In addition to the development of new IL-resistant enzymes, a variety of methods have been developed to stabilize enzymes in ILs [136]. Most of them have been applied to cellulases. One of these methods is the preparation of microemulsions with ILs that can reduce the dehydration effect of ILs on enzymes. To reduce toxicity and deactivation, microemulsions of water in [BMIM][PF6] were stabi­lized using the surfactant Triton X-100. Lignin peroxidases from Phanerochaete chrysosporium and laccases from Trametes versicolor could oxidize 2,6-dime — thoxyphenol and o-phenylenediamine in these microemulsions. The highest activities for lignin peroxidases and laccases were 13 and 33 qmol/l/min, respectively. In contrast, the same enzymes had negligible catalytic activity in pure or water-saturated [BMIM][PF6] [128].

Another approach to stabilize cellulases is their immobilization on a substrate. For example, immobilization on a poly(ethylene glycol) substrate increased activity of cellulases from Trichoderma reesei in 0.05 M citrate buffer and [BMIM][Cl], compared to the free enzyme [121]. Cellulase from Trichoderma reesei immobilized on 150 qm particles suffered no inhibition in 20 vol.% of [MMIM][DMP], N, N-dimethylethanolammonium lactate, and N, N-dimethylet- hanolammonium acetate. In contrast, reducing sugar yields decreased in 20 vol.% of [MMIM][MeSO4], [BMIM][Cl], [BMIM][PF6], and [BMPy][Cl], by 36, 28, 37, and 34%, respectively [44].

Celluclast immobilized onto a polymeric support (Amberlite XAD4) was coated with the hydrophobic ILs 1-butyl-3-methylimidazolium bis(tri — fluoromethylsulfonyl)imide ([BMIM][Tf2N]) or butyltrimethylammonium bis(trifluoromethylsulfonyl)imide ([N1114][Tf2N]). The activity of these coated cellulases were assessed as a function incubation time in [BMIM][Cl] and in [N1114][Tf2N]/[BMIM][Cl] mixtures at different molar ratios. The hydrophobic IL coating slowed the deactivation effect from [BMIM][Cl]. It was believed that the hygroscopicity of water-immiscible ILs can keep the enzymes hydrated and prevent their unfolding. The polymeric support may act as a water reservoir to preserve the cellulase activity. The cellulase activity decreased with increasing [N1114][Tf2N] concentration, most likely due to the restricted access of cellulose to the coated enzyme [137].