Efficient kinetic resolution of (R,S)-1-trimethylsilylethanol via lipase-mediated enantioselective acylation in ionic liquids

Efficient enantioselective acylation of (R,S)-1-trimethylsilylethanol {(R,S)-1-TMSE} with vinyl acetate catalyzed by immobilized lipase from Candida antarctica B (i.e., Novozym 435) was successfully conducted in ionic liquids (ILs). A remarkable enhancement in the initial rate and the enantioselectivity of the acylation was observed by using ILs as the reaction media when compared to the organic solvents tested. Also, the activity, enantioselectivity, and thermostability of Novozym 435 increased with increasing hydrophobicity of ILs. Of the six ILs examined, the IL C4MIm.PF6 gave the fastest initial rate and the highest enantioselectivity, and was consequently chosen as the favorable medium for the reaction. The optimal molar ratio of vinyl acetate to (R,S)-1-TMSE, water activity, and reaction temperature range were 4:1, 0.75, and 40 -50 degrees C, respectively, under which the initial rate and the enantioselectivity (E value) were 27.6 mM/h and 149, respectively. After a reaction time of 6 h, the ee of the remaining (S)-1-TMSE reached 97.1% at the substrate conversion of 50.7%. Additionally, Novozym 435 was effectively recycled and reused in C4MIm.PF6 for five consecutive runs without substantial lose in activity and enantioselectivity. The preparative scale kinetic resolution of (R,S)-1-TMSE in C4MIm.PF6 is shown to be very promising and useful for the industrial production of enantiopure (S)-1-TMSE.     

Markedly improving Novozym 435-mediated regioselective acylation of 1-beta-D-arabinofuranosylcytosine by using co-solvent mixtures as the reaction media

A comparative study was made of Novozym 435-catalyzed regioselective acylation of 1-beta-D-arabinofuranosylcytosine with vinyl propionate for the preparation of the 5'-O-monoester in eleven co-solvent mixtures and three pure polar solvents. Novozym 435 displayed low or no acylation activity toward 1-beta-D-arabinofuranosylcytosine in pure polar solvents, although those solvents can dissolve the nucleosides well. When a hexane-pyridine co-solvent system was adopted, both the initial rate and the substrate conversion were enhanced markedly. The polarity of co-solvent mixtures had significant effect on the reaction. Among the solvent mixtures investigated, the higher the polarity of the solvent mixture, the lower the initial reaction rate and the substrate conversion. It was also found that the acylation was dependent on the hydrophobic solvent content, the water activity and the reaction temperature. The most suitable co-solvent, initial water activity, and reaction temperature were hexane-pyridine (28:72, v/v), 0.07, and 50 degrees C, respectively. Under these conditions, the initial rate, the substrate conversion and the regioselectivity were as high as 91.1 mM h(-1), >97% and >98%, respectively, after a reaction time of 6 h. Among the reaction mediums examined, the lowest apparent activation energy was achieved with hexane-pyridine (28:72, v/v), in which Novozym 435 also exhibited good thermal stability.     

Synthesis of methyl (R)-3-(4-fluorophenyl)glutarate via enzymatic desymmetrization of a prochiral diester

An efficient procedure for enzymatic desymmetrization of the prochiral dimethyl 3-(4-fluorophenyl)glutarate (3-DFG) in an aqueous–organic phase was successfully developed to prepare methyl (R)-3-(4-fluorophenyl)glutarate ((R)-3-MFG). Novozym 435 was selected as a highly efficient biocatalyst through lipase screening. The effects of various parameters in terms of co-solvent and its concentration, buffer pH, ionic strength and reaction temperature, on the reaction were investigated. It was found that 0.2 M phosphate buffer (pH 8.0) containing 20% MTBE (v/v) was the optimum reaction medium, and the optimum reaction temperature was 30 °C. Under the optimized reaction conditions, (R)-3-MFG was obtained in 95.6% ee value and 92.6% yield after 64 h when the concentration of 3-DFG and Novozym 435 were 200 mmol/l and 20 g/l respectively. Furthermore, Novozym 435 showed an excellent operational stability, retaining above 95% of the initial activity and enantioselectivity after 10 cycles of reaction. The developed method has a potential to be used for efficient enzymatic production of (R)-3-MFG.     

Efficient anti-Prelog enantioselective reduction of acetyltrimethylsilane to (R)-1-trimethylsilylethanol by immobilized Candida parapsilosis CCTCC M203011 cells in ionic liquid-based biphasic systems

ABSTRACT: BACKGROUND: Biocatalytic asymmetric reductions with whole cells can offer high enantioselectivity, environmentally benign processes and energy-effective operations and thus are of great interest. The application of whole cell-mediated bioreduction is often restricted if substrate and product have low water solubility and/or high toxicity to the biocatalyst. Many studies have shown that a biphasic system is often useful in this instance. Hence, we developed efficient biphasic reaction systems with biocompatible water-immiscible ionic liquids (ILs), to improve the biocatalytic anti-Prelog enantioselective reduction of acetyltrimethylsilane (ATMS) to (R)-1-trimethylsilylethanol {(R)-1-TMSE}, which is key synthon for a large number of silicon-containing drugs, using immobilized Candida parapsilosis CCTCC M203011 cells as the biocatalyst. RESULTS: It was found that the substrate ATMS and the product 1-TMSE exerted pronounced toxicity to immobilized Candida parapsilosis CCTCC M203011 cells. The biocompatible water-immiscible ILs can be applied as a substrate reservoir and in situ extractant for the product, thus greatly enhancing the efficiency of the biocatalytic process and the operational stability of the cells as compared to the IL-free aqueous system. Various ILs exerted significant but different effects on the bioreduction and the performances of biocatalysts were closely related to the kinds and combination of cation and anion of ILs. Among all the water-immiscible ILs investigated, the best results were observed in 1-butyl-3-methylimidazolium hexafluorophosphate (C4mim * PF6)/buffer biphasic system. Furthermore, it was shown that the optimum substrate concentration, volume ratio of buffer to IL, buffer pH, reaction temperature and shaking rate for the bioreduction were 120 mM, 8/1 (v/v), 6.0, 30 degreesC and 180 r/min, respectively. Under these optimized conditions, the initial reaction rate, the maximum yield and the product e.e. were 8.1 mumol/min gcwm, 98.6 % and >99 %, respectively. The efficient whole-cell biocatalytic process was shown to be feasible on a 450-mL scale. Moreover, the immobilized cells remained around 87 % of their initial activity even after being used repeatedly for 8 batches in the C4mim * PF6/buffer biphasic system, exhibiting excellent operational stability. CONCLUSIONS: For the first time, we have successfully utilized immobilized Candida parapsilosis CCTCC M203011 cells, for efficiently catalyzing anti-Prelog enantioselective reduction of ATMS to enantiopure (R)-1-TMSE in the C4mim * PF6/buffer biphasic system. The substantially improved biocatalytic process appears to be effective and competitive on a preparative scale.     

Novozym 435-catalyzed regioselective benzoylation of 1-β-d-arabinofuranosylcytosine in a co-solvent mixture of C4MIm·PF6 and pyridine

Regioselective acylation of 1-β-d-arabinofuranosylcytosine (ara-C), using vinyl benzoate (VB) as acyl donor and Novozym 435 as catalyst, was carried out in various reaction media including pure organic solvents, organic solvent mixtures, and ionic liquid (IL)-containing systems. Although the reaction was highly regioselective in all the media assayed, remarkable enhancement of substrate conversion was achieved with a co-solvent mixture of 1-butyl-3-methylimidazolium hexafluorophosphate (C₄MIm·PF₆) and pyridine as the reaction medium, compared with other media tested. Additionally, the results demonstrated that the anions of ILs had a significant effect on the initial rate and substrate conversion. To better understand the reaction performed in IL-containing system, several variables were examined. The optimum molar ratio of VB to ara-C, initial water activity, temperature and shaking rate were 25:1, 0.11, 40°C and 250rpm, respectively. Under these optimum reaction conditions, the initial rate, substrate conversion, and regioselectivity were 0.49mMmin^?1, 99.4 and 99%, respectively. The product of the lipase-catalyzed reaction was characterized by ¹³C NMR and was shown to be 5′-O-benzoyl ara-C.     

Efficient enantioselective reduction of 4′-methoxyacetophenone with immobilized Rhodotorula sp. AS2.2241 cells in a hydrophilic ionic liquid-containing co-solvent system

The biocatalytic enantioselective reduction of 4′-methoxyacetophenone to (S)-1-(4-methoxyphenyl)ethanol was successfully conducted in a hydrophilic IL-containing co-solvent system using immobilized Rhodotorula sp. AS2.2241 cells. Of all the tested ILs, the best results were observed with the novel IL 1-(2′-hydroxy)ethyl-3-methylimidazolium nitrate (C₂OHMIM·NO₃), which showed a good biocompatibility with the cells and increased the cell membrane permeability moderately, thus improving the efficiency of the bioreduction. To better understand the bioreduction, several crucial influential variables were also examined. The optimal C₂OHMIM·NO₃ content, buffer pH, reaction temperature and substrate concentration were 5.0% (v/v), 8.5, 25 °C and 12 mM, respectively. Under the optimized conditions, the initial reaction rate, the maximum yield and the product e.e. were 9.8 μmol/h g_cell, 98.3% and >99%, respectively, which are much better than the results previously reported. The established biocatalytic system has proven to be highly effective for the reduction of other aryl ketones. Also, the cells exhibited excellent operational stability in the presence of C₂OHMIM·NO₃. Moreover, the ILs can accumulate within the cells, suggesting that ILs are likely to interact with the related enzymes within the cells.     

有机介质中脂肪酶催化外消旋苯甘氨酸甲酯的对映体选择性氨解反应

由水解酶催化的酯的对映体选择性水解和醇解反应 ,已在外消旋物质的拆分中得到广泛应用[1 ] 。近年来的一些研究表明 ,某些水解酶还可催化一些非天然酰基受体的转化 ,如过氧化氢、烷基胺、联胺和氨等。这些非天然酰基受体的转化反应在多肽的合成及手性化合物的拆分中显示出巨大的应用前景。其中 ,以氨为酰基受体的酶促氨解反应 ,是继酶促对映体选择性水解、酯化及转酯反应之后的另一制备光学纯化合物的新反应[2 ] 。目前国际上对这一新反应的研究尚属起步 ,国内未见有对该反应研究的报道。(Ｄ ,Ｌ) 苯甘氨酸是半合成 β 内酰胺类抗生素的重…     

Efficient asymmetric reduction of 4-(trimethylsilyl)-3-butyn-2-one by Candida parapsilosis cells in an ionic liquid-containing system

Hydrophilic ionic liquids (ILs) were employed as green solvents to construct an IL-containing co-solvent system for improving the asymmetric reduction of 4-(trimethylsilyl)-3-butyn-2-one by immobilized Candida parapsilosis cells. Among 14 hydrophilic ILs examined, 1-(2'-hydroxyl)ethyl-3-methylimidazolium nitrate (C(2)OHMIM·NO(3)) was considered as the most suitable IL for the bioreduction with the fastest initial reaction rate, the highest yield and the highest product e.e., which may be due to the good biocompatibility with the cells. For a better understanding of the bioreduction performed in the C(2)OHMIM·NO(3)-containing co-solvent system, the effects of several crucial variables were systematically investigated. The optimal C(2)OHMIM·NO(3) content, substrate concentration, buffer pH, co-substrate concentration and temperature were 10% (v/v), 3.0 mmol/L, 5.0, 98.1 mmol/L and 30°C, respectively. Under the optimal conditions, the initial reaction rate, the maximum yield and the product e.e. were 17.3 μmol/h g(cell), 95.2% and >99.9%, respectively, which are much better than the corresponding results previously reported. Moreover, the immobilized cells remained more than 83% of their initial activity even after being used repeatedly for 10 batches in the C(2)OHMIM·NO(3)-containing system, exhibiting excellent operational stability.     

A two-step, one-pot enzymatic synthesis of ampicillin from penicillin G potassium salt

A two-step, one-pot synthesis of ampicillin from penicillin G potassium salt (PGK) in aqueous buffer/organic co-solvent has been achieved. Ethylene glycol (EG) was chosen as the organic co-solvent. Factors including co-solvent content, enzyme loading, reaction temperature and substrate concentration were investigated. The optimum conditions were as follow: pH 8.0 phosphate buffer solution, 50% EG (v/v), 25 °C, 100 mM PGK and 300 mM d-phenylglycine methyl ester (D-PGM), 43.2 IU/ml IPA-750. The maximum yield was 57.3% after a reaction time of 17 h. It is the first report about the synthesis of ampicillin from penicillin G potassium salt in one-pot combining the enzymatic hydrolysis and the subsequent enzymatic condensation, and the novel methodology will have important application in the β-lactam antibiotics industry.     

Kinetic resolution of racemic alpha-methyl-beta-propiothiolactone by lipase-catalyzed hydrolysis

Kinetic resolution of racemic alpha-methyl-beta-propiothiolactone (rac-MPTL) using lipases in organic solvent was studied. The lipase from Pseudomonas cepacia (PCL) showed the highest (S)-enantioselectivity (E > 100), and cyclohexane containing 1% (v/v) buffer was identified as the best reaction medium for maintaining high enantioselectivity as well as high reaction rate. While the substrate inhibition was not observed up to 300 mM rac-MPTL, severe product inhibition was observed even at 50 mM racemic 3-mercapto-alpha-methyl propionic acid (rac-MMPA), which made the use of high substrate concentration difficult. To overcome the product inhibition, the products, (R)-MMPA, were neutralized by addition of a dilute basic solution. Although the resolution reaction proceeded further by the base titration, the enantioselectivity of the reaction decreased as a result of nonenantioselective hydrolysis of rac-MPTL in the basic solution. Under these conditions, 200 mM rac-MPTL was successfully resolved to above 95% ee(S) with 53% conversion.     

Microwave-assisted enzymatic resolution of (R,S)-2-octanol in ionic liquid

Combination use of microwave irradiation (MW) as heating mode and ionic liquid (IL) as reaction medium in enzymatic resolution of (R,S)-2-octanol with vinyl acetate as the acyl donor through transesterification by Novozym 435 was investigated. A synergistic effect of MW and IL [EMIM][NTf2], which was screened as the best reaction medium for this reaction, on improving enzyme activity and enantioselectivity was observed. The activity and enantioselectivity of Novozym 435 in [EMIM][NTf2] under MW were much higher than that in solvent free system under conventional heating, in solvent free system under MW, and in [EMIM][NTf2] under conventional heating, respectively. A systematic screening and optimization of the reaction parameters in [EMIM][NTf2] under MW were performed. Under the optimum conditions, 50% yield of (S)-2-octanol with 99% enantiomeric excess was obtained in 6 h. Furthermore, increased thermal stability and reusability of Novozym 435 under the combination use of MW and IL condition were also observed.     

Optimization of lipase-catalyzed glucose ester synthesis in ionic liquids

Lipase-catalyzed esterification of glucose with fatty acids in ionic liquids (ILs) mixture was investigated by using supersaturated glucose solution. The effect of ILs mixture ratio, substrate ratio, lipase content, and temperature on the activity and stability of lipase was also studied. The highest yield of sugar ester was obtained in a mixture of 1-butyl-3-methylimidazolium trifluoromethanesulfonate ([Bmim][TfO]) and 1-methyl-3-octylimidazolium bis[(trifluoromethyl)-sulfonyl]amide ([Omim][Tf₂N]) with a volume ratio of 9:1, while Novozym 435 (Candida antarctica type B lipase immobilized on acrylic resin) showed the optimal stability and activity in a mixture of [Bmim][TfO] and [Omim][Tf₂N] with a 1:1 volume ratio. Reuse of lipase and ILs was successfully carried out at the optimized reaction conditions. After 5 times reuse of Novozym 435 and ILs, 78% of initial activity was remained.     

Lipase catalysed synthesis of sebacic and phthalic esters

The enzymatic synthesis and hydrolysis of alkyl sebacates and o-, m-, p-phthalates were studied. Biosyntheses were conducted through alcoholysis of dimethyl phthalates and dimethyl sebacate with 2-ethylhexanol and 3,5,5-trimethylhexanol in a solvent-free medium, using lipases from Candida antarctica (Novozym 435), Rhizomucor miehei (Lipozyme IM) and Porcine pancreas (PPL). It was found that the synthesis and hydrolysis of sebacic acid esters were characterised by a satisfactory rate, however, by low enantioselectivity. The yield of synthesis of di-3,5,5-trimethylhexyl sebacate catalysed by Novozym 435 at 50 °C was 84%, after 20 h of reaction. The degree of conversion, 62.9% after 350 h, was obtained for alcoholysis reaction of dimethyl m-phthalate with 3,5,5-trimethylhexanol. For the enzymes used, no activity was detected at all on both the synthesis and hydrolysis of di-2-ethylhexyl o-phthalate and di-3,5,5-trimethylhexyl o-phthalate.     

Enhanced production of fructose palmitate by lipase-catalyzed esterification in ionic liquids

For the enhancement of enzyme activity, application of ultrasound irradiation on lipase-catalyzed esterification of fructose with palmitic acid in ionic liquids (ILs) mixture containing supersaturated fructose solution was investigated. In the mixture of [Bmim][TfO] and [Omim][Tf₂N] (1:1, v/v), 1.44 times higher enzyme activity (29.2 μmoL/min/g) was achieved under ultrasound irradiation. Besides, ultrasound irradiation enhanced enzyme stability in viscous ILs mixture. After 5 times reuse of Novozym 435 and ILs mixture, 84.4% of initial enzyme activity was remained under ultrasound irradiation, while the residual activity using magnetic stirring only method was 76.2%. These results show that enzymatic reaction in viscous ILs mixture under ultrasound irradiation is an effective method for enzyme activity, as well as, enzyme stability resulting in economic competitiveness of green process.     

Lipase-catalyzed synthesis of glucose fatty acid ester using ionic liquids mixtures

Novozym 435-catalyzed synthesis of 6-O-lauroyl-d-glucose in ionic liquids (ILs) was investigated. The highest lipase activity was obtained in water-miscible [Bmim][TfO] which can dissolve high concentration of glucose, while the highest stability of lipase was shown in hydrophobic [Bmim][Tf(2)N]. The optimal activity and stability of lipase could be obtained in [Bmim][TfO] and [Bmim][Tf(2)N] mixture (1:1, v/v). Specifically, the activity of lipase was increased from 1.1 to 2.9 micromolmin(-1)g(-1) by using supersaturated glucose solution in this mixture, compared with reaction using saturated solution. After 5 times reuse of lipase, 86% of initial activity was remained in this mixture, while the residual activity in pure [Bmim][TfO] was 36%. Therefore, the productivity obtained by using ILs mixtures was higher than those in pure ILs.     

Cascade synthesis of chiral block copolymers combining lipase catalyzed ring opening polymerization and atom transfer radical polymerization

The enantioselective polymerization of methyl-substituted epsilon-caprolactones using Novozym 435 as the catalyst was investigated. All substituted monomers could be polymerized except 6-methyl-epsilon-caprolactone (6-MeCL), which failed to propagate after ring opening. Interestingly, an odd-even effect in the enantiopreference of differently substituted monomers was observed. The combination of 4-methyl-epsilon-caprolactone with Novozym 435 showed good enantioselectivity also in bulk polymerization and resulted in enantiomerically enriched P((S)-4-MeCL) (eep up to 0.88). Subsequently, a novel initiator combining a primary alcohol to initiate the ring opening polymerization and a tertiary bromide to initiate atom transfer controlled radical polymerization (ATRP) was synthesized, and showed high initiator efficiencies (> 90%) in the ring opening polymerization of 4-methyl-epsilon-caprolactone in bulk. In addition, the enantioselectivity was retained (E = 11). By using Ni(PPh3)2Br2 as the ATRP catalyst, Novozym 435 could be effectively inhibited at the desired conversion of 4-methyl-epsilon-caprolactone, thus ensuring a high enantiomeric excess in the polymer backbone. At the same time, Ni(PPh3)2Br2 catalyzed the ATRP of methyl methacrylate resulting in the formation of P((S)-4-MeCL-b-MMA) block copolymers. By this combination of two inherently different polymerization reactions, chiral P((S)-4-MeCL-b-MMA) block copolymers can be conveniently obtained in one pot without intermediate workup.     

Optimized enzymatic synthesis of ascorbyl esters from lard using Novozym 435 in co-solvent mixtures

A mild and efficient method for the conversion of fatty acid methyl esters from lard into ascorbyl esters via lipase-catalyzed transesterification in co-solvent mixture is described. A solvent engineering strategy was firstly applied to improve fatty acid ascorbyl esters production. The co-solvent mixture of 30% t-pentanol:70% isooctane (v/v) was optimal. Response surface methodology (RSM) and central composite design (CCD) were employed to estimate the effects of reaction parameters, such as reaction time (12–36 h), temperature (45–65 °C), enzyme amount (10–20%, w/w, of fat acid methyl esters), and substrate molar ratio of fatty acid methyl esters to ascorbic acid (8:1–12:1) for the synthesis of fatty acid ascorbyl esters in co-solvent mixture. Based on the RSM analysis, the optimal reaction conditions were determined as follows: reaction time 34.32 h, temperature 54.6 °C, enzyme amount 12.5%, substrate molar ratio 10.22:1 and the maximum conversion of fatty acid ascorbyl esters was 69.18%. The method proved to be applicable for the synthesis of ascorbyl esters using Novozym 435 in solvent.     

Enzymatic desymmetrization of 3-(4-fluorophenyl)glutaric anhydride through enantioselective alcoholysis in organic solvents

The enzymatic desymmetrization of 3-(4-fluorophenyl)glutaric anhydride (3-FGA) was investigated through lipase-catalyzed enantioselective alcoholysis in organic solvents. An immobilized Lipase B from Candida Antarctica (Novozym 435) was found to be an efficient biocatalyst for the enantioselective alcoholysis of 3-FGA. Methyl tert-butyl ether (MTBE) and methanol were chosen as the suitable reaction medium and acyl acceptor, respectively. The optimum reaction temperature, molar ratio of methanol to 3-FGA and 3-FGA concentration were 25°C, 2:1 and 100 mM, respectively. Under these conditions, complete conversion was achieved and methyl (S)-3-(4-fluorophenyl)glutarate ((S)-MFG) was obtained in a moderate ee value of 80%. Furthermore, the reaction was performed on a gram scale and the ee value of (S)-MFG was enriched to 96% after treatment with a toluene/hexane (2/1, v/v) mixture.     

Improved octyl glucoside synthesis using immobilized β-glucosidase on PA-M with reduced glucose surplus inhibition

A β-glucosidase extracted from bitter almond (Prunus dulcis var. amara) was immobilized on polyamine microspheres (PA-M) for catalytic octyl glucoside (OG) synthesis from glucose and octanol through reversed hydrolysis. The immobilization increased the activity of enzyme at pH 6.0–7.0, and the optimal reaction temperature for immobilized enzyme was identical to the free enzyme. The thermal stability and solvent tolerance of enzyme were increased by its immobilization. In the co-solvent system using 10% t-butyl alcohol and 10% (v/v) water, the yield of OG was increased by 1.7-fold compared to the yield from the system without co-solvent. Based on dynamic and Dixon plot analyses, the initial reaction velocity (V₀) increased approximately three-fold on immobilization and the OG synthesis was inhibited by surplus glucose. The inhibition dissociation constants for free and immobilized enzyme were 219?mM and 116?mM, respectively. A fed-batch mode was applied in the OG synthesis to minimize substrate inhibition. After 336?h of reaction, the OG yield and the conversion rate of glucose reached 134?mM and 59.6%, respectively. Compared to the batch operation, the fed-bath operation increased the OG yield and the conversion rate of glucose by 340% and 381%, respectively.     

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.