Direct observation of cellulase penetration in oven-dried pulp by confocal laser scanning microscopy

Hornification of cellulose is a well-known phenomenon that takes place during the drying process. Hornification involves a structural change in the cellulose that restricts the enzyme’s ability to access and saccharify the cellulose. We observed never-dried (ND), pressed (PR), and oven-dried (OD) softwood pulp during saccharification catalyzed by fluorescent labelled cellulase from Trichoderma reesei by confocal laser scanning microscopy. Initially cellulases were observed on outer surfaces and dislocations of all the ND, PR, and OD pulp fibers. It was clearly observed that over time that cellulases penetrated the cell walls of the ND pulp fiber from the outer surface, inner surface and cracks and remained in the cell walls. In contrast, they did not penetrate the cell walls of the OD pulp fiber and instead stayed on the cracks and ends of the shortened fibers. Mannan and xylan were found at dislocations of ND and OD fibers by immunolabelling microscopy. The results suggested that hemicellulose plays an important role in hornification at dislocations.