Enzymatic hydrolysis of cellulose in aqueous ionic liquids

Total enzymatic hydrolysis of the polysaccharides in lignocellulosic biomass to monosaccharides is currently a focus research area. The monosaccharides obtained from lignocellulose hydrolysis can be used for the production of platform chemicals and biofuels, most notably ethanol. One major challenge in the commercialization of lignocellulosic ethanol production is the recalcitrance of lignocellulosics towards enzymatic hydrolysis, necessitating efficient pretreatment of the lignocellulosic feedstock. Certain ionic liquids (ILs, salts with melting points below 100 °C) dissolve cellulose and even lignocellulosic biomass and are as such interesting candidates for pretreatment technology. However, cellulose-dissolving ILs have been found to severely inactivate the hydrolytic enzymes (cellulases) employed in cellulose hydrolysis. This work focuses on elucidating how certain ILs affect the action of cellulases in cellulose hydrolysis. The main emphasis was on the action of purified monocomponent Trichoderma reesei cellulases, but some commercial cellulase preparations were also studied in IL matrices. Hydrolysis experiments were made in solutions containing up to 90% of the two cellulose-dissolving ILs 1-ethyl-3-methylimidazolium acetate ([EMIM]AcO) and 1,3-dimethylimidazolium dimethylphosphate ([DMIM]DMP). The presence of increasing amounts of IL led to decreasing yields of solubilised saccharides in enzymatic hydrolysis. Depending on the IL and cellulase, no soluble saccharides were released in hydrolysis matrices containing over 40–50% IL. There were clear differences in the severity of the effects of different cellulose-dissolving ILs on cellulase action. [EMIM]AcO was generally more harmful for cellulase action than [DMIM]DMP. Pure [EMIM]AcO completely inactivated T. reesei endoglucanase in 4 h in residual activity measurements, whereas pure [DMIM]DMP supported considerable cellulase activity for at least three days. These results were confirmed by time curves of microcrystalline cellulose (MCC) hydrolysis in matrices containing the two ILs. Cellulose-dissolving ILs based on carboxylate salts of the organic superbases 1,1,3,3-tetramethylguanidine (TMG) and 1,5-diazabicyclo[4.3.0]non-5-ene (DBN) have recently become available. These compounds are distillable under relatively mild conditions and are thus recyclable. However, these ILs were found to be at least as harmful for cellulase action as the studied imidazolium-based ILs and did thus not offer any benefits in terms of enzyme compatibility. T. reesei endoglucanases were unable to reduce the molecular weight of MCC in buffer or in any aqueous matrix containing IL, except in 90% (v/v) [DMIM]DMP in which the MCC was partially dissolved. Cellulose-dissolving ILs were found to be basic in aqueous solution. According to the results in this work, the pH increase caused by IL basicity was not the main reason for the observed cellulase inactivation. Cellulases with confirmed activity at high pH did not perform better than acidic or neutral cellulases in IL solutions. Some indications were however obtained that cellulase thermostability may be associated with better activity in cellulose-dissolving ILs. The studied ILs were found to have very detrimental effects on saccharide analytics. A capillary electrophoresis (CE) method was developed for the analysis of mono- and oligosaccharides in matrices containing ILs. With this CE method, the yields and product distribution of cello-oligomers produced in the hydrolysis experiments could be determined. It was found that the presence of ILs shifted the product distribution to larger cello-oligomers for some cellulases. The CE method was also used to monitor the hydrolysis of cello-oligomers with Aspergillus niger β-glucosidase in IL matrices. This β-glucosidase was found to be very IL sensitive. ILs were found to affect the cellulose binding of T. reesei cellulases. The cellulase binding to MCC in solutions with [DMIM]DMP and [EMIM]AcO was studied with radiolabeled T. reesei Cel5A (endoglucanase II) and Cel7A (cellobiohydrolase I) and their respective core domains. Cel7A was able to bind to MCC with its core domain, whereas it was shown that Cel5A was very dependent on its CBM for efficient substrate binding. High cellulose binding affinity was not necessary for all the cellulases in order for them to be hydrolytically active. [EMIM]AcO interfered more with cellulase substrate binding than [DMIM]DMP. The binding ability of the T. reesei carbohydrate-binding modules (CBMs) was very IL sensitive.