Candida antarctica lipase B (CALB, 10.9 U∙mg^-1), horseradish peroxidase (HRP, 150 U∙mg^-1), bis-tris-propane (BTP), guaiacol and ethyl butyrate were purchased from Sigma-Aldrich. Ethyl butyrate and n-butanol were analytical reagents and were dried by 3A molecular sieves before use. All other chemicals and reagents were of analytical grade. 1-buthyl-3-methyl-imidazolium chloride ([BMIM]Cl) and 1-(1-hydroxyethyl)-3-methyl-imidazolium tetrafluoroborate ([C₂OHMIM] [BF₄]) were synthesised according to published procedures [11] . [C₂OHMIM] [BF₄] was checked for the absence of chloride and acid. The IL was passed through a neutral alumina column, dried at 50˚C under reduced pressure for more than 18 h, and stored under dry N₂.

The ¹H NMR and ¹³C NMR spectra were obtained on a Brüker AV-400 Fourier transform NMR spectrometer. ¹H NMR spectra were referenced to tetramethylsilane in CDCl₃.

Titration was carried out using a custom-built autotitrator. Place a magnetic stirring bar in the beaker and set the beaker over a magnetic stirrer. An appropriate amount of EDTA acid (1.0 mmol) was dissolved in 20 mL of water. Immerse the electrodes in the solution and start the stirring. 0.1 M [BMIM] [OH] solution was added by a peristaltic pump (Shanghai Hu Xi analysis instrument factory Co., Ltd., model HL-2) running at 1.0 mL∙min⁻¹. The pH of the mixture was recorded with a pH meter (ORION, model 828) interfaced to a computer.

An aqueous solution of 1-buthyl-3-methyl-imidazolium hydroxide ([BMIM]OH) was prepared by passing [BMIM]Cl solution through a column filled with anion exchange resin, as described in the literature [2] [11] . The aqueous [BMIM]OH solution was then neutralized with EDTA acid in a beaker and the pH of the solution was adjusted to 3.90, 6.50, 8.50 or 9.80. The solution was evaporated at 50˚C under reduced pressure to give a viscous liquid, which was then vacuum dried at 50˚C for 18 h to afford EDTA IL buffer. NMR spectra of EDTA IL buffer: pH 9.80 ¹H NMR (400 MHz, D₂O) δ: 7.368 (s, 4H, im-H), 7.320 (s, 4H, im-H), 4.088 (t, 8H, -CH₂), 3.784 (s, 12H, im-CH₃), 3.447 (s, 8H, N-CH₂-COO-), 3.033 (s, 4H, N-CH₂-CH₂-N), 1.721 - 1.758 (m, 8H, -CH₂), 1.199 - 1.217 (m, 8H, -CH₂), 0.794 - 0.831 (t, 12H, -CH₃); ¹³C NMR (400MHz, D₂O) δ: 175.703, 135.599, 123.385, 122.119, 57.238, 51.069, 49.200, 35.547, 31.212, 18.706, 12.600. pH 3.90 ¹H NMR (400 MHz, D₂O) δ: 8.067 (s, 2H, im-H), 7.368 (s, 2H, im-H), 7.328 (s, 2H, im-H), 4.085 (t, 4H, -CH₂), 3.796 (s, 6H, im-CH₃), 3.782 (s, 8H, N-CH₂-COO-), 3.580 (s, 4H, N-CH₂-CH₂-N), 1.717 - 1.754 (m, 4H, -CH₂), 1.195 - 1.214 (m, 4H, -CH₂), 0.790 - 0.827 (t, 6H, -CH₃); ¹³C NMR (400 MHz, D₂O) δ: 170.500, 135.822, 123.4495, 122.169, 57.939, 51.569, 49.232, 35.602, 31.235, 18.721, 12.624.

CALB (1.2 mg) was dissolved in 500 μL of [C₂OHMIM][BF₄] (30 mg of EDTA IL buffer was added in the case of buffered medium) in a 5 mL flask. 110 μL (0.83 mmol) ethyl butyrate and 110 μL (1.21 mmol) n-butanol and 50 μL nonane (internal standard) were added. The reaction mixture was stirred at 40˚C in oil bath for 3 h. After the reaction was complete, the products was decanted from [C₂OHMIM][BF₄]. The organic phase was analyzed with a gas chromatograph equipped with an FID and a capillary column (SE-30, 30 m × 0.32 mm × 0.25 μm). The residual reactant mixture in IL phase was removed in vacuum at 40˚C for more than 1 h. The new cycle was restarted by addition of fresh substrate.

HRP activity was measured by following the H₂O₂-dependent oxidation of guaiacol at 470 nm. Guaiacol stock solutions (1.0 mM) were prepared by dissolving guaiacol in 0.1 M buffer. For assays done in the presence of Hg²⁺ ions, appropriate amounts of Hg(NO₃)₂ stock solution were mixed with 0.1 M buffer and the pH was readjusted whenever required. H₂O₂ stock solutions (300 mM) were prepared daily by appropriate dilution of 30% H₂O₂ in distilled water. HRP solutions (10 μg/ml) were prepared by dissolving the enzyme in distilled water. The assay was performed by mixing 500 μL guaiacol stock solutions with 15 μL of HRP solution (final concentration 7.8 nM) and the mixture was then incubated at 298 K for 30 min. The reaction was started by adding 15 μL of 300 mM H₂O₂. The initial velocity (v₀) of the oxidation of guaiacol was determined from linear plotting of the absorbance versus time, using an extinction coefficient of 2.66 × 10 ⁴ M ⁻¹∙cm⁻¹ for guaiacol-derived oxidation product.

All assays were carried out at 298 K using a UV-Vis spectrophotometer (Unico, UV2800) which cell was connected to a thermostat.

The titration profile of EDTA with [BMIM]OH in water expressed 3 buffering-like regions, which are centered at pH 3.90, 6.50 and 8.50, respectively (Figure 1).

Thus, we considered that it is possible to synthesize new ILs of buffering ability by neutralization of aqueous solutions of [BMIM]OH with aqueous solutions of EDTA at pH 3.90, 6.50 and 8.50, as illustrated in Scheme 1.

The structure of EDTA IL buffers were shown in Scheme 1. The IL buffers were synthesized as previously reported [2] [11] and characterized by NMR. The characterization data of EDTA IL at pH 3.90 and pH 9.80 are consistent with the expected compositions and structures. The pH, the dilution value, and buffer values of EDTA IL buffers were summarized in Table 1. It can be seen that the ILs synthesized possess buffering ability and can be used for controlling the ionization of enzymes in both aqueous and non-aqueous media.

Scheme 1. Synthesis of EDTA IL buffer.

The structure of EDTA IL buffers, [ BMIM ] n + [ EDTA ] n − were shown in Scheme 1. The parameters were measured at room temperature. ^aDilution value is defined as the change of pH on dilution with an equal volume of water. ^bThe buffer value is defined as the number of moles of strong base required to change the pH of one liter of solution by one unit.

Lipase-mediated transesterification is one of the economically viable clean technology for flavor ester production [29] . To test the activity of CALB lipase in dissolved form in ILs, we examined the CALB-catalyzed transesterification of ethyl butyrate with n-butanol. All the reactions were performed under the same conditions, 40˚C and 300 rpm, in this study to eliminate any temperature or mixing effects. The results showed that CALB afforded lower substrate conversion in pure [C₂OHMIM][BF₄] (8.7%). In contrast, CALB exhibited great transesterification activity in the buffered [C₂OHMIM][BF₄] (see Figure 2), indicating that the buffer was responsible for the rate enhancement.

The above results showed that the lipase activity is greatly affected by the IL and IL buffer. This is because the ionization constant of ionizable groups of the lipase is greatly affected by the solvent [30] . Thomazeau et al. reported that the acid HNTf₂ is more acidic in [BMIM][NTf₂] and [BMIM][BF₄] than in water

[31] . Thus, when CALB transfer from water to [C₂OHMIM][BF₄], the ionization state of the lipase will be changed, resulting in decrease of lipase activity. And addition of EDTA IL buffer can regulate the ionization state of ionizable groups of the lipase to the appropriate state. One can conclude that enzyme activity in ILs is also buffer dependent as in aqueous systems.

The recycling ability of the soluble CALB in buffered [C₂OHMIM][BF₄] under the above reaction conditions is illustrated in Figure 2. After each reaction, the reactant mixture in the upper layer and CALB-IL in the lower layer were separated by decantation. The CALB-IL phase was then evacuated to remove residual reactant mixture and charged for the next reaction without adding any new enzyme, IL, and buffer. The lipase activity dropped significantly during recycling in the presence of EDTA IL buffer of pH 3.90, suggesting that the lipase was gradually denatured in the acidic environment. However, the conversion of ethyl butyrate can maintain 75% of its initial value after 10 runs in the presence of EDTA IL buffer of pH 6.50 or pH 8.50, indicating that the dissolved CALB in the above buffered [C₂OHMIM][BF₄] is very stable.

Oxidative stress causes uncontrolled oxidation, resulting in the progressive deterioration and the collapse of organs and systems in the living organisms [32] . Peroxidases are important detoxifying enzymes serving to rid cells of excess

H₂O₂, Horseradish peroxidase (HRP) is one of the best characterized peroxidases [33] . The effects of Hg²⁺ on HRP can be easily assayed through activity of H₂O₂-dependent oxidation, which can be assayed spectrophotometrically at 470 nm using the H₂O₂-dependent oxidation of guaiacol.

Figure 3 showed that EDTA IL buffers as Hg²⁺ chelators protected HRP against Hg²⁺-induced denaturation and precipitation. Higher pH favored the protection, while at lower pH the protection diminished. Because the concentration of the deprotonated conjugate base of EDTA increases with the increase of pH of solution, its subsequent increased binding to Hg²⁺ causes the protection of HRP against Hg²⁺-induced denaturation. On the contrary, there is almost no Hg²⁺ chelators in BTP buffer system, thereby, Hg²⁺ inhibition occurs. This chelating effect undoubtedly explained the difference in Figure 3 and Figure 4.