Although ILs have proven to be ideal solvents for biomass pretreatment and homogeneous chemical catalytic conversion of biomass into monosugars, the process still suffered a shortage of high cost cellulose regeneration. Considering the fact that ILs are also regarded as ideal solvents for biocatalysis due to their unique advantages compared to conventional solvents, researchers are devoting to develop an integrated process of pretreatment and enzymatic hydrolysis in one batch, which will eliminate the need to recover the regenerated lignocellulosic materials, and will lead to a more economic and environmentally friendly conversion process for bio-energy production [5]. It is rational to postulate that ILs are potentially ideal media for the enzymatic conversion of cellulose and ligno — cellulosic materials into sugar. However, carbohydrate-dissolving ILs are typically composed of Cl-, dca-, HCOO-, — OAc, i. e., anions which form strong hydrogen bonds with the carbohydrate. These interactions facilitate the dissolution of bio­mass, but denaturation of enzymes can be a problem which hinders the enzymatic conversion of dissolved cellulose in ILs. To overcome this obstacle, the design and synthesis of enzyme-compatible ionic liquids which are capable of dissolving cellulose, and do not considerably deactivate enzymes is essentially necessary. In addition, factors such as IL polarity, IL network, ion kosmotropicity, viscosity, hydrophobicity, the enzyme dissolution, surfactant effect, etc., may also influence the catalytic performance of enzymes [76]. To improve the enzyme solubility and activity in ILs, various attempts have been made, including immobilized enzymes, microemulsions, whole cells catalysis, multi-phase partitioning (TPP) reaction, the use of additives (NaHCO3, Na2CO3, or triethylamine), enzyme-coated micro­crystals, and lipase lyophilization with cyclodextrins [77].

In 2008, Kamiya et al. first reported a one-batch enzymatic process for the saccharification of cellulose in aqueous-IL [1-methyl-3-methyl-imidazolium] [Diethyl phosphate] system, which showed initial information on the potential of [1-methyl-3-methyl-imidazolium] [Diethyl phosphate] as the solvent for in situ pretreatment and enzymatic hydrolysis of lignocellulosic materials in ILs media [78]. Further study by Yang et al. with the diethyl phosphate-based ionic liquids showed that ultrasonic pretreatment could enhance the in situ enzymatic sac­charification of cellulose in aqueous-ionic liquid media, as a result 95.5% con­version of cellulose could be obtained [79]. Furthermore, they also found that the pretreatment of corn cob in 1-methyl-3-methylimidazolium dimethylphosphite ([Mmim]DMP) in view of its biocompatibility with both lignocellulose solubility and cellulase activity (more than 70% saccharification rate), did not bring negative effects on saccharification, cell growth, and accumulation of lipid of R. opacus ACCC41043 [80].

It is well recognized that ILs can be designed with different cation and anion combinations, which allows the possibility of tailoring reaction solvents with specific desired properties, and these unconventional solvent properties of ILs provide the opportunity to carry out many important biocatalytic reactions that are impossible in traditional solvents. In order to avoid denaturing enzyme, Zhao et al. designed a series of glycol-substituted cation and acetate anion ILs that are able to dissolve carbohydrates but do not considerably inactivate the enzyme (immobi­lized lipase B from Candida Antarctica). The ILs could dissolve more than 10% (wt) cellulose and up to 80% (wt) D-glucose. The transesterification activities of the lipase in these ILs are comparable with those in hydrophobic ILs [81]. Garcia et al. reported a class of biocompatible and biodegradable cholinium-based ILs, the cholinium alkanoates, which showed a highly efficient and specific dissolution of the suberin domains from cork biopolymers. These results are almost more efficient than any system reported so far [82]. However, they did not perform the in situ conversion experiments in these ILs. Bose et al. employed tryptophyl fluo­rescence and DSC to investigate the reactivity and stability of a commercial mixture of cellulases in eight ILs. Only 1-methylimidazolium chloride (mim Cl) and tris-(2-hydroxyethyl) methylammonium methylsulfate (HEMA) provided a medium hydrolysis [83]. Although we can conclude that high concentrated ILs can make the enzyme lose its activity, there are still many new ILs or enzymes that show good biocompatability or IL-tolerance. These results provide us a green approach to the production of biofuels. At present, it is evident that the pretreat­ment of lignocellulose in ILs is a good choice for the fast enzymatic hydrolysis of cellulose.

With the aim to search for cellulose hydrolyzing enzymes that are stable in ILs, in 2009, Pottkamper et al. applied metagenomics for the identification of bacterial cellulases that are stable in ILs. By screening metagenomic libraries, 24 novel cellulase clones were identified and tested for their performance in the presence of ILs. Most enzyme clones showed only very poor or no activities. Three enzyme clones,(i. e.,. pCosJP10, pCosJP20, and pCosJP24) were moderately active and stable in the presence of 1-butyl-1-methyl-pyrrolidinium trifluoromethanesulfo — nate. The corresponding genes of these environment-derived cosmids were similar to known cellulases from Cellvibrio japonicus and a salt-tolerant cellulase from an uncultured microorganism. It was found that the most active protein (CelA10) belonged to GH5 family cellulases and was active at IL concentrations of up to 30% (v/v). Recombinant CelA10 was extremely tolerant to 4 M NaCl and KCl. In addition, improved cellulase variants of CelA10 were isolated in a directed evolution experiment employing SeSaM-technology. The analysis of these vari­ants revealed that the N-terminal cellulose binding domain played a pivotal role for IL resistance [84]. Meanwhile, Datta et al. found that both hyperthermophilic enzymes were active on [Emim] [OAc] pretreated Avicel and corn stover. Furthermore, these enzymes could be recovered with little loss in activity after exposure to 15% [Emim] [OAc] for 15 h. These results demonstrated the potential of using IL-tolerant extremophilic cellulases for hydrolysis of IL-pretreated lig — nocellulosic biomass and for biofuel production [85].