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Ionic liquids (ILs) with various structures and properties have been extensively investigated in many biocatalytic reactions and processes. However, although hydrophobic ILs tend to stabilize the insoluble (suspended) enzymes, they usually have low hydrogen-bonding basicity with solutes, which limits the solubility of many substrates (such as D-glucose, ascorbic acid, and cellulose). In contrast, hydrophilic ILs (such as those based on chloride, acetate and dicyanamide) are able to dissolve many of these substances that are not quite soluble in common organic solvents. Unfortunately, enzymes are not always active in these hydrophilic media due to strong interactions (such as H-bonding) between proteins and ILs. To resolve this dilemma, we recently synthesized new acetate-based ILs carrying a long alkyloxyalkyl chain in their cations, and found that these ether-functionalized solvents are lipase-compatible and can dissolve considerable amounts of D-glucose and cellulose (Green Chem., 2008, 10, 696). In this study, we further observed that these ILs could dissolve high concentrations of lipase B from Candida antarctica (CALB) (> 5 mg/mL at 50 • C), as well as other substrates including amino acids and betulinic acid. Therefore, these novel media offer new opportunities for carrying out homogeneous enzymatic reactions, which is practically important for large substrate molecules. In this article, we further confirmed the lipase compatibility of these ILs through the transesterification between ethyl butyrate and 1-butanol. The second derivative infrared spectra of CALB suggest the conservation of secondary structures of proteins in these ILs. We further investigated these ether-functionalized ILs in two important biocatalytic reactions: enzymatic synthesis of methyl-phthalate of betulinic acid, and CALB-catalyzed synthesis of D-glucose fatty acid esters. These substrates are not very soluble in conventional organic solvents, but very soluble in ILs, which improved the catalytic efficiency of these reactions. Moderate to high conversions were achieved in both reactions.

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The pretreatment of cellulose using ionic liquids (ILs) has been shown to be an effective method for improving the enzymatic hydrolysis of cellulose; this technique affords a fast and complete saccharification of cellulose into reducing sugars (Dadi et al., Biotechnol Bioeng. 2006; 95:904-910; Liu and Chen, Chinese Sci Bull. 2006; 51:2432-2436; Zhao et al., J Biotechnol. 2009; 139:47-54). Motivated by these advances, this study examines the effect of IL-pretreatment on the enzymatic hydrolysis of purified xylan (as a model system of hemicellulose) and switchgrass (as a real lignocellulose). The IL-pretreatment resulted in no improvement in the hydrolysis of xylan. The likely reason is that pure xylan has a low degree of polymerization (DP), and is readily biodegraded even without any pretreatment. However, in real cellulosic materials (such as switchgrass), xylan is entrapped within the cellulosic matrix, and cannot be conveniently accessed by enzymes. Our data demonstrate that the IL-pretreatment of switchgrass significantly improved the enzymatic saccharification of both cellulose (96% D-glucose yield in 24 h) and xylan (63% D-xylose yield in 24 h). The compositional analysis of switchgrass suggests a lower lignin content after IL-pretreatment. In addition, the infrared spectrum of regenerated switchgrass indicates a lower substrate crystallinity, whereas the enzyme adsorption isotherm further implies that the regenerated substrate is more accessible to enzymes. This study has further confirmed that IL-pretreatment is an effective tool in enhancing the enzymatic hydrolysis of cellulosic biomass, and allowing a more complete saccharification.

Ionic liquids (ILs) are being explored as solvents for the enzymatic methanolysis of triglycerides. However, most available ILs (especially hydrophobic ones) have poor capability in dissolving lipids, while hydrophilic ILs tend to cause enzyme inactivation. Recently, we synthesized a new type of ether-functionalized ionic liquids (ILs) carrying anions of acetate or formate; they are capable of dissolving a variety of substrates and are also lipase-compatible (Green Chem., 2008, 10, 696-705). In the present study, we carried out the lipase-catalyzed transesterifications of Miglyol oil 812 and soybean oil in these novel ILs. These ILs are capable of dissolving oils at the reaction temperature (50 degrees C); meanwhile, lipases maintained high catalytic activities in these media even in high concentrations of methanol (up to 50% v/v). High conversions of Miglyol oil were observed in mixtures of IL and methanol (70/30, v/v) when the reaction was catalyzed by a variety of lipases and different enzyme preparations (free and immobilized), especially with the use of two alkylammonium ILs 2 and 3. The preliminary study on the transesterification of soybean oil in IL/methanol mixtures further confirms the potential of using oil-dissolving and lipase-stabilizing ILs in the efficient production of biodiesels.