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.
Circular dichroism spectroscopy was used to measure the thermal unfolding of bovine pancreatic ribonuclease A (RNase A) with various concentrations of guanidine hydrochloride (GuHCl). A red shift in transition midpoint temperatures, T
m, occurred with increasing concentration of the strong protein denaturant. van Hoff enthalpy changes, ΔH°, were calculated and plotted as a function of T
m to determine the heat capacity change, ΔC
p, for denaturation. A value of 4.02 ± 0.02 kJ mol–1 K–1 was calculated from d( ΔH)/d(ΔT
m). Reported values for ΔC
p range from 4.2 to 9.6 kJ mol–1 K–1. The shift in T
m for RNase A with increasing concentration of GuHCl suggests that the protein is undergoing substantial changes in its secondary structure.
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