MICROCRYSTALLINE CELLULOSE-HYDROLYZING CELLULASE (ENDO-CELLULASE) FROM TRICHODERMA REESEI CDU-11

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1 J. Gen. App!. Microbiol., 37, (1991) MICROCRYSTALLINE CELLULOSE-HYDROLYZING CELLULASE (ENDO-CELLULASE) FROM TRICHODERMA REESEI CDU-11 KIHACHIRO OGAWA,* DAISUKE TOYAMA AND NOBORU FUJI! Applied Microbiology Laboratory, Department of Agricultural Chemistry, Faculty of Agriculture, Miyazaki University, Miyazaki , Japan (Received February 18, 1991) A microcrystalline cellulose (Avicel)-hydrolyzing cellulase (an endocellulase) was isolated from a preparation of Trichoderma reesei CDU-11 and purified successively by DEAE-Sepharose CL 6B and Concanavalin A-Sepharose column chromatography, and preparative electrophoresis and on Sephadex G-100 gel filtration. The cellulase preparation showed a single protein band on SDS-PAGE slab and gel disc electrophoresis. Its molecular weight was estimated to be 64,000 by Sephadex G-100 gel filtration and 67,000 by SDS slab electrophoresis. The isoelectric point was at ph 4.75 on polyacrylamide gel electric focusing. This cellulase hydrolyzed more specifically water insoluble microcrystalline cellulose such as Avicel rather than water soluble cellulose such as carboxymethyl-cellulose. It produced glucose, cellobiose and cellotriose from cellotetraose and cellohexaose, and it produced a significant amount of glucose and cellobiose from Avicel. Based on these results, it appears that the cellulase should be regarded as an endo-type cellulase, although it hydrolyzes Avicel relatively easier than CM-cellulose. We obtained no evidence that this cellulase is an exo-type one named cellobiohydrolase (CBH) in the systematic nomenclature. Although there has been much investigation of the enzymatic degradation of cellulose (S, 8, 9,11,12,14,16,19), the mode of cellulase action on it is still incompletely understood; ambiguous nomenclature has formally been employed. At present, a mutant strain, Trichoderma reesei CDU-11 is the best available source of active cellulase. It was known that a cellulase preparation from Trichoderma reesei CDU-11 solubilizes highly ordered cellulose such as Avicel (1). The Faculty * Address reprint of Agriculture, requests to: Dr. Kihachiro Ogawa, Department of Agricultural Chemistry, Miyazaki University, Miyazaki , Japan. 249

2 250 OGAWA, TOYAMA, and FUJII VOL. 37 cellulolytic enzyme system produced by the fungus has extensively been fractionated by several researchers (2, 4,18), and it is believed that at least three different types of enzymes are involved in the complete breakdown of native cellulose to glucose. Two of the enzymes were named 1,4-$-D-glucan glucanohydrolase (EC ) (cellulase, endo-l,4-,0-d-glucanase) and 1,4-/3-D-glucan cellobiohydrolase (EC ) (exo-cellobiohydrolase, cellulase 1,4-$-cellobiosidase or 1,4-18-cellobiohydrolase, CBH). The third is j3-glucosidase (EC ). The CBH is defined as an enzyme which hydrolyzes 1,4-,3-D-glucosidic linkages in cellulose and cellotetraose, releasing cellobiose from the non-reducing ends of the chains (20). The present paper describes the purification and the hydrolysis of a cellulase component capable of decomposing a microcrystalline cellulose. This cellulase appears to have the enzymatic properties of CBH. MATERIALS AND METHODS Enzyme source. A crude enzyme preparation obtained from a mutant strain, T. reesei CDU-11, was provided by Kyowa Hakko Co., Ltd. Substrates. Avicel PH-101 was provided by Asahi Kasei Co., Ltd. Cellooligosaccharides were prepared from Avicel PH-101 by the method previously described (13). Determination of enzyme activity. Carboxymethyl-cellulose (CM-cellulose) and Avicel-saccharifying activities were assayed by the method previously described (13). The reducing sugar was measured by the Somogyi-Nelson method (10,15). The f3-glucosidase was assayed in the same way as with CM-cellulosesaccharifying using 1% salicin (Wako Pure Chem. Ind. Ltd.) solution at 45 C. One unit of enzyme activity in these assays was defined an amount of enzyme which produced l,amol of reducing sugar as glucose per min. Electrophoresis. Electrophoresis under SDS denaturing conditions was done using Laemmli's technique (6). Determination of molecular weight. The molecular weight of the purified enzyme was measured by SDS PAGE by Laemmli's technique (6), calibration being made with standard proteins (Bio-Rad Lab.) such as phosphorylase b (MW 97,400), bovine serum albumin (MW 66,200), ovalbumin (MW 45,000), carbonic anhydrase (MW 31,000), soybean trypsin inhibitor (MW 21,500) and lysozyme (MW 14,000). The molecular weight of the enzyme was also estimated by a gel filtration method using a Sephadex G-100 column (3). Paper chromatography. Paper chromatography was carried out on Toyo filter paper No. 50 in the solvent system of n-butanol : acetic acid : water (4: 1:1) by a multiple ascending method (3 times), and spots were detected by spraying with acetone-silver nitrate reagents (17). Determination of the isoelectric point. The isoelectric point of the enzyme was determined by polyacrylamide gel isoelectric focusing (IFF) method reported by the Instruction Manual (Model 111 Mini IFF Cell, Bio-Rad Lab.).

3 1991 Endo-cellulase of Trichoderma reesei 251 Determination of protein. Protein was determined by the method of Lowry et al. (7) using crystalline bovine serum albumin as a standard. Assay for cellooligosaccharides. One milliliter of enzyme solution in 0.1 M acetate buffer, ph 5.0 containing 0.5% cellooligosaccharides was incubated at 45 C for 30 min and then samples of the reaction mixture were withdrawn. The sugar in the hydrolyzates were analyzed by high-performance liquid chromatography (HPLC). A 10,01 sample of the supernatant was applied to a Shimadzu HPLC instrument (Shimadzu corporation) equipped with a SHIM-Pack SCR-101-C column (7.9 m/m X 30 cm) equilibrated with a solvent system of water. Assay for Avicel. One milliliter of enzyme solution in 0.1 M acetate buffer containing 5% Avicel suspension was incubated at 45 C for 6-48 h with 0.02% sodium azide. Portions of the reaction mixture were withdrawn at regular intervals. The supernatants of these hydrolyzates were analyzed by HPLC and paper chromatography. Assay for CM cellulose. A reaction mixture composed of 18 ml of a 1 % CM-cellulose solution and 2 ml of enzyme solution was incubated in an Ostwald viscometer (No. 5) at ph 5.0 and at 30 C for 10 to 40 min. RESULTS AND DISCUSSION Purification of enzyme One hundred milliliters of the enzyme solution (2,044 mg) were loaded on a column (2.5 X 30 cm) of DEAE-Sepharose CL 6B equilibrated with distilled water. After washing the column with 100 ml of water, the column was eluted with a linear gradient of ionic strength from 0 to 0.5 M acetate buffer, ph 5.0, with a total volume of 1,000 ml. Cellulases having Avicel-saccharifying activity were fractionated into P 1 to P4 fractions with different rates of cellulase activities toward Avicel and CM-cellulose (Fig. 1). P4 (fraction Nos , ml) was then precipitated with ethanol chilled at -3 C. The precipitated enzyme protein was dissolved in 10 ml of 0.1 M acetate buffer, ph 5.0, and applied to a column (2.5 X 30 cm) of DEAE-Sepharose CL 6B equilibrated with the same buffer. The column was first washed with 500 ml of the same buffer, and eluted with a linear ph gradient (440 ml of ph 5.0 and 440 ml of ph 3.8 of 0.1 M same buffer). A cellulase solution obtained from fractions 48 to 55 seemed almost pure in view of the elution pattern even at this step of purification. This fraction was concentrated with a collodion bag and dialyzed against distilled water. Forty milliliters of the solution was subsequently purified by preparative electrophoresis using Rotofor (Bio-Rad Lab.). The eluates (Nos. 6-15) showed a single protein band by SDS-PAGE electrophoresis. The fractions were collected and freeze-dried. The powder was then dissolved in 4 ml of a 0.1 M acetate buffer, ph 6.0 which contained 1 M NaCI, 1 mm MnCl2, and 1 mm CaC12. For further purification, the cellulase solution was applied to a column (1.5 X 26.5 cm) of Concanavalin A-Sepharose equilibrated with the same buffer, and eluted at a 20 ml/h flow rate. Each eluate (10 ml) was collected

4 252 OGAWA, TOYAMA, and FUJII VOL. 37 traction no. l lu mi Fig. 1. Fractionation of cellulases from a T reesei CDU-11 preparation on a column (2.5 X 30 cm) of DEAE-Sepharose CL 6B. Each fraction (10 ml) was collected at a 20 ml/h flow rate. Fraction P4 shown at the top of the peak ( ) was pooled., CM-cellulose saccharifying activity; 0, Avicel saccharifying activity; 0, /3- glucosidase activity,...., protein; S, sample; D, washing with distilled water; G, start of gradient elution. Fig. 2. Purification of P4 on column (0.9 X 17.5 cm) of Concanavalin A- Sepharose 4B. The column was equilibrated with 0.1 M acetate buffer, ph 6.0 which contained 1 M NaCI, 1 mm MnCl2, 1 mm CaC12. The enzyme solution (305.2) which had been equilibrated by dialysis in a collodion bag against the same equilibrating buffer, was applied to the column in a cold room and then washed with the same buffer solution. After the 32nd tube, the column was eluted with a 0.5% (w/v) methyl-a-dmannopyranoside in the above buffer. The symbols are the same as in Fig. 1.

5 1991 Endo-cellulase of Trichoderma reesei 253

6 254 OGAWA, TOYAMA, and FUJII VOL. 37 and assayed for enzyme activities. After the 32nd tube, the column was eluted with a 0.5% (w/v) methyl-a-d-mannopyranoside in the same buffer, Q-glucosidase activity was not detected in these fractions (Fig. 2). Combined fractions (Nos , 60 ml) were concentrated in a collodion bag, and 1 ml thereof was applied to a Sephadex G-100 column (1.4 x 60.5 cm) which had been equilibrated with 0.1 M acetate buffer, ph 5.0. The column was washed with same buffer. Eluates (Nos , 30 ml, P4-2) were used as the purified enzyme solution after dialyzing with distilled water (P4-2-1). The enzyme preparation (P4-2-1) showed no /3-glucosidase activity. This fraction is a cellulase component having the highest Avicelsaccharifying activity per unit of CM-cellulose-saccharifying activity of all cellulase components of T, reesei CDU-11. Fig. 3. Molecular weight measurement of purified cellulase (P4-2-1) by SDS- PAGE slab electrophoresis. Electrophoresis was done using 22.2% acrylamide gel and 0.2% SDS. The gel was stained for protein with Coomassie Brilliant Blue R-250. E, P4-2-1; St, standard; Lane 1, rabbit muscle phosphorilase b (97,400); Lane 2, bovine serum albumin (BSA, 66,200); Lane 3, hen egg white ovalbumin (45,000); Lane 4, bovine carbonic anhydrase (31,000). Fig. 4. Molecular weight of purified cellulase (P4-2-1) determined with a Sephadex G-100 column (1.4>< 60.5 cm) eluted with 0.1 M acetate buffer, ph 5.0. A, cytochrome c (horse heart, 12,400); B, carbonic anhydrase (bovine erythrocytes, 29,000); C, albumin (bovine serum, 66,000); E, purified Avicelase; D, alcohol dehydrogenase (yeast, 150,000).

7 1991 Endo-cellulase of Trichoderma reesei 255 Homogeneity of purified cellulase (P4-2-1) An adequate amount of P4-2-1 was subjected to SDS-PAGE slab electrophoresis (Fig. 3). The cellulase preparation was evidently electrophoretically homogeneous. The purification steps for the enzyme are summarized in Table 1. As shown in Table 1, the purified cellulase P4-2-1 had low specific activity toward crystalline cellulose, Avicel. The drop in specific activity of the cellulase component may be due to the loss of the synergistic effects with other cellulases, such as the one with higher CM-cellulose-hydrolyzing activity and /3-glucosidase. Molecular weight of the cellulase The molecular weight of cellulase was estimated chromatographically by the Sephadex G-100 column (1.4>< 96 cm). As shown in Fig. 4, the molecular weight of the cellulase was estimated to be 64,000. Almost the same molecular weight, 67,000, was also obtained by SDS-PAGE electrophoresis. Isoelectric point The ioelectric point of the cellulase was estimated to be ph Hydrolysis of cellooligosaccharides The HPLC elution patterns of enzymatic hydrolyzates of cellotetraose and cellohexaose with the purified cellulase are shown in Fig. 5 and Table 2. This cellulase hydrolyzed these cellooligosaccharides, but did not attack cellobiose, even in 6 h of incubation. The products of hydrolysis from cellotetraose and cellohexaose were glucose, cellobiose and cellotriose. It is noteworthy that a significant amount of glucose using these with the cellooligosaccharides with even-number glucose units was produced in addition to cellobiose and cellotriose. However, no glycosyltransferase activity of this cellulase was detected in a mixture consisting of 2% cellobiose and 1 % glucose, even after 24 h of incubation (Fig. 6). Hydrolysis of crystalline cellulose, Avicel The hydrolysis products from Avicel with the purified cellulase were identified by HPLC. As shown in Fig. 7 and Table 3, cellobiose and glucose were the main products in the hydrolyzate. The ratio of glucose to cellobiose during hydrolysis Table 2. Relative product ratios of hydrolyzates from cellotetraose and cellohexaose by purified cellulase.

8 256 OGAWA, TOYAMA, and FUJII VOL. 37 Fig. 5. HPLC elution pattern of cellooligosaccharide hydrolyzates by purified cellulase (P4-2-1). The details are given in the text. Gl, glucose; G2, cellobiose; G3, cellotriose; G4, cellotetraose; G6, cellohexaose. Fig. 6. Nonexistence of transglycosylase action of purified cellulase (P4-2-1). The details are shown in the text. G1, glucose; G2, cellobiose; G3, cellotriose. increased with the incubation period. Since the reaction rate of purified cellulase to highly ordered cellulose such as Avicel is very slow, it is difficult to detect the intermediary products. So learn the mode of action of this cellulase, cellooligosaccharides seemed to be a desirable substrate rather than highly ordered cellulose. From these results, it appears that this cellulase essentially attacked the cellulosic substrates randomly, and should indicate an endo-l,4-$-glucanase (endo-type cellulase) according to the definition of cellulase (20). Table 3. Relative product ratios of hydrolyzates from Avicel by purified cellulase.

9 1991 Endo-cellulase of Trichoderma reesei 257 rig. I. rig, a. Fig. 7. HPLC elution pattern of Avicel hydrolyzate by purified cellulase (P4-2-1). The details are shown in the text. G1, glucose; G2, cellobiose; G3, cellotriose. Fig. 8. Relationship between fluidity and CM-cellulose by purified cellulase (P4-2-1). The details are shown in the text. reducing sugar during the hydrolysis of Hydrolysis of CM cellulose The random hydrolysis of substrates by this cellulase was also shown by the relationship between the decrease in viscosity and the increase in the reducing sugars from CM-cellulose. The purified cellulases have a straight-line slope (l/'/ r reducing sugar) with a roughly 45 angle, as shown in Fig. 8. This suggests that the cellulase should be an endo-1s-1,4-glucanase. If CMC is hydrolyzed by a cellulase in an exo fashion, the slope against the abscissa must be smaller angle, since the viscosity drop of CM-cellulose would be very small compared with the simultaneous production of reducing sugar. In this sense, the cellulase obtained in the present study should be regarded as an endo type although it appeared to be a kind of CBH in view of its substrate specificity; its capacity to hydrolyze Avicel was relatively much larger than that of CM-cellulose. In addition, at an early stage of the hydrolysis, the hydrolysis products from CM-cellulose by the purified cellulase were also identified by paper-chromatography as a mixture of glucose, cellobiose and even cellotriose. These results may reinforce the idea that the mode of action of this enzyme toward CM-cellulose is of a typical endo-type. It is concluded in view of the present study that the CBH from the fungus Trichoderma reported by Wood (18) and Berghem et al. (4) may be cellulases in the same category as the cellulase obtained in the present work. Recently, it has been

10 258 OGAWA, TOYAMA, and FUJII VOL. 37 suggested that a cellulase which was derived from a mesophilic fungus, strain Y-94, and readily hydrolyzed Avicel should be classified as a specific endo-glucanase rather than an exo-glucanase (21). We suggest that a cellulase purified by us from T. reesei, which appears so far to be a CBH according to the nomenclature used, should belong to endo-$-1,4- glucanase. According to the nomenclatural definition, CBH must produce only cellobiose in the hydrolyzate of cellulose and cellotetraose. However, the CBH reported by Wood (18) and Berghem et al. (4), always produced considerable glucose in addition to cellobiose. Previously (13) we showed that Avicel-saccharifying cellulase hydrolyzes Avicel 33.7 to 66.5 times more readily than other CM-cellulose cellulases do. These Avicel-saccharifying cellulases also produced considerable glucose and cellobiose from cellotetraose and even cellotriose from cellohexaose. The results also suggested that all cellulases derived from T viride attacked the cellulose substrates randomly. Since we could find out no other cellulase fraction which hydrolyzes Avicel relatively much more easily than the CM-cellulose from T. reesei, we concluded that there is no such cellulases as reported by Wood and Berghem et al. REFERENCES 1) Ado, Y., Production of cellulase by Trichoderma reesei. Biosci. Ind., 47, (1989). 2) Andreotti, R. E., Mandels, M., and Roche, C., Effect of some fermentation variables on growth and cellulase production by Trichoderma reesei QM 9414, proc. bioconversion symp., IIT Delhi, (1977). 3) Andrews, P., Estimation of the molecular weight of proteins by Sepnadex gel filtration. Biochem. J., 91, (1964). 4) Berghem, L. E. and Pettersson, L. G., The mechanism of enzymatic cellulose degradation- Purification of cellulolytic enzyme from Trichoderma viride active on highly ordered cellulose. Eur. J. Biochem., 37, (1973). 5) Berghem, L. E. R., Pettersson, L. G., and Axio-Fredriksson. The mechanism of enzymatic cellulose degradation-characterization and enzymat~:- properties of a js-1,4-glucan cellobiohydrolase from Trichoderma viride. Eur. J. Biochem., 53, (1975). 6) Laemmli, U. K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, (1970). 7) Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J., Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193, (1951). 8) Murao, S., Sakamoto, R., and Arai, M., Purification of two Avicelase from Aspergillus aculeatus No. F-50. Agric. Biol. Chem., 49, (1985). 9) Nakamura, K. and Kitamura, K., Purification and some properties of a cellulase active on crystalline cellulose from Cellulomonus uda. J. Ferment. Technol., 61, (1983). 10) Nelson, N., A photometric adaptation of the Somogyi method for the determination of glucose. J. Biol. Chem., 153, (1952). 11) Okada, G., Nisizawa, K., and Suzuki, H., Cellulase component from Trichoderma viride. J. Biochem., 63, (1968). 12) Ogawa, K., Toyama, H., and Toyama, N., Native cellulose hydrolyzing cellulase of Trichoderma reesei. J. Ferment. Technol., 60, (1982). 13) Ogawa, K., Some properties of purified cellulases from Trichoderma viride. Bull. Fac. Agric.,

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