Changes of MCC Surface during Enzymatic Hydrolysis

Abstract

The existence of the crystalline region of cellulose is a serious problem for enzymatic hydrolysis. How does the cellulase hydrolyze cellulose ? How does the cellulase begin to hydrolyze cellulose, especially the crystalline region ? AFM observations and FTIR analyses were performed on bacterial cellulose microcrystalline cellulose (BMCC), which was reacted on a cover-glass at 40°C with a solution of the Trichoderma crude cellulase, Trichoderma cellobiohydrolase I (CBHI (Cel7A)), its cellulose binding module (CBM), and the fusion CBM with Thioredoxin (Trx), respectively. Each molecular weight of the CBHI, fusion CBM and Trx was 70000-80000, 21000, and 17200, respectively. After the reaction for 10 and 30 min, AFM images of the residues were obtained by tapping mode under water. Fibrillation was observed on each residue after the enzymatic treatment, furthermore, narrowed tip and/or smoothed surface were observed after the crude cellulase treatment only. It was interesting that the increase of the molecular weight of enzymes seemed to be accompanied by an increase of the depth of the cracks within the sub-element of the fibrillated BMCCs. If the size of the molecular weight corresponds to the size of the protein, the difference of the degree of fibrillation of MCC may be caused by the existence of the protein which can be connected by CBM. From the above-mentioned result, it is estimated that the enzymatic hydrolysis of BMCC begins as follows; CBM adsorbs the BMCC surface, the crack is generated, then the catalytic module of large volume expands the crack like a wedge, and therefore the site where an enzyme can attack increase. © 2010, The Japan Wood Research Society. All rights reserved.