Cellulases operate optimally at a specific temperature, and the introduction of IL can shift the optimal temperature. One of the cellulase identified by metagenomics exhibited an optimum activity at 55°C in McIllvaine buffer (0.2 M Na2HPO4 with 0.1 M citric acid, pH 6.5). In 1-ethyl-3-methylimidazolium trifluoroacetate ([EMIM][TfAc]) and 1-butyl-1-methylpyrrolidinium trifluoromethanesulfonate ([BMPy][CF3SO3]), the optimum temperature shifted to 37 and 20°C, respectively [126].

Increasing the temperature from 50 to 60-70°C can also accelerate the deac­tivation of cellulases from T. reesei in [EMIM][OAc] [91]. The stability of mix­tures of Celluclast 1.5 l and Novozyme 188 was tested in the presence of [EMIM][OAc] at concentrations ranging from 5 to 30% (volume/volume) in citrate buffer (pH 4.8) with poplar and Avicel [120]. When the hydrolysis was conducted at 4°C for a [EMIM][OAc] concentration of 30%, the activity of the cellulase mixture after 24 h remained above 70% of the activity without [EMIM][OAc]. At 50°C, the drop in cellulase activity dropped further to 31% of the control activity after 24 h in a 30% [EMIM][OAc] solution [120].

Enzyme activity is also pH-dependent [126, 127]. Celluclast 1.5 l hydrolyzes cellulose at an optimum pH between 4.5 and 5. No cellulase activity was observed for pH values below 2 or above 8 [127]. Three cellulases identified with metagenomic libraries have optimal pH values of 5, 7, and 7.5 [126]. The oxi­dation of o-phenylenediamine by lignin peroxidase was most effective at pH 3.2 [128]. A deviation from the optimal pH induced by the introduction of ILs can cause the deactivation of cellulases. The pH of the wood/IL mixture is affected not only by the IL concentration but also IL composition and structure [129]. Increasing the concentration of 1,3-dimethylimidazolium dimethylphosphate [MMIM][DMP] from 0 to 0.5 vol.% in the enzyme solution led to an increase of the pH from 4.8 (optimum for hydrolysis) to 6.5 [67]. Mixtures of water with ILs based on an imidazolium cation and a BF — anion have a pH that increases with the length of the alkyl chain on the cation. The addition of hydroxyl groups increases the acidity of the IL [129]. The pH can also vary during the biomass reaction with the IL. Measurements in wheat straw and pine wood solution in [EMIM][Cl], [BMIM][Cl], and [EMIM][OAc] showed a drop in pH over time. A HPLC analysis showed the formation and the accumulation of acetic acid, which comes from the hydrolysis of acetate groups in the biomass [47].