The Mechanism of Enzymatic Cellulose Degradation

Purification of a Cellulolytic Enzyme from Trichoderma viride Active on Highly Ordered Cellulose

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Abstract

  • 1A cellulolytic enzyme (“C1” enzyme) has been isolated from a commercial cellulase preparation derived from culture filtrates of the fungus Trichoderma viride.
  • 2The 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.
  • 3A yield of 144 mg enzyme was obtained per 100 g commercial cellulase.
  • 4The isolated enzyme was homogeneous in polyacrylamide gel electrophoresis at pH 5.0 and at pH 8.0 by isoelectric focusing in a polyacrylamide gel and also in the ultracentrifuge.
  • 5No enzyme activity towards carboxymethylcellulose could be detected in the purified material under the assay conditions used. Similarly, there was no β-glucosidase activity.
  • 6The purified enzyme was associated with 3.3% 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-HCl.
  • 7Crystalline cellulose (Avicel), phosphoric acid-swollen Avicel and cellotetraose were degraded by the enzyme and in each case the principle reaction product was cellobiose.
  • 8Evidence indicates that the purified enzyme is a β-1,4-glucan cellobiohydrolase.

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