Purification and properties of a β-1, 4-glucan 4- glucanohydrolase from Trichoderma viride

A β-1,4-glucan 4-glucanohydrolase or 3.2.1.4. from Trichoderma viride was purified 39-fold over the crude enzyme and completely freed of hydrocellulase, β-1,4-exoglucanase, and aryl-β-D-glucosidase activities by the application of Avicel column adsorption chromatography, alkali swollen cellulose column adsorption chromatography, precipitation with ammonium sulfate, rechromatography on alkali swollen cellulose column, and glass paper electrophoresis at pH 4 in that order. The optimum temperature was 60°C. for the action of this purified enzyme on both amorphous cellulose and CMC for a period of 1 hour incubation. An activation energy of 5.1 Kcal per mole for amorphous cellulose and 6.4 Kcal per mole for CMC between 40° to 60°C. was observed. The optima pH appeared to be 4.2 for amorphous cellulose and 5.0 for CMC. The purified enzyme involved neither activator nor cofactor nor inhibitor in its action, and it was found to be stable at pH 6 to 7 at room temperature for a period of two hours. The sedimentation coefficient of this purified enzyme was 3.5S. It contained no cysteine and only a trace of methionine. From K_m determinations, cellulodextrins with DP above 6 and amorphous cellulose appeared to be the optimum substrates for this purified enzyme, but it did not attack hydrocellulose. A more or less random attack was indicated from the study of mode of action on reduced cellopentaose. Evidence that medium-size chain oligosaccharides might be intermediates in the decomposition of amorphous cellulose by the purified enzyme was obtained. Cellotriose in addition to cellobiose and glucose was found among the principal products from the hydrolysis of both G₅H and amorphous cellulose. The difference in the role of three main cellulase components obtained from T. viride was discussed in connection with the concept of the complete decomposition of cellulose by a multiple enzyme system, and a scheme of the possible mechanism of decomposition of cellulose by the cellulase system of T. viride was proposed.