Development and adjustment of helper proteins for easier hydrolysis and adsorption

The crystalline structure of cellulose necessitates large quantities of hydrolytic enzymes, which is one of the major limitations for the biorefinery industry. One way to pretreat cellulose is to apply non-hydrolyzing helper proteins such as members of the glycosyl hydrolase family 61 (GH61), 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 such a new fusion helper protein, which is capable of enhancing the activity of cellulases through reducing the crystallinity of cellulose. Its corresponding gene, fusion of aspen tree expansin with carbohydrate binding domain (CBD) from the Trichoderma reesei, was cloned and heterologously expressed in yeast. Expansin-CBD fusion protein binds tightly to cellulose, and reduces crystallinity of cellulose by using X-ray diffractometry analysis, which can be a crucial contributor to the improvement of enzymatic degradation rate. In addition to synergistic activity, we investigated putative function of linkers by comparing adsorption capacity on cellulose of several linker variants in terms of length and amino acid composition. Both of them play a role on cellulose adsorption, interestingly, linker length functions as a crucial factor rather than amino acid composition. We demonstrate that cellulosic substrate becomes susceptible to the action of commercial lignocellulolytic enzymes, β-glucosidase and cellulase, following treatment with expansin-CBD fusion protein and suggest the idea of optimal linker for effective adsorption.