Ionic liquid tolerant hyperthermophilic cellulases for biomass pretreatment and hydrolysis

One of the main barriers to the enzymatic hydrolysis of cellulose results from its highly crystalline structure. Pretreating biomass with ionic liquids (IL) increases enzyme accessibility and cellulose recovery through precipitation with an anti-solvent. For an industrially feasible pretreatment and hydrolysis process, it is necessary to develop cellulases that are stable and active in the presence of small amounts of ILs co-precipitated with recovered cellulose. However, a significant decrease in cellulase activity in the presence of trace amounts of ILs has been reported in the literature, necessitating extensive processing to remove residual ILs from the regenerated cellulose. Towards that end, we have investigated the stability of hyperthermophilic enzymes in the presence of the IL 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]) and compared it to the industrial benchmark Trichoderma viride ( T. viride) cellulase. The endoglucanase from a hyperthermophilic bacterium, Thermatoga maritima, and a hyperthermophilic archaeon, Pyrococcus horikoshii, were over expressed in E. coli and purified to homogeneity. Under their optimum conditions, both hyperthermophilic enzymes showed significantly higher [C2mim][OAc] tolerance than T. viride cellulase. Using differential scanning calorimetry we determined the effect of [C2mim][OAc] on protein stability and our data indicates that higher concentrations of IL correlated with lowered protein stability. Both hyperthermophilic enzymes were active on [C2mim][OAc] pretreated Avicel and corn stover. Furthermore, these enzymes can be recovered with little loss in activity after exposure to 15% [C2mim][OAc] for 15 h. These results demonstrate the potential of using IL-tolerant extremophilic cellulases for hydrolysis of IL-pretreated lignocellulosic biomass, for biofuel production.