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. 2019 Sep 4;4(12):15234–15239. doi: 10.1021/acsomega.9b02118

“Water-like” Dual-Functionalized Ionic Liquids for Enzyme Activation

Hua Zhao 1,*, Glenn A Harter 1, Caden J Martin 1
PMCID: PMC6751713  PMID: 31552369

Abstract

graphic file with name ao9b02118_0005.jpg

By mimicking the water structure to improve the enzyme activity, we designed imidazolium (Im)-based ionic liquids (ILs) functionalized with both ether and tert-alcohol groups (e.g., [CH3(OCH2CH2)n-Im-t-BuOH][Tf2N]). This unique combination of the “water-like” structure enabled very high transesterification (synthetic) activities for immobilized lipase B from Candida antarctica, which are up to 2–4 folds higher than nonfunctionalized “classical” ionic liquids (such as [BMIM][Tf2N]) and up to 40–100% higher than diisopropyl ether and tert-butanol. Fluorescence emission spectra confirmed the general protein structural preservation in these tailored ionic solvents. In addition, functionalized ILs showed high thermal stabilities, which are comparable with diisopropyl ether but much higher than tert-butanol.

1. Introduction

Polyols and sugars (e.g., sorbitol and trehalose) are known protein-stabilizing compensatory solutes in aqueous solutions.1,2 For instance, the thermal stability of glucose dehydrogenase in aqueous polyols was improved by the type of polyols in the sequence of glycerol < erythritol < xylitol < sorbitol.3 Polyols and sugars can increase the water’s surface tension, leading to preferential hydration of proteins in aqueous solutions.1 Similarly, some alcohols and ethers as nonaqueous media have been found highly compatible with enzymes following different mechanisms. tert-Butanol is a common solvent for lipase-catalyzed transesterification and ammoniolysis reactions, producing relatively high enzyme activities; thus, many studies compared enzyme activities in other media such as ionic liquids (ILs) with that in tert-butanol.4−10 Our recent study11 suggested that Novozym 435 (immobilized lipase B from Candida antarctica (CALB)) was more active in tert-butanol than in a number of ILs including ether-functionalized ILs, as assayed by the transesterification reaction between ethyl sorbate and 1-propanol. In addition, tert-butanol is less inhibitory to the enzyme and less reactive as a substrate than 1-butanol (a primary alcohol).4 Molecular dynamics simulations of CALB indicate that lipase structure in tert-butanol resembles that in three-site model (TIP3P) water; specifically, the high enzyme compatibility of tert-butanol can be attributed to several factors including high protein flexibility in tert-butanol, preservation of entrance size for the substrate as well as the enzyme’s binding pocket size, and maintenance of hydrogen bonding of Ser105 with His224 (in the active site of CALB, Ser105–His224–Asp187 is the so-called “catalytic triad”12,13).14

Some ethers are also benign solvents for nonaqueous biocatalysis. Diisopropyl ether often leads to high lipase activities in enzymatic transesterification reactions.15−18 Ethers (such as 2,2-dimethoxypropane and 2-ethoxyethyl ether) have been found compatible with cross-linked enzyme aggregates of Penicillin G acylase resulting in high conversions in enzymatic acylation of 6-aminopenicillanic acid and d-phenylglycine amide.19 For this reason, ether groups have been incorporated into IL structures to develop enzyme-compatible ionic solvents.20,21

However, enzyme activities are typically lower in nonaqueous organic solvents than in aqueous solutions by several magnitudes.22,23 As an example, proteases (i.e., α-chymotrypsin and subtilisin) were 104–105 folds less active in neat octane than in water.22 To develop “water-like” nonaqueous solvents, we combined both features of tert-alcohols and ethers to design dual-functionalized ILs, hypothesizing that the tailored structures offer both hydrogen-bond-donating (−OH) and -accepting (R–O–R) properties and create “water-like” environments for enzymes.

ILs functionalized with ether groups have been synthesized and utilized in enzymatic reactions.20,21,24 However, neither tert-alcohol-functionalized ILs nor ILs dual-functionalized with alcohol and ether groups have been developed for biocatalysis, although tert-alcohol-functionalized imidazolium mesylate (CH3SO3−) ILs were evaluated as effective phase-transfer catalysts (PTCs) in substitution reactions including fluorination with high chemoselectivity.25−27 These mesylate salts also exhibited antimicrobial and antibiofilm activities.28 Furthermore, the Chi and Shinde groups29,30 covalently attached tert-alcohol-functionalized ILs onto polystyrene, resulting in reusable solid PTCs for efficient aliphatic nucleophilic substitutions by alkali metal salts. The present study aims to synthesize dual-functionalized ILs by grafting both tert-alcohol and ether groups to the cation core (such as imidazolium) and hypothesizes that these ILs have high compatibility with the lipase (CALB), as assayed by a transesterification reaction.

2. Results and Discussion

2.1. Preparation of Dual-Functionalized ILs and Their Compatibility with CALB

Dual-functionalized ILs carrying both tert-alcohol and ether groups on their cations were synthesized by a three-step reaction strategy (Scheme 1): (a) nucleophilic attack of isobutylene oxide by imidazole; (b) nucleophilic substitution of tert-butanol-grafted imidazole with alkoxyalkyl halide; (c) metathesis to replace halide anion with Tf2N– resulting in a hydrophobic IL. To prepare tert-alcohol-substituted imidazole, the Arnold group31 mixed an equal molar mixture of imidazole and epoxide in a high pressure ampoule at 50 °C for 12 h; later, this method was further expanded to the reactions of various epoxides with imidazole or benzimidazole.32 Following this approach, a number of tert-alcohol-functionalized imidazolium mesylate ILs were synthesized.25−27 The drawback of this solventless approach of reacting imidazole with epoxides is the formation of yellow/brown byproduct(s), which is difficult to remove. Takemura et al.33 reacted benzimidazole with 2-methyl-2-phenyloxirane in ethanol with the addition of sodium acetate by refluxing for 8 h. We adopted this method by refluxing imidazole and isobutylene oxide in ethanol in the presence of sodium acetate; sodium acetate became partially soluble in ethanol and acted as a base to promote the ring-opening attack of less substituted carbon in epoxide. Thus, this method is more regioselective than other approaches. The structures and characterizations of our dual-functionalized ILs are shown in the Supporting Information.

Scheme 1. Three-Step Synthesis of Dual-Functionalized ILs.

Scheme 1

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The synthetic activity of Novozym 435 (immobilized C. antarctica lipase B (CALB)) in these ILs was assessed by a highly sensitive synthetic reaction of ethyl sorbate with 1-propanol (Scheme 2), which was developed and evaluated by our group earlier.11,34,35 As illustrated in Table 1, the lipase in tert-butanol (trial 1) and diisopropyl ether (trial 2) showed high synthetic activities (6.23–8.57 μmol min–1 g–1 CALB). Due to their high compatibility with enzymes, these two organic solvents have been used as “golden standards” in the literature for comparing enzyme activities.4−10,15−18 Common enzyme-compatible ILs (i.e., [BMIM][PF6], [BMIM][Tf2N], and [BMIM][BF4]) only afforded lipase activities in the range of 3.41–5.12 μmol min–1 g–1 CALB (trials 3–5 in Table 1). In these “classical” ILs, enzyme activities are usually similar to or only slightly higher than that in tert-butanol4,5,7,8 and are typically below the activity in diisopropyl ether.15,18 A single functionalization, tert-butanol-grafted [Et-Im-t-BuOH][Tf2N] (trial 6), afforded a high lipase activity (8.04 μmol min–1 g–1 CALB) that is comparable with diisopropyl ether. A longer alkyl chain-grafted IL [Bu-Im-t-BuOH][Tf2N] (trial 7) remains as a solid at the reaction temperature (50 °C) and, thus, could not be used as a solvent for this reaction. In contrast, several dual-functionalized ionic liquids (trials 8, 11, 12, 14, and 15) led to very high CALB activities in the range of 9.06–12.36 μmol min–1 g–1 CALB (with >99% selectivities, see Scheme 2), which are up to 2–4 folds higher than those in nonfunctionalized “classical” ionic liquids, nearly double the activity in tert-butanol, and over 40% higher than that in diisopropyl ether. This degree of enzyme activation has not been observed in the literature. 2-Methyl-substituted imidazolium IL (trial 16) showed a lower enzyme activity of 6.97 μmol min–1 g–1 CALB, but it is still comparable with tert-butanol. The water content has a drastic impact on lipase activity as assayed by this transesterification reaction; when comparing trials 8, 9, and 10 (0.02–0.05 wt % water) and trials 12 with 13 (0.01–0.02 wt % water), we found CALB activities decreased drastically from 12.36 to 5.66 and from 9.06 to 6.26 μmol min–1 g–1 CALB, respectively, even with a small increase in water content. Therefore, a fine control of water content in ionic solvents is critical for achieving high transesterification activities.

Scheme 2. Transesterification Reaction between Ethyl Sorbate and 1-Propanol Catalyzed by a Lipase.

Scheme 2

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Table 1. Lipase-Catalyzed Transesterification between Ethyl Sorbate and 1-Propanola.

trial solvent (water, wt %)b viscosity at 30 °C (mPa s)c density at 30 °C (g cm–3)c enzyme activity (μmol min–1 g–1 CALB) selectivity
1 tert-butanol (0.02) 4.31 (25 °C)36 0.7887 (20 °C)36 6.23 >99%
2 diisopropyl ether (0.02) 0.29937 0.71337 8.57 >99%
3 [BMIM][PF6] (0.01) 205.8 1.362 3.87 86.6
4 [BMIM][Tf2N] (0.01) 41.4 1.430 5.12 >99%
5 [BMIM][BF4] (0.03) 84.638 1.19838 3.41 >99%
6 [Et-Im-t-BuOH][Tf2N] (0.02) 173.0 1.435 8.04 >99%
7 [Bu-Im-t-BuOH][Tf2N] (0.01) solid solid –d –d
8 [CH3OCH2CH2-Im-t-BuOH][Tf2N] (0.02) 303.0 (71.5 at 50 °C) 1.421 (1.406 at 50 °C) 12.36 >99%
9 [CH3OCH2CH2-Im-t-BuOH][Tf2N] (0.03)     7.71 >99%
10 [CH3OCH2CH2-Im-t-BuOH][Tf2N] (0.05)     5.66 >99%
11 [CH3CH2OCH2CH2-Im-t-BuOH][Tf2N] (0.02) 231.7 1.390 10.54 >99%
12 [(CH3)2CHOCH2CH2-Im-t-BuOH][Tf2N] (0.01) 274.0 1.357 9.06 >99%
13 [(CH3)2CHOCH2CH2-Im-t-BuOH][Tf2N] (0.02)     6.26 >99%
14 [CH3(OCH2CH2)2-Im-t-BuOH][Tf2N] (0.02) 299.7 1.407 9.68 >99%
15 [CH3(OCH2CH2)3-Im-t-BuOH][Tf2N] (0.02) 325.5 1.378 10.18 >99%
16 [CH3OCH2CH2-MIm-t-BuOH][Tf2N] (0.02) 415.9 1.415 6.97 >99%
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a

The transesterification reaction was conducted by adding ethyl sorbate (5 mM) and 1-propanol (0.67 M) in 1.0 mL of the solvent with the presence of 20 mg of Novozym 435 at 50 °C.

b

A coulometric Karl Fischer titrator was used to measure the water contents at 22 °C with a Hydranal Coulomat AG as the analyte.

c

An Anton Paar SVM 3000 viscometer was used to determine the dynamic viscosity and density data at 30 °C (except noted otherwise).

d

The reaction mixture solidified and no sample could be taken.

One aspect of these dual-functionalized ILs that needs improvement is their relatively high dynamic viscosities (∼300 mPa s at 30 °C, see Table 1) although it was not problematic for the current transesterification reaction at 50 °C (much less viscous, e.g., [CH3OCH2CH2-Im-t-BuOH][Tf2N] has a viscosity of 71.5 mPa s at 50 °C); however, these viscosity values appear much higher than those of [BMIM][Tf2N] (41.4 mPa s at 30 °C) and [CH3OCH2CH2-Im-Et][Tf2N] (33.1 mPa s at 30 °C).11 It is known that the covalent addendum of an ether group to an ionic liquid usually reduces its viscosity, whereas the addition of an alcohol group increases the viscosity (mainly due to hydrogen bonding).39 Our current effort is underway to fine-tune these IL structures to lower their viscosities while maintaining their high enzyme compatibility.

2.2. Fluorescence Spectra and Thermal Stability of CALB in ILs

To assess protein structural changes in different solvents, fluorescence emission spectroscopy is a convenient technique. After CALB in phosphate buffer (pH 7.5, 20 mM) was excited at 280 nm, the emission maximum was observed at 313 nm due to both tryptophan and tyrosine residues (Figure 1), which is used as a baseline spectrum for native enzyme structure. One challenge in measuring fluorescence emission spectra of enzymes in hydrophobic solvents is their poor solubility in these media. Therefore, we added 10 μL of aqueous lipase (20 mg mL–1 CALB) into each solvent (1.0 mL) instead of enzyme power for a number of considerations: (1) the final water content in the mixture was kept low (1%, v/v) after adding aqueous droplets; (2) aqueous lipase was easier to disperse in hydrophobic solvents as small droplets than powder enzyme; (3) the amount of CALB used was low (0.2 mg mL–1 overall); this low quantity of lipase can be easily dispersed by using aqueous lipase than the powder. Following this approach, all other CALB samples in different solvents were excited at 280 nm, and the solvent emission spectra were deducted (see Figures S1–S4 in the Supporting Information). Since CALB in aqueous droplets was dispersed in hydrophobic solvents (such as Tf2N–-based ILs in Figure 1), the enzyme is not homogeneously distributed in hydrophobic solutions when comparing with the case of lipase being fully solubilized in aqueous solutions or hydrophilic ILs, which could cause different spectrum profiles and signal-to-noise ratios. For this reason, the change in fluorescence intensity maximum in hydrophobic solvents is not a warrant of enzyme denaturation, but the shift in emission maximum becomes more meaningful. As illustrated in Figure 1, CALB displayed the emission maximum at 315 nm in tert-butanol and 305 nm (blue shift) in diisopropyl ether, which explains high lipase activities in these two solvents; a more red shift in emission maximum (336 nm) in [CH3CH2OCH2CH2-MIm-t-BuOH][Tf2N] than that (322 nm) in [CH3CH2OCH2CH2-Im-t-BuOH][Tf2N] correlates with a lower CALB activity in the former IL since the redshift often implies the protein denaturation.40 However, fluorescence emission spectra could not fully explain why the lipase was much more active in some of these functionalized ILs than in organic solvents.

Figure 1.

Figure 1

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Effect of solvents on fluorescence emission spectra of CALB (0.2 mg mL–1 of free CALB dissolved or suspended in each solvent; see fluorescence spectrophotometer conditions in the Section 4.4).

In addition to producing high enzyme activities, functionalized ILs offer unique thermal stability for the lipase. As illustrated in Figure 2, the thermal stability of Novozym 435 was compared after its incubation at 50 °C for 24–48 h in tert-butanol, diisopropyl ether, [CH3OCH2CH2-Im-Et][Tf2N], or [CH3OCH2CH2-Im-t-BuOH][Tf2N]. The residual enzyme activity after 24–48 h of incubation at 50 °C in t-butanol was only 17–19%; in fact, the clear solution became cloudy after 24 h in tert-butanol implying the dissociation of CALB from the beads or the partial dissolution of the acrylic beads. In contrast, the thermal stability of lipase in diisopropyl ether and two ILs was significantly higher: 81–92% residual activities after 24 h and 65–69% residual activities after 48 h. Therefore, the thermal stability of CALB in functionalized ILs is much higher than that in tert-butanol. This finding is consistent with our earlier study41 suggesting that short-chain-glycol-grafted ILs were able to increase the lipase’s thermal stability.

Figure 2.

Figure 2

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Thermal stability of Novozym 435 in various solvents. Reaction conditions: a closed vial containing 20 mg of Novozym 435 and 1.0 mL of the solvent was placed in the 50 °C oil bath for 24 or 48 h under gentle agitation. At the end of incubation, the mixture was cooled to room temperature, followed by the addition of ethyl sorbate (50 μL, 100 mM in 1-propanol). The reaction mixture was stirred in an oil bath at 50 °C. The lipase activity was determined following the procedures in the Section 4.3.

3. Conclusions

Water-like dual-functionalized ILs were synthesized by grafting two functional groups (ether and tert-alcohol) onto the imidazolium ring. These unique ionic solvents were found highly compatible with lipase CALB in terms of high enzyme activities and stabilities. Fluorescence emission spectra confirmed the characteristic protein peak being generally maintained in dual-functionalized ILs.

4. Experimental Section

4.1. Materials

Enzymes purchased from Sigma-Aldrich (St. Louis, MO) include C. antarctica lipase B (CALB) as a recombinant from Aspergillus oryzae (catalog #62288, Lot #BCBP3380V) and Novozym 435 (CALB immobilized on the acrylic resin) (Catalog #L4777, Lot #SLBW1544). 2-Bromoethyl methyl ether and 2-bromoethyl ethyl ether were products of BeanTown Chemical (Hudson, NH). Lithium bis(trifluoromethylsulfonyl)imide (Li[Tf2N]) was obtained from Matrix Scientific (Columbia, SC). 1-Ethyl-3-(2-methoxyethyl)imidazolium bis(trifluoromethylsulfonyl)imide ([MeOCH2CH2-Im-Et][Tf2N]) was prepared by our earlier study.24

4.2. Synthesis of Dual-Functionalized ILs

The synthesis of a tert-butanol- and ether- dual-functionalized ionic liquid (IL) was illustrated as an example. The first step was to graft tert-butanol group onto the imidazolium ring, which was a modification of a literature method.33 Imidazole (∼10 g, 1 equiv) and 2,2-dimethyloxirane (1.2 equiv) were mixed in 100 mL of ethanol along with the addition of sodium acetate (1.1 equiv). After refluxing for 24 h, the reaction mixture was cooled first in the air to room temperature and was further cooled in an ice bath. The salt was removed by vacuum filtration, followed by vacuum evaporation of ethanol from the filtrate. The precipitated salt was further filtered off (when needed, dichloromethane was added to dilute the mixture). The crude product was dissolved in 100 mL of dichloromethane and dried by anhydrous Na2SO4. After the salt was filtered off and the solvent was evaporated under vacuum, a slightly yellow liquid product was obtained. (Note: N-(2-hydroxy-2-methylpropyl)imidazole is very soluble in water; therefore, the extraction of the organic phase by water to purify the product is proven to be inefficient.)

The second step involved the alkylation of tert-butanol-grafted imidazole to become an ionic salt. tert-Butanol-grafted imidazole (∼10 g, 1.0 equiv) was mixed with 1.1 equiv alkyl halide (such as 2-bromoethyl methyl ether) in 100 mL of anhydrous acetonitrile. The reaction mixture was wrapped by an aluminum foil to minimize light-initiated reactions and refluxed for 24 h. At the end of the reaction, the solvent was removed under vacuum, the halide compound was rinsed by diethyl ether twice (50 mL each time).

The third step was to convert halide to Tf2N– anion. Into the aqueous solution of halide salt (∼10 g, 1.0 equiv), aqueous lithium bis(trifluoromethylsulfonyl)imide (Li[Tf2N], 1.05 equiv) was added in drops at room temperature. The mixture was stirred for 30 min and then sat still for a complete phase separation; the bottom hydrophobic layer was dissolved in dichloromethane. The mixture in a separatory funnel was extracted by deionized water several times until no halide in the aqueous phase could be detected by the silver nitrate test. After drying the organic layer by sodium sulfate and evaporating dichloromethane, the hydrophobic IL was further rinsed by n-heptane twice (50 mL each). The trace amount of n-heptane was removed from IL by vacuum evaporation. The purified IL in a small beaker was dried in a vacuum oven (80 °C with 25 mmHg vacuum) for 1 week. The structure of the dried IL was determined by 1H and 13C NMR, MS spectra (see the Supporting Information).

4.3. Lipase-Catalyzed Transesterification between Ethyl Sorbate and 1-Propanol

In a capped reaction vial, 50 μL of 1-propanol containing 100 mM ethyl sorbate was mixed with 1.0 mL of IL (or other solvents). The overall concentrations of ethyl sorbate and 1-propanol in the reaction mixture were 5 mM and 0.67 M, respectively. After adding 20 mg of Novozym 435, the reaction mixture was incubated and stirred in an oil bath at 50 °C. Periodically in the first hour (each 15 min), 50 μL of the reaction mixture was taken and diluted with 1.0 mL of methanol, followed by centrifugation for 2 min. Then, the supernatant was transferred to an autosampler vial for the high-performance liquid chromatography (HPLC) analysis. The concentration of propyl sorbate was computed from its integrated area using the standard curve of ethyl sorbate (as propyl sorbate is not commercially available). Due to potential migration of traces of sorbic acid and sorbate ester from Novozym 435 beads,34 control experiments were performed without the addition of ethyl sorbate but with 50 μL of 1-propanol. For this reason, all reported lipase activities were initial reaction rates after deducting the control rates. The properly diluted reaction mixtures were analyzed by LC-20AD Shimadzu HPLC using a SPD-20A UV–visible dual-wavelength detector and an autosampler. The HPLC column used was a Phenomenex Kinetex C18 column (100 mm × 4.6 mm in dimension, 2.6 μm in particle size). The flow rate was set at 1.0 mL min–1. The isocratic eluent consisted of 60:40 (v/v) methanol/water; the UV–visible detection wavelength was 258 nm.

4.4. Fluorescence Emission Spectra of Free CALB in ILs

A Hitachi F-2500 fluorescence spectrophotometer was used to capture the fluorescence emission spectra. The excitation of CALB solution at 280 nm produced the emission of tyrosine and tryptophan residues near 300 nm. Free CALB (10 μL, 20 mg mL–1 dissolved in pH 7.5, 20 mM phosphate buffer) was mixed with 1.0 mL of the solvent (phosphate buffer, organic solvent, or IL) in a microcentrifuge tube through a gentle turning and shaking. The overall lipase concentration was 0.2 mg mL–1. The enzyme mixture was placed in a quartz cuvette (1.0 mL volume). Fluorescence spectra were collected at room temperature (22 °C) except the case of t-butanol at 30 °C (t-butanol has a low melting point of 25.8 °C).36 Both the excitation monochromator slit and the emission monochromator slit width were set at 5 nm. Other parameters include the photomultiplier voltage at 400 V and the response time of 0.08 s. The emission spectra were measured from 250 to 400 nm with a scanning rate of 300 nm min–1. The emission spectrum of each solvent itself was obtained under the same condition and subtracted from the lipase emission spectrum in the same solvent.

Acknowledgments

Acknowledgment is made to the donors of The American Chemical Society Petroleum Research Fund (PRF #60077-ND4) for partial support of this research. H.Z. acknowledges the startup fund support and Faculty Research and Publications Board (FRPB)’s publication fund award, both of which were provided by the University of Northern Colorado. The ESI-MS characterizations were completed by the Central Instrument Facility (CIF) at Colorado State University.

Supporting Information Available

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.9b02118.

  • NMR and MS characterizations of ionic liquids (ILs), the structures of [Et-Im-t-BuOH][Tf2N], [Bu-Im-t-BuOH][Tf2N], [CH3OCH2CH2-Im-t-BuOH][Tf2N] and other dual-functionalized ILs; fluorescence emission spectra of CALB in different solvents, including t-butanol, diisopropyl ether, [MeOCH2CH2-Im-t-BuOH][Tf2N], [MeOCH2CH2-MIm-t-BuOH][Tf2N] (PDF)

The authors declare no competing financial interest.

Supplementary Material

ao9b02118_si_001.pdf (796.3KB, pdf)

References

  1. Kaushik J. K.; Bhat R. Thermal stability of proteins in aqueous polyol solutions: Role of the surface tension of water in the stabilizing efect of plyols. J. Phys. Chem. B 1998, 102, 7058–7066. 10.1021/jp981119l. [DOI] [Google Scholar]
  2. Kaushik J. K.; Bhat R. Why Is trehalose an exceptional protein stabilizer? An analysis of the thermal stability of proteins in the presence of the compatible osmolyte trehalose. J. Biol. Chem. 2003, 278, 26458–26465. 10.1074/jbc.M300815200. [DOI] [PubMed] [Google Scholar]
  3. Obón J. M.; Manjón A.; Iborra J. L. Comparative thermostability of glucose dehydrogenase from Haloferax mediterranei. Effects of salts and polyols. Enzyme Microb. Technol. 1996, 19, 352–360. 10.1016/S0141-0229(96)00028-2. [DOI] [Google Scholar]
  4. Madeira Lau R.; van Rantwijk F.; Seddon K. R.; Sheldon R. A. Lipase-catalyzed reactions in ionic liquids. Org. Lett. 2000, 2, 4189–4191. 10.1021/ol006732d. [DOI] [PubMed] [Google Scholar]
  5. Madeira Lau R.; Sorgedrager M. J.; Carrea G.; van Rantwijk F.; Secundo F.; Sheldon R. A. Dissolution of Candida antarctica lipase B in ionic liquids: effects on structure and activity. Green Chem. 2004, 6, 483–487. 10.1039/b405693k. [DOI] [Google Scholar]
  6. Zhang K.-P.; Lai J.-Q.; Huang Z.-L.; Yang Z. Penicillium expansum lipase-catalyzed production of biodiesel in ionic liquids. Bioresour. Technol. 2011, 102, 2767–2772. 10.1016/j.biortech.2010.11.057. [DOI] [PubMed] [Google Scholar]
  7. van Rantwijk F.; Secundo F.; Sheldon R. A. Structure and activity of Candida antarctica lipase B in ionic liquids. Green Chem. 2006, 8, 282–286. 10.1039/B513062J. [DOI] [Google Scholar]
  8. Toral A. R.; de los Ríos A. P.; Hernández F. J.; Janssen M. H. A.; Schoevaart R.; van Rantwijk F.; Sheldon R. A. Cross-linked Candida antarctica lipase B is active in denaturing ionic liquids. Enzyme Microb. Technol. 2007, 40, 1095–1099. 10.1016/j.enzmictec.2006.08.027. [DOI] [Google Scholar]
  9. Royon D.; Daz M.; Ellenrieder G.; Locatelli S. Enzymatic production of biodiesel from cotton seed oil using t-butanol as a solvent. Bioresour. Technol. 2007, 98, 648–653. 10.1016/j.biortech.2006.02.021. [DOI] [PubMed] [Google Scholar]
  10. Degn P.; Pedersen L. H.; Duus J. O.; Zimmermann W. Lipase-catalysed synthesis of glucose fatty acid esters in tert-butanol. Biotechnol. Lett. 1999, 21, 275–280. 10.1023/A:1005439801354. [DOI] [Google Scholar]
  11. Zhao H.; Kanpadee N.; Jindarat C. Ether-functionalized ionic liquids for nonaqueous biocatalysis: Effect of different cation cores. Process Biochem. 2019, 81, 104–112. 10.1016/j.procbio.2019.03.018. [DOI] [Google Scholar]
  12. Uppenberg J.; Hansen M. T.; Patkar S.; Jones T. A. The sequence, crystal structure determination and refinement of two crystal forms of lipase B from Candida antarctica. Structure 1994, 2, 293–308. 10.1016/S0969-2126(00)00031-9. [DOI] [PubMed] [Google Scholar]
  13. Uppenberg J.; Oehrner N.; Norin M.; Hult K.; Kleywegt G. J.; Patkar S.; Waagen V.; Anthonsen T.; Jones T. A. Crystallographic and molecular-modeling studies of lipase B from Candida antarctica reveal a stereospecificity pocket for secondary alcohols. Biochemistry 1995, 34, 16838–16851. 10.1021/bi00051a035. [DOI] [PubMed] [Google Scholar]
  14. Park H. J.; Park K.; Yoo Y. J. Understanding the effect of tert-butanol on Candida antarctica lipase B using molecular dynamics simulations. Mol. Simul. 2013, 39, 653–659. 10.1080/08927022.2012.758850. [DOI] [Google Scholar]
  15. Itoh T.; Akasaki E.; Kudo K.; Shirakami S. Lipase-catalyzed enantioselective acylation in the ionic liquid solvent system: Reaction of enzyme anchored to the solvent. Chem. Lett. 2001, 30, 262–263. 10.1246/cl.2001.262. [DOI] [Google Scholar]
  16. Itoh T.; Han S.; Matsushita Y.; Hayase S. Enhanced enantioselectivity and remarkable acceleration on the lipase-catalyzed transesterification using novel ionic liquids. Green Chem. 2004, 6, 437–439. 10.1039/b405396f. [DOI] [PubMed] [Google Scholar]
  17. Itoh T.; Matsushita Y.; Abe Y.; Han S.; Wada S.; Hayase S.; Kawatsura M.; Takai S.; Morimoto M.; Hirose Y. Increased enantioselectivity and remarkable acceleration of lipase-catalyzed transesterification by using an imidazolium PEG-alkyl sulfate ionic liquid. Chem. - Eur. J. 2006, 12, 9228–9237. 10.1002/chem.200601043. [DOI] [PubMed] [Google Scholar]
  18. Itoh T.; Nishimura Y.; Ouchi N.; Hayase S. 1-Butyl-2,3-dimethylimidazolium tetrafluoroborate: the most desirable ionic liquid solvent for recycling use of enzyme in lipase-catalyzed transesterification using vinyl acetate as acyl donor. J. Mol. Catal. B: Enzym. 2003, 26, 41–45. 10.1016/S1381-1177(03)00147-4. [DOI] [Google Scholar]
  19. Cao L.; van Langen L. M.; van Rantwijk F.; Sheldon R. A. Cross-linked aggregates of penicillin acylase: robust catalysts for the synthesis of .beta.-lactam antibiotics. J. Mol. Catal. B: Enzym. 2001, 11, 665–670. 10.1016/S1381-1177(00)00078-3. [DOI] [Google Scholar]
  20. Zhao H. Methods for stabilizing and activating enzymes in ionic liquids—A review. J. Chem. Technol. Biotechnol. 2010, 85, 891–907. 10.1002/jctb.2375. [DOI] [Google Scholar]
  21. Zhao H. Protein stabilization and enzyme activation in ionic liquids: specific ion effects. J. Chem. Technol. Biotechnol. 2016, 91, 25–50. 10.1002/jctb.4837. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Zaks A.; Klibanov A. M. Enzymatic catalysis in nonaqueous solvents. J. Biol. Chem. 1988, 263, 3194–3201. [PubMed] [Google Scholar]
  23. Klibanov A. M. Why are enzymes less active in organic solvents than in water?. Trends Biotechnol. 1997, 15, 97–101. 10.1016/S0167-7799(97)01013-5. [DOI] [PubMed] [Google Scholar]
  24. Zhao H.; Afriyie L. O.; Larm N. E.; Baker G. A. Glycol-functionalized ionic liquids for hight-emperature enzymatic ring-opening polymerization. RSC Adv. 2018, 8, 36025–36033. 10.1039/C8RA07733A. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Shinde S. S.; Lee B. S.; Chi D. Y. Synergistic effect of two solvents, tert-alcohol and ionic liquid, in one molecule in nucleophilic fluorination. Org. Lett. 2008, 10, 733–735. 10.1021/ol702679d. [DOI] [PubMed] [Google Scholar]
  26. Shinde S. S.; Chi H. M.; Lee B. S.; Chi D. Y. tert-Alcohol-functionalized imidazolium ionic liquid: catalyst for mild nucleophilic substitution reactions at room temperature. Tetrahedron Lett. 2009, 50, 6654–6657. 10.1016/j.tetlet.2009.09.064. [DOI] [Google Scholar]
  27. Shinde S. S.; Patil S. N.; Ghatge A.; Kumar P. Nucleophilic fluorination using imidazolium based ionic liquid bearing tert-alcohol moiety. New J. Chem. 2015, 39, 4368–4374. 10.1039/C5NJ00481K. [DOI] [Google Scholar]
  28. Navale G. R.; Dharne M. S.; Shinde S. S. Antibiofilm activity of tert-BuOH functionalized ionic liquids with methylsulfonate counteranions. RSC Adv. 2015, 5, 68136–68142. 10.1039/C5RA12854D. [DOI] [Google Scholar]
  29. Shinde S. S.; Lee B. S.; Chi D. Y. Polymer-supported protic functionalized ionic liquids for nucleophilic substitution reactions: superior catalytic activity compared to other ionic resins. Tetrahedron Lett. 2008, 49, 4245–4248. 10.1016/j.tetlet.2008.04.151. [DOI] [Google Scholar]
  30. Shinde S. S.; Patil S. N. One molecule of ionic liquid and tert-alcohol on a polystyrene-support as catalysts for efficient nucleophilic substitution including fluorination. Org. Biomol. Chem. 2014, 12, 9264–9271. 10.1039/C4OB01781A. [DOI] [PubMed] [Google Scholar]
  31. Arnold P. L.; Rodden M.; Davis K. M.; Scarisbrick A. C.; Blake A. J.; Wilson C. Asymmetric Lithium(I) and copper(II) alkoxy-N-heterocyclic carbene complexes; crystallographic characterisation and Lewis acid catalysis. Chem. Commun. 2004, 1612–1613. 10.1039/b404614e. [DOI] [PubMed] [Google Scholar]
  32. Torregrosa R.; Pastor I. M.; Yus M. Solvent-free direct regioselective ring opening of epoxides with imidazoles. Tetrahedron 2007, 63, 469–473. 10.1016/j.tet.2006.10.055. [DOI] [Google Scholar]
  33. Takemura N.; Kuninobu Y.; Kanai M. Copper-catalyzed C-H alkoxylation of azoles. Org. Lett. 2013, 15, 844–847. 10.1021/ol303533z. [DOI] [PubMed] [Google Scholar]
  34. Zhao H.; Song Z. Migration of reactive trace compounds from Novozym 435 into organic solvents and ionic liquids. Biochem. Eng. J. 2010, 49, 113–118. 10.1016/j.bej.2009.12.004. [DOI] [Google Scholar]
  35. Zhao H.; Baker G. A.; Holmes S. New eutectic ionic liquids for lipase activation and enzymatic preparation of biodiesel. Org. Biomol. Chem. 2011, 9, 1908–1916. 10.1039/c0ob01011a. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Rumble J. R.CRC Handbook of Chemistry and Physics, 99th ed.; CRC Press, Taylor & Francis Group: New York, 2018. [Google Scholar]
  37. Meng X.; Wu J.; Liu Z. Viscosity and density measurements of diisopropyl ether and dibutyl ether at different temperatures and pressures. J. Chem. Eng. Data 2009, 54, 2353–2358. 10.1021/je8005369. [DOI] [Google Scholar]
  38. Harris K. R.; Kanakubo M.; Woolf L. A. Temperature and pressure dependence of the viscosity of the ionic liquid 1-butyl-3-methylimidazolium tetrafluoroborate: Viscosity and density relationships in ionic liquids. J. Chem. Eng. Data 2007, 52, 2425–2430. 10.1021/je700370z. [DOI] [Google Scholar]
  39. Tang S.; Baker G. A.; Zhao H. Ether- and alcohol-functionalized task-specific ionic liquids: attractive properties and applications. Chem. Soc. Rev. 2012, 41, 4030–4066. 10.1039/c2cs15362a. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Lozano P.; De Diego T.; Iborra J. L. Dynamic structure/function relationships in the α-chymotrypsin deactivation process by heat and pH. Eur. J. Biochem. 1997, 248, 80–85. 10.1111/j.1432-1033.1997.00080.x. [DOI] [PubMed] [Google Scholar]
  41. Zhao H.; Jones C. L.; Cowins J. V. Lipase dissolution and stabilization in ether-functionalized ionic liquids. Green Chem. 2009, 11, 1128–1138. 10.1039/b905388c. [DOI] [Google Scholar]

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