Cellulose hydrolysis and binding with Trichoderma reesei Cel 5 A and 1 Cel 7 A and their core domains in ionic liquid solutions

1 Ionic liquids (ILs) dissolve lignocellulosic biomass and have a high potential as pretreatment 2 prior to total enzymatic hydrolysis. ILs are, however, known to inactivate cellulases. In this 3 article, enzymatic hydrolysis of microcrystalline cellulose (MCC) and enzyme binding onto 4 the cellulosic substrate were studied in the presence of cellulose-dissolving ionic liquids. Two 5 different ionic liquids, 1,3-dimethylimidazolium dimethylphosphate ([DMIM]DMP) and 1-6 ethyl-3-methylimidazolium acetate ([EMIM]AcO), and two monocomponent cellulases, 7 Trichoderma reesei cellobiohydrolase Cel7A and endoglucanase Cel5A, were used in the 8 study. The role and IL sensitivity of the carbohydrate-binding module (CBM) were studied 9 by performing hydrolysis and binding experiments with both the intact cellulases, and their 10 respective core domains (CDs). Based on hydrolysis yields and substrate binding experiments 11 for the intact enzymes and their CDs in the presence of ILs, the function of the CBM 12 appeared to be very IL sensitive. Binding data suggested that the CBM was more important 13 for the substrate binding of endoglucanase Cel5A than for the binding of cellobiohydrolase 14 Cel7A. The CD of Cel7A was able to bind well to cellulose even without a CBM, whereas 15 Cel5A CD had very low binding affinity. Hydrolysis also occurred with Cel5A CD even if 16 this protein had very low binding affinity in all the studied matrices. Binding and hydrolysis 17 were less affected by the studied ILs for Cel7A than for Cel5A. To our knowledge, this is the 18 first systematic study of IL effects on cellulase substrate binding.