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During the second and final year of the ACS-PRF funded project, the following important tasks have been successfully accomplished:

1. 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 a further study, we 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. 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. The fluorescence spectra of CALB in ILs failed to provide information on protein structures, probably due to the interference of 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. (This part of research has been published in Green Chemistry, 2009, 11, 1128-1138.)

2. To extend the finding of new ionic liquids from the proposed research to renewable energy research, we further investigated the use of these ionic liquids in the production of biodiesel. Ionic liquids (ILs) have been 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 ˚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. 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. (This part of research has been accepted for publication in Applied Biochemistry and Biotechnology, doi: 10.1007/s12010-009-8717-6.)

3. Two undergraduate students (Vernecia N. Person and Janet V. Cowins) were supported by our university to participate in the research of this project. They assisted the PI in setting up experiments, collecting and analyzing samples. They are co-authors of several papers published by the PI. The research experience had a big impact on their choices of careers, and made them very competitive upon graduation. Both of them are now enrolled in the graduate program of chemistry.

4. This ACS-PRF grant is the PI's first external support. This grant has a significant impact on PI's research and his career. PI has not only accomplish the proposed research activities, but also extended the research into a new area in renewable energy. PI has published at least five papers in top peer-reviewed journals. These progresses allow the PI to be established in the field, and enable him to be very competitive in applying for other major grants.