(235c) Development of Helper Proteins for Improved Cellulose Decrystallization and Hydrolysis

To produce biofuels from cellulosic materials, the materials are pretreated and then subsequently hydrolyzed with cellulolytic enzymes for conversion to simple fermentable sugars. However, due to its partially crystalline structure, the complete hydrolysis of cellulose often requires a prohibitively large quantity of hydrolytic enzymes, which is one of the major limitations to its industrial scale up. To overcome this limitation, cellulose can be pretreated by non-hydrolyzing helper proteins, such as members of the glycosyl hydrolase family (GH), more specifically swollenins and expansins for facilitation of hydrolysis. Previously, we have found that cellulose-binding domains (CBDs) from cellobiohydrolases lower the crystallinity index (CrI), even when added to the hydrolysis medium without the catalytic domain (CD) (Hall et al., Bioresour Technol. 2011, 102, 2910-2915)
Here, we report on a new fusion helper protein, which is capable of enhancing the activity of cellulases through reducing the crystallinity of cellulose. This helper protein fuses aspen expansin with the carbohydrate binding domain (CBD) from Trichoderma reesei. The Expansin-CBD fused gene was cloned and heterologously expressed in yeast as well as expansin or CBD as a fusion protein with green fluorescent protein were expressed for comparison. Among these proteins, the expansin-CBD fusion protein binds tightly to cellulose, and reduces crystallinity of cellulose as was determined by X-ray diffraction studies. This observation portends a greatly enhanced enzymatic degradation rate. We further demonstrate that cellulosic substrate becomes susceptible to the action of commercial lignocellulolytic enzymes, -glucosidase and cellulase, following treatment with expansin-CBD fusion protein.