New methodology for the characterization of endoglucanase activity and its application on the Trichoderma longibrachiatum cellulolytic complex

Abstract

The cellulolytic complex of Trichoderma longibrachiatum was separated in nine fractions using FPLC. The avicelase, cellobiase, carboxymethylcellulase, and endoglucanase activities of these fractions were characterized. The endoglucanase activity was measured by a method that allows the determination of the variation in the degree of polymerization of the insoluble celluloses. This method, which is based on the measurement of the reducing power of the insoluble fibers, is proposed as a tool for the identification and characterization of the endoglucanases. Using this technique and H3PO4-swollen cotton as substrate, the kinetic parameters of two proteins that showed high specific endoglucanase activity (pI = 5.25; Mr = 55 kDa; and pI = 4.70; Mr = 70 kDa) were determined. The measurement of the degree of polymerization variation during digestion of Sigmacell gives evidence that the endoglucanase activity is located at the beginning of the reaction.