Cellulose Dissolution in Ionic Liquids: Influence of Ion Structure On β-Glucosidase Stability and Activity

Certain ionic liquids are now well known to be effective solvents for cellulose and therefore are of strong interest as solvents for biomass pretreatment and processing. After separating cellulose from raw biomass, enzymatic hydrolysis of the cellulose to D-glucose may be utilized. The influence of the ionic liquid in bulk or trace amounts on the enzymatic activity is not yet well known. Therefore, the effects of four ionic liquids – 1-butyl-3-methylimidazolium chloride ([BMIM]Cl), 1-butyl-3-methylimidazolium acetate ([BMIM]Ac), 1-ethyl-3-methylimidazolium chloride ([EMIM]Cl) and 1-ethyl-3-methylimidazolium acetate ([EMIM]Ac) – on ß-glucosidase stability and activity are compared with a range of inorganic salts with either chloride (Cl^-) or acetate (Ac^- or CH₃COO^-) anions. The ionic liquids changed the enzyme conformation in a very different way from the inorganic salts. Conformational changes of the enzyme did not directly result in the inactivation of the enzyme. When incubated with ß-glucosidase prior to the substrate addition, the four ionic liquids denatured the enzyme irreversibly. With the same anion (CH₃COO^- or Cl^-), the EMIM⁺ cation was more amicable to the enzyme than the BMIM⁺ cation. With the same EMIM⁺ cation, the Cl^- anion was more amicable to the enzyme than CH₃COO^-. However, with the same BMIM⁺ cation, the Cl^- and CH₃COO^- anions did not result in a significant difference in the enzyme stability. Further, [BMIM]Cl showed stronger inhibition of ß-glucosidase than [EMIM]Ac. These results indicate that the cation in an ionic liquid plays a more important role than the anion in deactivating ß-glucosidase. When the four ionic liquids were instead pre-incubated with the substrates (p-nitrophenyl-ß-D-glucopyranoside or cellobiose) before the enzyme addition, the highest hydrolysis efficiency was obtained with [EMIM]Cl. A higher hydrolysis efficiency was also noted for [BMIM]Cl than for the acetate ionic liquids suggesting that the interactions between the chloride based ionic liquids and the substrates evidently protected the enzyme somewhat from being denatured.