The role of carboxyl groups on the chitosanase and CMCase activity of a bifunctional enzyme purified from a commercial cellulase with EDC modification

The carboxyl groups of the bifunctional cellulase–chitosanase (CCBE), purified from a commercial cellulase prepared from Trichoderma viride were modified using the water-soluble carbodiimide 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide (EDC). The EDC modified CCBE lost 80–90% of its chitosnase activity and 20% of its carboxylmethyl cellulase (CMCase) activity; meanwhile, its conformation changed slightly, which altered the substrate binding affinity to chitosan, without affecting its binding to CMC. However, the modification did not alter the structure integrity. The dynamic analysis of modification indicated that the CCBE possessed two carboxylates essential for its chitosanase activity and one carboxyl group for its CMCase activity. One of the two carboxylates involved in chitosanase activity was deduced to be the proton donator, and the other may function for substrate recognition, while the only catalytic carboxyl group for CMCase activity probably also acted as a proton donator.