Purification and characterization of hemicellulolytic and cellulolytic enzymes and their interaction on bleached wood fiber

Considerable work is needed to improve our understanding of enzyme behavior under industrial treatment conditions. Several studies were performed to isolate and characterize hydrolytic enzymes to study their behavior on bleached wood fiber. Methods for purifying endoxylanases were reviewed, and problems with enzyme assay and purification techniques were identified. From the review, a simple method for purifying large quantities of xylanase to electrophoretic homogeneity was developed. In addition, a small T. reesei endoglucanase, EG V, was also purified and characterized for the first time. EG V showed evidence of a substrate binding domain, which makes it unique from other small endoglucanases that have been isolated.; The effect of fiber type, fiber history, and ionic strength on cellulase adsorption was investigated. Pure T. reesei exoglucanase (CBH I) and endoglucanase (EG II) enzymes showed increased adsorption onto softwood fibers compared to hardwood fibers. The adsorption of CBH I and EG II onto bleached kraft fiber was substantially decreased by drying of the fiber. High ionic strength increased the extent of adsorption of EG II and decreased the maximum adsorption of CBH I.; Adsorption of some T. reesei hemicellulases was also investigated. The presence of a cellulose binding domain (CBD) was the major factor which determined the binding of hemicellulases onto wood fiber. The binding of a mannanase with a CBD was greater than 90% of the total amount of enzyme added, while the binding of the mannanase core without the CBD was less than 20%. Xylanase adsorption (no binding domain) was less than 10%, and was only observed in the first few minutes of treatment. The mannanases exhibited higher adsorption for the softwood fibers, while xylanase adsorption was higher on the hardwood fibers. Drying decreased the adsorption of all three hemicellulases. Electrostatic interactions also appeared to play a large role in the adsorption of the hemicellulases without CBDs.; A micromethod was developed to efficiently measure water retention on a large number of samples. The use of several factorial designs showed that there were no statistically significant changes in water retention values after enzymatic treatment of both virgin and recycled fibers.