Penicillium expansum lipase-catalyzed production of biodiesel in ionic liquids

Penicillium expansum lipase (PEL) was used to catalyze biodiesel production from corn oil in [BMIm][PF₆]¹ (an ionic liquid, IL) and tert-butanol. Both systems were optimized in terms of MeOH/oil molar ratio, reaction temperature, enzyme loading, solvent volume, and water content. The high conversion obtained in the IL (86%) as compared to that in tert-butanol (52%) demonstrates that the IL is a superior solvent for PEL-catalyzed biodiesel production. Poor yields were obtained in a series of hydrophilic ILs. Addition of salt hydrates affected biodiesel production predominantly through the specific ion (Hofmeister) effect. The impact of methanol on both activity and stability of PEL in the IL and in hexane was investigated, in comparison to the results obtained by two commonly used lipases, Novozym 435 and Lipozyme TLIM. The results substantiate that while different lipases show different resistance to methanol in different reaction systems, PEL is tolerant to methanol in both systems.     

The role of different anions in ionic liquids on Pseudomonas cepacia lipase catalyzed transesterification and hydrolysis

Lipase Pseudomonas cepacia (PS) catalyzed transesterification of ethyl 3-phenylpropanoate with eleven alcohols was investigated in three ionic liquids [ILs], [Bmim]BF₄, [Bmim]PF₆, and [Bmim]Tf₂N, consisting of an identical cation and different anions. The yields were higher in hydrophobic ILs [Bmim]Tf₂N (55–96%) and [Bmim]PF₆ (22–95%), than in hydrophilic [Bmim]BF₄ (0–19%). The incubation of lipase PS in hydrophobic ILs for a period of 20–300 days at room temperature resulted in an increased yield of 62–98% in [Bmim]Tf₂N and 45–98% in [Bmim]PF₆, respectively. The lipase PS-hydrophobic IL mixture was recycled five times without any decrease in the yield of the products. In another set of experiments, the hydrolytic activity of the enzyme was determined after incubation in each of the three ILs and in hexane for 20 days at room temperature. It was found to be 1.8- and 1.6-fold higher in [Bmim]Tf₂N and [Bmim]PF₆, respectively, remained unchanged in [Bmim]BF₄ and was 1.6 times lower in hexane as compared to the non-incubated enzyme.     

Ionic liquids as a reaction medium for lipase-catalyzed methanolysis of sunflower oil

Ionic liquids, 1-butyl-3-methyl imidazolium hexafluorophosphate ([BMIm][PF₆]) and 1-ethyl-3-methyl imidazolium hexafluorophosphate ([EMIm][PF₆]), were used for the methanolysis of sunflower oil using Candida antarctica lipase (Novozyme 435) and gave yields of fatty acid methyl esters at 98–99% within 10 h. The optimum conditions of methanolysis in hydrophobic ionic liquids are 2% (w/w) lipase, 1:1 (w/w) oil/ionic liquid and 1:8 (mol/mol) oil/methanol at 58–60°C. Methanolysis using hydrophilic ionic liquids, 3-methyl imidazolium tetrafluoroborate ([HMIm][BF₄]) and 1-butyl-3-methyl imidazolium tetrafluoroborate ([BMIm][BF₄]), gave very poor yields. A hydrophobic ionic liquid thus protects the lipase from methanol. Recovered ionic liquids and lipase were used for four successive reaction cycles without any significant loss of activity.     

Catalytic performance of cross-linked enzyme aggregates of Penicillium expansum lipase and their use as catalyst for biodiesel production

Cross-linked enzyme aggregates (CLEAs) of lipase from Penicillium expansum (PEL) were prepared directly from fermentation broth, a more practical and economically viable procedure than the generally used methods that require purified or partially purified enzymes for CLEA preparation. A systematic study of the activity and stability of PEL-CLEAs was undertaken in aqueous solution, organic solvents, and ionic liquids (ILs). Immobilization of the enzyme resulted in a significantly enhanced stability in aqueous solution with regard to pH and temperature. PEL-CLEAs showed an improved activity in the IL [BMIm][PF₆] relative to that observed in hexane, both keeping increased with temperature (up to 90 °C in the IL and 60 °C in hexane). The effect of water content and water activity in these two nonaqueous media showed similar patterns as for the uncrosslinked enzyme. The half life of the CLEAs was higher in hydrophobic organic solvents (hexane and chloroform) than in aqueous solution, and presented a sigmoid relationship with the log P of the organic solvent tested. PEL-CLEAs catalyzed biodiesel production from microalgal oil in the IL [BMIm][PF₆] with a conversion of 85.7%, demonstrating that they can be taken as a promising catalyst for this application.     

α-Rhamnosidase and β-glucosidase expressed by naringinase immobilized on new ionic liquid sol-gel matrices: Activity and stability studies

Novel ionic liquid (IL) sol-gel materials development, for enzyme immobilization, was the goal of this work. The deglycosylation of natural glycosides were performed with α-l-rhamnosidase and β-d-glucosidase activities expressed by naringinase. To attain that goal ILs with different structures were incorporated in TMOS/Glycerol sol-gel matrices and used on naringinase immobilization.The most striking feature of ILs incorporation on TMOS/Glycerol matrices was the positive impact on the enzyme activity and stability, which were evaluated in fifty consecutive runs. The efficiency of α-rhamnosidase expressed by naringinase TMOS/Glycerol@ILs matrices increased with cation hydrophobicity as follows: [OMIM] > [BMIM] > [EMIM] > [C₂OHMIM] > [BIM] and [OMIM] ≈ [E₂-MPy] ? [E₃-MPy]. Regarding the imidazolium family, the hydrophobic nature of the cation resulted in higher α-rhamnosidase efficiencies: [BMIM]BF₄ ? [C₂OHMIM]BF₄ ? [BIM]BF₄. Small differences in the IL cation structure resulted in important differences in the enzyme activity and stability, namely [E₃-MPy] and [E₂-MPy] allowed an impressive difference in the α-rhamnosidase activity and stability of almost 150%. The hydrophobic nature of the anion influenced positively α-rhamnosidase activity and stability. In the BMIM series the more hydrophobic anions (PF₆⁻, BF₄⁻ and Tf₂N⁻) led to higher activities than TFA. SEM analysis showed that the matrices are shaped lens with a film structure which varies within the lens, depending on the presence and the nature of the IL.The kinetics parameters, using naringin and prunin as substrates, were evaluated with free and naringinase encapsulated, respectively on TMOS/Glycerol@[OMIM][Tf₂N] and TMOS/Glycerol@[C₂OHMIM][PF₆] and on TMOS/Glycerol. An improved stability and efficiency of α-l-rhamnosidase and β-glucosidase expressed by encapsulated naringinase on TMOS/Glycerol@[OMIM][Tf₂N] and TMOS/Glycerol@[C₂OHMIM][PF₆] were achieved. In addition to these advantageous, with ILs as sol-gel templates, environmental friendly processes can be implemented.     

Stereoselective synthesis of (R)-1-chloro-3(3,4-difluorophenoxy)-2-propanol using lipases from Pseudomonas aeruginosa in ionic liquid-containing system

Direct transesterification of (R,S)-1-chloro-3-(3,4-difluorophenoxy)-2-propanol (rac-CDPP) (a key intermediate in the synthesis of the chiral drug (S)-lubeluzole) with vinyl butyrate by lipases from Pseudomonas aeruginosa (P. aeruginosa) MTCC 5113 was performed in hexane with ionic liquids (ILs) 1-butyl-3-methyl imidazolium hexafluorophosphate [BMIm][PF₆] and 1-butyl-3-methyl imidazolium tetrafluoroborate [BMIm][BF₄] as co-solvents. The maximum conversion (>49%) and enantiomeric excess (ee > 99.9%) was achieved in 6 h of incubation at 30 °C with [BMIm][PF₆] as co-solvent in a two-phase system. The enzyme was able to perform with the same specificity even at 60 °C in the presence of ILs. It was possible to use lipases repeatedly for more than 10 times while still maintaining absolute enantioselectivity and reactivity. Stability studies on lipases from P. aeruginosa in ILs revealed the fact that the enzyme constancy and the reactivity in catalyzing transesterification of rac-CDPP into (S)-1-chloro-3-(3,4-difluorophenoxy)-2-butanoate was of the order of [BMIm][PF₆] > [BMIm][BF₄] in two-phase system.     

Enantioselective transesterification of racemic phenyl ethanol and its derivatives in organic solvent and ionic liquid using Pseudomonas aeruginosa lipase

The transesterification of 1-phenyl ethanol has been carried out using lipases from Pseudomonas aeruginosa MTCC 5113, to obtain chirally pure aryl ethanols with good yield and excellent enantioselectivity. The lipase from P. aeruginosa gave good conversion and moderate enantioselectivity (ee) in organic solvents, however, when the catalytic amount of ionic liquids were added in the reaction mixture, excellent enantioselectivity was obtained. Moreover, the change in enantiomer preference was seen in the presence of catalytic amount of ionic liquids. The findings revealed that hydrophobic ionic liquids (two-phase system) were the best solvents and 4-substituted aryl ethanols were the pre-eminent substrates for such type of reactions. The preparative scale (5 g) transesterification of 1-phenylethanol using lipases from P. aeruginosa yielded S-(−)-1-phenyl ethanol with 39% yield and >99% ee in hexane and 46% yield and >99% ee in [BMIm][PF₆].     

Enhanced transesterification of ethyl ferulate with glycerol for preparing glyceryl diferulate using a lipase in ionic liquids as reaction medium

Glyceryl diferulate (DFG) is a water-soluble ester of ferulic acid. A novel ionic liquid (IL) system for enzymatic transesterification of ethyl ferulate (EF) with glycerol to produce DFG was developed. Of three ILs with different anions (BF₄ ^?, PF₆ ^? and TF₂N^?) and cations (BDMIM, OMIM, HMIM, BMIM, and EMIM), EMIMTF₂N proved the best using a commercial lipase. It had a significant protective effect against thermal inactivation of the enzyme. High EF conversion (~100 %) and DFG yield (45 %) were achieved using 45 mg enzyme/ml; temperature, 70 °C; reaction time, 12 h.     

Enantioselective acylation of (RS)-phenylethylamine catalysed by lipases

The enzymatic acylation of (RS)-phenylethylamine with different acyl donors catalysed by lipases, was studied in organic solvents with different hydrophobicities and in mixtures with ionic liquids ((ILs); [BMIm][BF₄], [BMIm][SCN], [BMIm][Cl] and [BMIm][PF₆]). Using lipases from Candida antarctica B (CAL-B) and from Aspergillus niger higher conversion degrees and E-values were obtained with ethyl acetate as the acyl donor. When CAL-B was used as the biocatalyst, in a two-phase system formed by [BMIm][X]/dichloromethane or [BMIm][X]/chloroform, the selectivity was better than that obtained in pure organic solvents. The selectivity was found to be related to individual anions in ILs. In this reaction, the ion effectiveness in enhancing the enzyme selectivity followed the series: Cl⁻ > SCN⁻ > BF₄⁻ > PF₆⁻ in mixtures with dichloromethane, and PF₆⁻ > BF₄⁻ > SCN⁻ > Cl⁻ in mixtures with chloroform.     

Production of biodiesel fuel from soybean oil catalyzed by fungus whole-cell biocatalysts in ionic liquids

The methanolysis of soybean oil to produce a fatty acid methyl ester (ME, i.e., biodiesel fuel) was catalyzed by lipase-producing filamentous fungi immobilized on biomass support particles (BSPs) as a whole-cell biocatalyst in the presence of ionic liquids. We used four types of whole-cell biocatalysts: wild-type Rhizopus oryzae producing triacylglycerol lipase (w-ROL), recombinant Aspergillus oryzae expressing Fusarium heterosporum lipase (r-FHL), Candida antarctica lipase B (r-CALB), and mono- and diacylglycerol lipase from A. oryzae (r-mdlB). w-ROL gave the high yield of fatty acid methyl ester (ME) in ionic liquid [Emim][BF₄] or [Bmim][BF₄] biphasic systems following a 24 h reaction. While lipases are known to be severely deactivated by an excess amount of methanol (e.g. 1.5 Mequiv. of methanol against oil) in a conventional system, methanolysis successfully proceeded even with a methanol/oil ratio of 4 in the ionic liquid biphasic system, where the ionic liquids would work as a reservoir of methanol to suppress the enzyme deactivation. When only w-ROL was used as a biocatalyst for methanolysis, unreacted mono-glyceride remained due to the 1,3-positional specificity of R. oryzae lipase. High ME conversion was attained by the combined use of two types of whole-cell biocatalysts, w-ROL and r-mdlB. In a stability test, the activity of w-ROL was reduced to one-third of its original value after incubation in [Bmim][BF₄] for 72 h. The stability of w-ROL in [Bmim][BF₄] was greatly enhanced by cross-linking the biocatalyst with glutaraldehyde. The present study demonstrated that ionic liquids are promising candidates for use as the second solvent in biodiesel fuel production by whole-cell biocatalysts.     

Enzymatic synthesis of poly(ε-caprolactone) in monocationic and dicationic ionic liquids

Enzymatic polymerization can offer metal-free routes to polymer materials that could be used in biomedical applications. To take advantage of the unique properties of ionic liquids (ILs) for enzyme stability, monocationic ionic liquid (MIL) and dicationic ionic liquid (DIL) were used to promote the ring-opening polymerization of ε-caprolactone (ε-CL) using Candida antarctica lipase B as catalyst. Considering the molecular weight (M _n ) and reaction yield of the resulting polymer (PCL), high density and viscosity of ILs would be good, especially in the case of DIL. With the same total alkyl chain length, the density and viscosity of [C₄(C₆Im)₂][PF₆]₂ were higher than that of [C₁₂MIm][PF₆]. Using a lipase/CL/ILs ratio of 1:20:20 (by wt) for 48 h at 90 °C, the highest M _n and reaction yield of PCL were 26,200 g/mol and 62 % with [C₄(C₆Im)₂][PF₆]₂, while the M _n and reaction yield of PCL obtained in [C₁₂MIm][PF₆] were 11,700 g/mol and 37 %.     

Efficient enzymatic preparation of hydroxycinnamates in ionic liquids enhances their antioxidant effect on lipoproteins oxidative modification

Biocatalytic lipophilization of hydroxycinnamic acids was performed in several BF₄^? and PF₆^? imidazolium ionic liquids using immobilized lipases. The influence of various reaction parameters on the performance of the biocatalytic process was pointed out, using as model reaction the esterification of ferulic acid. The biocatalytic lipophilization strongly depended on the ion composition of ionic liquids used. Conversions and initial reaction rates were significantly higher in PF₆^? as compared with BF₄^? ionic liquids and commonly used organic solvents. The high enzyme stability and the relative solubility of substrate versus product in PF₆^? ionic liquids can account for the improved synthesis of lipophilic ferulates. These lipophilic derivatives, when used at a concentration of up to 400-fold lower than the parental compound, efficiently inhibited the oxidation of isolated LDL, HDL and total serum in vitro. Moreover, it has been shown for the first time that the lipophilic ferulates improve the antioxidant efficiency of the HDL_3c towards LDL in vitro oxidation.     

Conversion of a Rhizopus chinensis lipase into an esterase by lid swapping

In an effort to explore the feasibility of converting a lipase into an esterase by modifying the lid region, we designed and characterized two novel Rhizopus chinensis lipase variants by lid swapping. The substrate specificity of an R. chinensis lipase was successfully modified toward water-soluble substrates, that is, turned into an esterase, by replacing the hydrophobic lid with a hydrophilic lid from ferulic acid esterase from Aspergillus niger. Meanwhile, as a comparison, the lid of R. chinensis lipase was replaced by a hydrophobic lid from Rhizomucor miehei lipase, which did not alter its substrate specificity but led to a 5.4-fold higher catalytic efficiency (k*_cat/K*_m) toward p-nitrophenyl laurate. Based on the analysis of structure-function relationships, it suggests that the amphipathic nature of the lid is very important for the substrate specificity. This study provides new insight into the structural basis of lipase specificities and a way to tune the substrate preference of lipases.     

Water-in-ionic liquid microemulsion-based organogels as novel matrices for enzyme immobilization

The use of water-in-ionic liquid microemulsion-based organogels (w/IL MBGs) as novel supports for the immobilization of lipase B from Candida antarctica and lipase from Chromobacterium viscosum was investigated. These novel lipase-containing w/IL MBGs can be effectively used as solid phase biocatalysts in various polar and non-polar organic solvents or ILs, exhibiting up to 4.4-fold higher esterification activity compared to water-in-oil microemulsion-based organogels. The immobilized lipases retain their activity for several hours at 70°C, while their half life time is up to 25-fold higher compared to that observed in w/IL microemulsions. Fourier-transform infrared spectroscopy data indicate that immobilized lipases adopt a more rigid structure, referring to the structure in aqueous solution, which is in correlation with their enhanced catalytic behavior observed.     

Characterization of an organic solvent-tolerant lipase from Idiomarina sp. W33 and its application for biodiesel production using Jatropha oil

A halophilic strain W33 showing lipolytic activity was isolated from the saline soil of Yuncheng Salt Lake, China. Biochemical and physiological characterization along with 16S rRNA gene sequence analysis placed the isolate in the genus Idiomarina. The extracellular lipase was purified to homogeneity by 75 % ammonium sulphate precipitation, DEAE-Sepharose anion exchange and Sephacryl S-200 gel filtration chromatography. The molecular mass of the purified lipase was estimated to be 67 kDa by SDS-PAGE. Substrate specificity test indicated that it preferred long-chain p-nitrophenyl esters. Optimal lipase activity was found to be at 60 °C, pH 7.0–9.0 and 10 % NaCl, and it was highly active and stable over broad temperature (30–90 °C), pH (7.0–11.0) and NaCl concentration (0–25 %) ranges, showing excellent thermostable, alkali-stable and halotolerant properties. Significant inhibition by diethyl pyrocarbonate and phenylarsine oxide was observed, implying histidine and cysteine residues were essential for enzyme catalysis. In addition, the lipase displayed high stability and activity in the presence of hydrophobic organic solvents with log P _ow ≥ 2.13. The free and immobilized lipases produced by Idiomarina sp. W33 were applied for biodiesel production using Jatropha oil, and about 84 and 91 % of yields were achieved, respectively. This study formed the basic trials conducted to test the feasibility of using lipases from halophile for biodiesel production.     

Growth of Escherichia coli, Pichia pastoris and Bacillus cereus in the Presence of the Ionic Liquids [BMIM][BF4] and [BMIM][PF6] and Organic Solvents

The influence of the two most commonly used ionic liquids (1-butyl-3-methyl imidazolium tetrafluoroborate, [BMIM][BF₄], 1-butyl-3-methyl imidazolium hexafluorophosphate, [BMIM][PF₆]) and three selected organic solvents (dimethylsulfoxide, ethanol, methanol) on the growth of Escherichia coli, Pichia pastoris and Bacillus cereus was investigated. [BMIM][BF₄] was toxic at 1% (v/v) on all three microorganisms. The minimal inhibitory concentration (MIC) of [BMIM][BF₄] on E. coli growth was between 0.7 and 1% (v/v). In contrast, [BMIM][PF₆] was less toxic for P. pastoris and B. cereus, whereas E. coli was not able to tolerate [BMIM][PF₆] (MIC value: 0.3–0.7% v/v). Growth of P. pastoris was unaffected by [BMIM][PF₆] at 10% (v/v). Similar results were found for dimethylsulfoxide. Thus, ionic liquids (ILs) can have substantial inhibitory effects on the growth of microorganisms, which should be taken into account for environmental reasons as well as for the use of ILs as co-solvents in biotransformations. Revisions requested 2 November 2005; Revisions received 20 December 2005     

Optimization of novel halophilic lipase production by Fusarium solani strain NFCCL 4084 using palm oil mill effluent

Among different sources of lipases, fungal lipases have continued to attract a wide range of applications. Further, halophilic lipases are highly desirable for biodiesel production due to the need to mitigate environmental pollution caused as result of extensive use of fossil fuels. However, currently, the high production cost limits the industrial application of lipases. In order to address this issue, we have attempted to optimize lipase production by Fusarium solani NFCCL 4084 and using palm oil mill effluent (POME) based medium. The production was optimized using a combinatory approach of Plackett-Burman (PB) design, one factor at a time (OFAT) design and face centred central composite design (FCCCD). The variables (malt extract, (NH₄)₂SO₄, CaCl₂, MgSO₄, olive oil, peptone, K₂HPO₄, NaNO₃, Tween-80, POME and pH) were analyzed using PB design and the variables with positive contrast coefficient were found to be K₂HPO₄, NaNO₃, Tween-80, POME and pH. The significant variables selected were further analyzed for possible optimum range by using OFAT approach and the findings revealed that K₂HPO₄, NaNO₃, and Tween-80 as the most significant medium components, and thus were further optimized by using FCCCD. The optimum medium yielded a lipase with an activity of 7.8 U/ml, a significant 3.2-fold increase compared to un-optimized medium. The present findings revealed that POME is an alternative and suitable substrate for halophilic lipase production at low cost. Also, it is clearly evident that the combinatory approach employed here proved to be very effective in producing high activity halophilic lipases, in general.     

Combination of two lipases more efficiently catalyzes methanolysis of soybean oil for biodiesel production in aqueous medium

A combination of two lipases was employed to catalyze methanolysis of soybean oil in aqueous medium for biodiesel production. The two lipase genes were cloned from fungal strains Rhizomucor miehei and Penicillium cyclopium, and each expressed successfully in Pichia pastoris. Activities of the 1,3-specific lipase from R. miehei (termed RML) and the non-specific mono- and diacylglycerol lipase from P. cyclopium (termed MDL) were 550 U and 1545 U per ml respectively, and enzymatic properties of these supernatant of fermentation broth (liquid lipase) were stable at 4 °C for >3 months. Under optimized conditions, the ratio of biodiesel conversion after 12 h at 30 °C, using RML alone, was 68.5%. When RML was assisted by addition of MDL, biodiesel conversion ratio was increased to >95% under the same reaction conditions. The results suggested that combination of lipases with different specificity, for enzymatic conversion of more complex lipid substrates, is a potentially useful strategy for biodiesel production.     

Characterization of an organic solvent-tolerant lipase from Haloarcula sp. G41 and its application for biodiesel production

A haloarchaeal strain G41 showing lipolytic activity was isolated from the saline soil of Yuncheng Salt Lake, China. Biochemical and physiological characterizations along with 16S rRNA gene sequence analysis placed the isolate in the genus Haloarcula. Lipase production was strongly influenced by the salinity of growth medium with maximum in the presence of 20 % NaCl or 15 % Na₂SO₄. The lipase was purified to homogeneity with a molecular mass of 45 kDa. Substrate specificity test revealed that it preferred long-chain p-nitrophenyl esters. The lipase was highly active and stable over broad ranges of temperature (30–80 °C), pH (6.0–11.0), and NaCl concentration (10–25 %), with an optimum at 70 °C, pH 8.0, and 15 % NaCl, showing thermostable, alkali-stable, and halostable properties. Enzyme inhibition studies indicated that the lipase was a metalloenzyme, with serine and cysteine residues essential for enzyme function. Moreover, it displayed high stability and activation in the presence of hydrophobic organic solvents with log P _ow?≥?2.73. The free and immobilized lipases from strain G41 were applied for biodiesel production, and 80.5 and 89.2 % of yields were achieved, respectively. This study demonstrated the feasibility of using lipases from halophilic archaea for biodiesel production.     

Biosynthesis of glycyrrhetic acid 3-O-mono-β-d-glucuronide catalyzed by β-d-glucuronidase with enhanced bond selectivity in an ionic liquid/buffer biphasic system

The bond selective hydrolysis of glycyrrhizin (GL) to glycyrrhetic acid 3-O-mono-β-d-glucuronide (GAMG) catalyzed by recombinant β-d-glucuronidase from Escherichia coli BL21 (PGUS-E) was successfully performed in an ionic liquid (IL)/buffer biphasic system. Five ILs were analyzed, however, a hydrophobic IL 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM]PF₆) showed the best biocompatibility with PGUS-E. An obvious enhancement in the initial reaction rate, substrate conversion, GAMG yield and chemical bond selectivity (S_cb) was observed using 40% (v/v) [BMIM]PF₆/buffer as the reaction medium when compared to the acetate buffer medium. Under the optimized conditions (pH 6.0, temperature 50 °C, substrate concentration 6 mM and shaking speed 200 rpm), the initial reaction rate, the GAMG yield and the S_cb reached 3.15 mM h⁻¹, 74.36% and 98.12%, respectively. The recyclability of [BMIM]PF₆ was also studied and found to be reusable for five batches with high recovery percentage (≥92%). Furthermore, the desired product and byproduct were easily separated since they were distributed in different phases. Additionally, higher V_max (3.14 versus 2.24 mM h⁻¹), lower apparent K_m (1.21 versus 1.80 mM) and E_a (25.97 versus 32.60 kJ mol⁻¹) were achieved in [BMIM]PF₆/buffer biphasic system than that in monophasic buffer system.