= 4-

C 2

10

4

0.2 0.4 0.6

MOBILITY0.8 1.0

FIG. 4. Estimation of the molecular weights of the endoglu-canases by sodium dodecyl sulfate-polyacrylamide gel electropho-resis. Symbols (molecular weights of standard proteins): 1, myo-globin (17,800); 2, ovalbumin (45,000); 3, bovine serum albumin(67,000); 4, gammaglobulin (160,000); A, Avicel-unadsorbableendoglucanase (63,000); B, parent-strain endoglucanase (75,000); C,Avicel-adsorbable endoglucanase (128,000). Experimental detailsare described in the text.

filter paper and Avicel. No reducing sugar was detected inreaction mixtures incubated with both enzymes. The modeof disintegration of Avicel with Avicel-adsorbable endoglu-canase was observed under a scanning electron microscope(Fig. 6). Photo A shows an intact fibril of Avicel. Photo Billustrates the form of an Avicel fibril in stationary incubationat 50°C for 24 h with Avicel-adsorbable endoglucanase. TheAvicel fibrils were cracked, cleaved, and broken into innerlayers, developing many thin-sectioned pieces. Photo Cshows in detail the exposed sections, on which micells withfree chain ends were observed. Thus, cellulose chains be-came more accessible to cellobiohydrolase. Avicel-unadsorbable endoglucanase could only attack the surface ofmicrocrystalline cellulose (Fig. 6D).

(vii) Synergistic effects in hydrolysis of crystalline cellulose.

0

: 20I-

C=)

, 40

60

I--

I.,

V 80a-

FIG. 3. Gel disc electrophoretic patterns of Avicel-adsorbableendoglucanase from mutant no. 140 (A) and Avicel-unadsorbableendoglucanase from mutant no. 191 (B). Polyacrylamide (7.5%) gelcolumn, pH 8.3. About 10 ,ug of endoglucanase preparations was

used, and 2 mA per column (0.5 by 8 cm) was applied for 120 min.Staining was done with 0.005% Coomassie brilliant blue R-250.

0 5 10 15 20 25 30

TIME IN MINUTESFIG. 5. The time courses of the decreases in viscosity of CM-

cellulose during hydrolysis by purified Avicel-adsorbableendoglucanase (0) and Avicel-unadsorbable endoglucanase (0).The substrate was dissolved at 0.5% final concentration in 0.1 Macetate buffer, pH 5.0. Viscosity was measured at 50°C.

gn. 4-1- =0

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FIG. 6. Electron micrograph of Avicel incubated with endoglucanases. (A) An intact fibril of Avicel; (B) an Avicel fibril incubated withAvicel-adsorbable endoglucanase at 50'C for 24 h; (C) the detail of fibrils disintegrated with Avicel-adsorbable endoglucanase; (D) an Avicelfibril incubated with Avicel-unadsorbable endoglucanase at 50'C for 24 h. Bars, 10 jim. The reaction mixture, containing 200 mg of Avicel,5 ml of acetate buffer (pH 5.0), and 5 ml of Avicel-adsorbable endoglucanase solution (2.0 U/mil) or Avicel-unadsorbable endoglucanase (2.1U/ml), was incubated at 50°C in a stationary state and then centrifuged. The precipitates were collected, washed, and dried in a critical-pointdryer (JEOL) and used as samples for scanning electron microscopy.

Avicel-adsorbable endoglucanase from mutant no. 140 ex-hibited a highly synergistic effect with a commercial prepa-ration of T. reesei cellulase (Sumyzyme C; Shin-NihonKagaku Kogyo Co.) in the hydrolysis of crystalline cellu-lose. Addition of Avicel-adsorbable endoglucanase led to

acceleration of the hydrolysis rate by more than twofold(Fig. 7). Prepared cellulase from T. reesei hydrolyzed Avicel80% as glucose in a static reaction; however, the combina-tion of this cellulase and Avicel-adsorbable endoglucanasehydrolyzed it 93% under the same conditions, the hydrolysis

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60 -

20

20

0 20 40 60 80 100TIME IN HOURS

FIG. 7. Synergistic effects of Avicel-adsorbable endoglucanaseand Avicel-unadsorbable endoglucanase with T. reesei cellulase inthe hydrolysis of Avicel. Symbols: 0, mixed preparation of Avicel-adsorbable endoglucanase and Sumyzyme C; 0, mixed preparationof Avicel-unadsorbable endoglucanase and Sumyzyme C; 0,

Sumyzyme C; A, Avicel-adsorbable endoglucanase; A, Avicel-unadsorbable endoglucanase. The reaction mixture, containing 100mg of substrate in 5 ml of buffer and 5 ml of a mixed enzymesolution, was incubated at 50°C in a stationary state. Sumyzyme C(3 ml; 3.9 U/ml) plus Avicel-adsorbable endoglucanase (2 ml; 2.5U/ml) or Avicel-unadsorbable endoglucanase (2 ml; 2.5 U/ml) wereused as the mixed enzyme solution. The hydrolysis curves obtainedwere compared with that of the control (5 ml of Sumyzyme C, 5 mlof Avicel-adsorbable endoglucanase or Avicel-unadsorbable endo-glucanase).

rate of the latter was larger than before, and Avicel-unadsorbable endoglucanase exhibited a less synergisticeffect than did Avicel-adsorbable endoglucanase.

DISCUSSION

Reese et al. (8) proposed a two-step process, with an initialactivation step followed by the hydrolyzation step, as themechanism of enzymatic hydrolysis of cellulose. Activationwas caused by a nonhydrolytic enzyme called C1. Hydroly-sis of the activated cellulose was then effected by hydrolyticCx enzyme. However, the existence of a nonhydrolyticenzyme has not been confirmed. Eriksson (3) proposed thata nonhydrolytic step involving an oxidative mechanismmight proceed in the first reaction, thus creating a substratesusceptible to hydrolysis. This paper proposes the existenceof two types of endoglucanase, a novel type of Avicel-adsorbable endoglucanase that could intensively disintegrateAvicel and the ordinary type of Avicel-unadsorbable,nondisintegrating endoglucanase.The Avicel-adsorbable endoglucanase (molecular weight,

128,000) from a protease-negative mutant disintegrated notonly the surface of Avicel fibrils but the inner part of fibrils,removing thin sections layer by layer and newly exposingglucan chains, although no reducing sugar was detected inthe reaction mixture. The thin sections consisted of micellswith free chain ends, from which cellobiohydrolase may split

off cellobiose. This process might be the "initial activationstep" of Reese's hypothesis. In this way, the Avicel-disintegrating endoglucanase and cellobiohydrolase actedsynergistically, forming reducing sugar more rapidly. Thistype of endoglucanase played an important role in the initialstep of hydrolysis on native cellulose and made the fibrilsmore accessible to cellobiohydrolase.On the other hand, Avicel-nondisintegrating endoglu-

canase (molecular weight, 63,000) from a protease-positivemutant degraded glucan chains only on the surface ofcellulose fibrils, not leading to removal of the inner layer.The endoglucanases could be designated as Avicel-disintegrating endoglucanase C1 and Avicel-nondisinte-grating endoglucanase C,, according to Reese's hypothesis.As reported previously, fungal o-amylase and glucoamylasewere both divided into raw-starch-digesting and raw-starch-nondigesting enzymes. The raw-starch-digesting glucoam-ylase involved a specific structure of a "raw starch-affinitysite" (5) essential for digestion of raw starch, but theraw-starch-nondigesting glucoamylase lost the affinity siteby limited proteolysis, although both enzymes hydrolyzedgelatinized and soluble starch in the same manner. Theexistence of two types of endoglucanase was thus under-stood as in the case of the multiplicity of fungal amylases.Details of the properties of the novel type of Avicel-disintegrating endoglucanase C1 will be reported subsequent-ly.

LITERATURE CITED1. Davis, B. J. 1964. Disc electrophoresis-Il. Method and appli-

cation to human serum proteins. Ann. N.Y. Acad. Sci. 121:404-427.

2. Enari, T. M. 1983. Microbial cellulases, p. 188-199. In W. M.Fogarty (ed.), Microbial enzymes and biotechnology. AppliedScience Publishers, London.

3. Eriksson, K.-E. 1975. An oxidative mechanism in the hydrolysisof cellulose, p. 263. In M. Bailey, T.-M. Enari, and M. Linko(ed.), Symposium on enzymatic hydrolysis of cellulose. SITRA,Helsinki.

4. Hayashida, S., and P. Q. Flor. 1981. Raw starch-digestiveglucoamylase productivity of protease-less mutant from Asper-gillus awamori var. Kawachi. Agric. Biol. Chem. 45:2675-2681.

5. Hayashida, S., S. Kunizaki, M. Nakao, and P. Q. Flor. 1982.Evidence for raw starch-affinity site on Aspergillus awamoriglucoamylase I. Agric. Biol. Chem. 46:83-89.

6. Klein, G. 1932. Methoden zur quantitativen Zuckerbestimmungnach Reduktionsverfahren. Handbuch der Pflanzenanalyse II,spezielle Analyse, p. 786. Wien Verlag von Julius Springer,Vienna.

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