Tuesday, May 3, 2011
The binding of cellulases to cellulose is an important step in hydrolysis of cellulosic material. The hydrolysis has been observed to be more pronounced at sites where cellulose microfibrils are misaligned, called dislocations (Nyholm, 2001). Thygesen et al. (2010) showed that an Humicola insolens endoglucanase preferred binding to dislocations. Here we report a study using CLSM on the binding to filter paper fibers of an exoglucanase, Cel7A, and a CBM-missing variant of this enzyme, Cel7A Core. CLSM was used for identification of areas where the enzymes bound. Cel7A was found to have no preference for binding to dislocations. Cel7A Core in general showed the same tendency although occasionally it was observed within dislocations.
Experiments where enzyme binding was followed by washing steps indicated that Cel7A Core binding was weaker than that of Cel7A. Further, sequential experiments indicated that a fraction of bound Cel7A was removed when Cel7A Core was added. Interestingly, such removal did not occur if Cel7A was added instead of Cel7A Core. The limited resolution of the CLSM (pixel size: approximately 120 × 120 nm2), however, did not allow a firm conclusion as to the co-localization of Cel7A and Cel7A Core.