The Mechanism of Enzymatic Cellulose Degradation

1. A β‐glucosidase (cellobiase) has been isolated from a commercial cellulase preparation derived from culture filtrates of the fungus Trichoderma viride. 2. The crude material was pre‐fractionated by chromatography on Bio‐Gel P‐10 and by DEAE‐Sephadex chromatography as previously described [1]. Further fractionation methods utilized in the present paper included ammonium sulfate precipitation, SE‐Sephadex chromatography, isoelectiic focusing, and chromatography on Bio‐Gel P‐60. 3. A yield of 53 mg enzyme was obtained per 100 g commercial cellulase. The specific activity of the purified enzyme represented an approximately 60‐fold increase from that of the commercial product. 4. The isolated enzyme was homogeneous in polyacrylamide gel electrophoresis at pH 8.0 by isoelectric focusing in a polyacrylamide gel and also in free zone electrophoresis. 5. No enzyme activity towards Avicel (microcrystalline cellulose) or towards carboxymethyl‐cellulose could be detected in the purified material under the assay conditions used. 6. A molecular weight of 47000 was determined for the enzyme by dodecylsulfate‐polyacrylamide gel electrophoresis. The same molecular weight value was obtained by chromatography of the native enzyme on Bio‐Gel P‐100 and also by chromatography of the reduced and alkylated enzyme on Sepharose 6B in 6 M guanidine‐HCl. 7. The amino‐acid analysis showed that the enzyme was rich in aromatic (9.5%) and acidic (19.0%) amino acids. The half‐cystine content was 6 residues per molecule indicating 3 disulfide bridges, since no free sulfhydryl group was detectable. The purified enzyme contained no carbohydrate (as hexose). 8. The enzyme was isoelectric at pH 5.74 (10 °C) and the electrophoretic mobility was determined to be 1.49 × 10−5 cm2 s−l V− at pH 8.0. 9. The Km‐values for the enzyme towards the substrates p‐ nitrophenyl‐β‐d‐glucoside, cellobiose, and cellotetraose were found to be 0.28, 1.5, and 0.35 mM, respectively.