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1.A cellulolytic enzyme ("C," enzyme) has been isolated from a commercial cellulase preparation derived from culture filtrates of the fungus Trichoderma viride.2. The purification method is a four-step procedure including chromatography on Bio-Gel P-10, DEAE-Sephadex chromatography, isoelectric focusing and chromatography on Bio-Gel P-60.3. A yield of 144 mg enzyme was obtained per 100 g commercial cellulase. 4. The isolated enzyme was homogeneous in polyacrylamide gel electrophoresis at pH 5.0 and a t pH 8.0 by isoelectric focusing in a polyacrylamide gel and also in the ultracentrifuge. 5.No enzyme activity towards carboxymethylcellulose could be detected in the purified material under the assay conditions used. Similarly, there was no p-glucosidase activity.6. The purified enzyme was associated with 3.301, carbohydrate and is assumed to be a glycoprotein. The enzyme was isoelectric at pH 3.79 (10 "C). A molecular weight of 46000 was determined by chromatography of the reduced and alkylated enzyme on a calibrated column of Sepharose 6B in 6 M guanidine-HC1. 7.Crystalline cellulose (Avicel), phosphoric acid-swollen Avicel and cellotetraose were degraded by the enzyme and in each case the principle reaction product was cellobiose.8. Evidence indicates that the purified enzyme is a /?-I,.l-glucan cellobiohydrolase.

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1. The cellulolytic enzyme (p-1 ,4-glucan cellobiohydrolase) isolated from culture filtrates of the fungus Trichoderma viride as described previously by us was further characterized as regards chemical, physico-chemical and enzymatic properties.2. The amino-acid composition revealed a high content of acidic amino acids (21.0%) in contrast to a low content of basic amino acids (5.5 %). The half-cystine content was 16 residues per molecule indicating the presence of 8 disulfide bridges, since no free sulfhydryl group was detectable. Alanine was found to be the N-terminal amino acid. 3. The enzyme contained 9.2 %, carbohydrate, which is most probably covalently bound to the protein. The constituent carbohydrates were identified as mannose, glucose and galactose.4. A molecular weight of 41 800 was determined by sedimentation equilibrium analysis. The data from the amino-acid and the carbohydrate analyses indicated a molecular weight of about 42 000.5. The pH-optimum for the enzyme was about 4.8 and thermostability studies showed that the enzyme was completely denatured after 3-min incubation at 78 "C.6. Experimental results demonstrated the enzyme to be a p-1,4-glucan cellobiohydrolase.7. The enzyme was capable of about 80 % degradation of microcrystalline cellulose within 72 h, if the reaction products (mainly cellobiose) were continuously removed by ultrafiltration. The rate of degradation of microcrystalline cellulose by the enzyme could be enhanced by addition of P-glucosidase. The activity of the enzyme was also increased by addition of endo-glucanase.

The sequence of the approx. 490 amino acid residues of the main 1,4-&&1can CellobiohydroI~e (CBH I) (EC 3.2.1.91) from culture filtrates of the fungus Trichoder~ reesei QM 9414 has been establIshed by automatic liquid phase Edman degradation. Peptides obtained by chemical and enzymatic cleavage of the reduced and S-carboxymethylated protein were isolated by a combination of gel filtration and highperformance liquid chromatography. The amino-terminus of the single polypeptide chain is blocked by a pyroglutamyl residue. Most of the neutral carbohydrate present in the glycoprotein is bound within a short region near the carboxyl-terminus. Three attachment sites of glucosamine residues have also been established. Cellulase CeliobiohydrokseTrichoderma reesei Amino acid sequence