Cellulose hydrolysis is the result of the synergistic action of three different types of cellulases: endoglucanases that cleave b-1,4-glycosidic bonds on cellulose chains, cellobiohydrolases that convert long cellulose chains into cellobiose, and b-glu — cosidases that convert cellobiose into glucose [118, 119]. The mechanisms underlying cellulase activity on a heterogeneous substrate, such as lignocellulosic biomass, is still under investigation [72, 119]. Multiple models have been devel­oped to understand the multiple steps involved in cellulose hydrolysis: adsorption of cellulases on the substrate, formation of the enzyme-substrate complex, loca­tion and hydrolysis of b-glycosidic bonds, desorption of the enzyme, synergy between endoglucanases, cellobiohydrolases, and b-glucosidases [119].

Once biomass is regenerated from its IL solution, it can still contain traces of IL that can reduce cellulase activity [72]. Several studies have focused on the stability of commercial cellulases in various ILs and their saccharification yields on purified cellulose substrates and native biomass. Celluclast 1.5L (cellulases from Tricho — derma reesei) and Novozyme 188 (b-glucosidase from Aspergillus niger) retained 76 and 63% of their original activity on carboxymethylcellulose after incubation at 50°C for 24 h in 15 and 20% [EMIM][OAc] solutions, respectively [120]. The activity of Celluclast 1.5L was also assessed on a-cellulose in [MMIM][DMP], [AMIM][Cl], [BMIM][Cl], and [EMIM][OAc] at a 10 vol.% concentration. The activity in these ILs was between 70 and 85% lower than the activity in sodium acetate buffer at pH 4.8 [67]. An increase in the IL concentration led to an increase in the IL viscosity by a factor of 4 [67]. The activity of cellulases from Tricho — derma reesei on cellulose azure was found to decrease dramatically with low concentrations (22 mM) of [BMIM][Cl] or [BMIM][BF4] [121]. No saccharifi­cation of Avicel cellulose was observed with cellulases from Trichoderma reesei in 60 vol.% [EMIM][DEP] [122]. The activity of cellulases from Aspergillus niger decreased with incubation time in [BMIM][Cl] and [BMIM][Cl] concentration

[123] . It is important to note at this point that variations of 20% in cellulase activity were observed between different Celluclast 1.5L lots from the same manufacturer [67].

Despite the partial deactivation of cellulases in ILs, reducing sugar yields were still higher after IL pretreatment with low residual IL concentrations, due to the improved access of enzymes to the cellulose in biomass. For example, the cellu­lase mixture of Celluclast 1.5L and Novozyme 188 still converted 45% of the cellulose contained in a solution of 0.6% [EMIM][OAc]-pretreated yellow poplar with 15% [EMIM][OAc]. The conversion rate was much higher than for the untreated yellow poplar (11%) [120]. The activity of the same mixture was also assessed on purified cellulose substrates: an Avicel solution in [EMIM][OAc] and untreated Avicel in citrate buffer. After enzymatic hydrolysis for 24 h at 50°C, 91% of the [EMIM][OAc]-pretreated Avicel was converted to glucose, while only 49% of the untreated Avicel was converted [120]. With cellulases from Trichoderma reesei in 20 vol.% [EMIM][DEP], 70% of the cellulose was con­verted to cellobiose or glucose, a conversion rate that was higher than the untreated Avicel (about 33%). A comparison with [EMIM][OAc] using the same procedure yielded conversion rates that were half of those with the diethylphosphate anion [122].

The stability of another commercial cellulase, GC 220, a mixture of endoglu — conases and cellobiohydrolases from Trichoderma reesei was assessed in eight different ionic liquids. With the exception of tris-(2-hydroxyethyl)methylammo — nium methylsulfate (HEMA), the fluorescence of the trytophyl marker on the cellulases was quenched in the other ILs that included several imidazolium-based ILs, suggesting denaturation of the enzymes. The cellulase activity was measured spectroscopically in a citrate buffer (pH 4.8) and in the eight ILs using cellulose azure as the substrate. Cellulase activity was detected only in the ILs 1-methyl — imidazolium chloride ([MIM][Cl]) and HEMA, but it was significantly lower than in the buffer. The cellulases remained active even after 2 h in these two ILs at 65°C [124].

The tolerance of cellulases produced by Penicillium janthinellum to ionic liq­uids was tested by incubating the extracted enzymes in an aqueous solution of [BMIM][Cl] of concentration ranging from 10 to 50%, and then measuring their residual activity on different substrates (filter paper Whatman no. 1, carboxy — methylcellulose, xylan solution or p-nitro phenyl b-D-glucopyranoside). After incubation in 10% ionic liquid for 5 h, the cellulases retained at least 80% of their activity on all substrates. At a higher concentration of 50%, the residual activity decreased significantly to reach below 20% for all substrates [125].

The tolerance of cellulase-producing bacteria from termites to [BMIM][Cl] was studied by characterizing their growth in [BMIM][Cl] at concentrations ranging from 0.1 to 10 vol.%. The three bacteria that were the most effective at cellulase production could tolerate [BMIM][Cl] at concentrations smaller than 1.0 vol.%. No growth was observed for concentrations larger than 5 vol.%. For two of the bacteria, the growth rates were unchanged for concentrations smaller than 1.0 vol.%. [118].

Cellulases are deactivated in ILs through multiple mechanisms. Stability and unfolding of the cellulases were studied by differential scanning calorimetry. Thermal unfolding was irreversible in the citrate buffer with a broad transition peak between 60 and 75 °C. The ILs [MIM][Cl] and HEMA improved the stability of the cellulases with the shift of the transition temperatures above 75 °C. The low activity in HEMA compared to the buffer was attributed to the high viscosity of HEMA [124]. Cellulase activity also decreased when the viscosity of the enzyme solution without IL was increased with polyethylene glycol [67].

Deactivation was attributed to the dehydrating environment introduced with [BMIM][Cl] that causes the denaturation of the enzyme. This conclusion was supported by fluorescence spectra of the cellulase in [BMIM][Cl] and various denaturants, such as the surfactant sodium dodecylsulfate and urea [121]. Cellulase deactivation in [BMIM][Cl] was similar to the deactivation in NaCl solutions at high concentrations above 0.35 M, suggesting that interactions between the enzymes and the IL charged species also play a role in the denaturation of the enzymes [67, 121]. Enzyme activity can be recovered when the IL was diluted with buffer solution [67].