Tyrosinase activity in ionic liquids

Activity of mushroom tyrosinase was studied in three ionic liquids, 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIm][PF₆]), 1-butyl-3-methylimidazolium tetrafluoroborate ([BMIm][BF₄]) and 1-butyl-3-methylimidazolium methylsulfate ([BMIm][MeSO₄]), and was compared to that in chloroform. Kinetic parameters of the enzyme were determined and the results indicate that the enzyme in ionic liquids basically follows the same catalytic mechanism as in water, and that the ionic liquids may affect the enzyme activity by direct interacting with the enzyme and thus hindering the E–S binding due to their high hydrophilicity and polarity.     

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     

Retention factors and resolutions of amino benzoic acid isomers with some lonic liquids

Ionic liquids in the form of organic salts are being widely used as new solvent media. In this paper three positional isomers,o-amino benzoic acid,m-amino benzoic acid, andp-amino benzoic acids were separated with four different ionic liquids as mobile phase additives using high performance liquid chromatography (HPLC). The following ionic liquids were used: 1-butyl-3-methylimidazolium tetrafluoroborate ([BMIm][BF₄]), 1-ethyl-3-methylimidazolium tetrafluoroborate ([EMIm][BF₄]), 1-ethyl-3-methylimidazolium methylsulfate ([EMIm][MS]), and 1-octyl-3-methylimidazolium methylsulfate ([OMIm][MS]). The effects of the alkyl group length on the imidazolium ring and its counterion, and the concentrations of the ionic liquids on the retention factors and resolutions of amino benzoic acid isomers were tested. The results of the separations with ionic liquids as the eluents were better than those without ionic liquids. Excellent separations of the three isomers were achieved using 2.0≈8.0 mM/L [OMIm][MS] and 1.0≈8.0 mM/L [EMIm][MS] as the eluent modifiers.     

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.     

Effect of replacing [NTf2] by [PF6] anion on the [BMIm][NTf2] ionic liquid confined by gold

The effect of replacing bis(trifluoromethylsulphonyl)imide ([NTf₂]) by hexafluorophosphate ([PF₆]) in room temperature ionic liquid (IL) 1-butyl-3-methylimidazolium bis(trifluoromethylsulphonyl)imide ([BMIm][NTf₂]) confined between two gold interfaces is herein reported through molecular dynamics simulations using all-atom non-polarisable force-fields. Five systems were studied ranging from pure [BMIm][NTf₂] to pure [BMIm][PF₆], with [PF₆] molar fractions of 0, 0.125, 0.25, 0.375 and 0.5. Special attention was drawn to investigate the impact of the [PF₆] anion on the IL, in particular on the first layers of the liquid in close contact with the solid gold surface.     

A density functional theory study on the interactions between dibenzothiophene and tetrafluoroborate-based ionic liquids

The interactions between dibenzothiophene (DBT) and N-butyl-N-methylimidazolium tetrafluoroborate ([BMIM][BF₄]), N-butyl-N-methylmorpholinium tetrafluoroborate ([Bmmorpholinium][BF₄]), N-butyl-N-methylpiperdinium tetrafluoroborate ([BMPiper][BF₄]), N-butyl-N-methylpyrrolidinium tetrafluoroborate ([BMPyrro][BF₄]), and N-butylpyridinium tetrafluoroborate ([BPY][BF₄]) were investigated using density functional theory approach. Geometric, electron, and topological properties were analyzed using natural bond orbital, atoms in molecules theory, and noncovalent interaction methods in order to understand intermolecular interactions between DBT and ionic liquids. The result shows that hydrogen bond and van der Waals interactions are widespread in all the ionic liquids-DBT systems. Ion-π interactions between DBT and cation or anion are also observed, while π⁺-π interactions are only found in the [BMIM][BF₄]-DBT and [BPY][BF₄]-DBT systems. The order of interaction energy is [BPY][BF4]-DBT > [BMIM][BF₄]-DBT >> [BMPiper][BF₄]-DBT > [BMPyrro][BF₄]-DBT > [BMmorpholinum][BF₄]-DBT. The energies between DBT and the two ionic liquids containing aromatic cations are significantly higher.     

Optimization of (R,S)-1-phenylethanol kinetic resolution over Candida antarctica lipase B in ionic liquids

The kinetic resolution of racemates constitutes one major route to manufacture optically pure compounds. The enzymatic kinetic resolution of (R,S)-1-phenylethanol over Candida antarctica lipase B (CALB) by using vinyl acetate as the acyl donor in the acylation reaction was chosen as model reaction. A systematic screening and optimization of the reaction parameters, such as enzyme, ionic liquid and substrates concentrations with respect to the final product concentration, were performed. The enantioselectivity of immobilized CALB commercial preparation, Novozym 435, was assayed in several ionic liquids as reaction media. In particular, three different ionic liquids: (i) 1-butyl-3-methylimidazolium hexafluorophosphate [bmim][PF₆], (ii) 1-butyl-3-methylimidazolium tetrafluoroborate [bmim][BF₄] and (iii) 1-ethyl-3-methylimidazolium triflimide [emim][NTf₂] were tested. At 6.6% (w/w) of Novozym 435, dispersed in 9.520 M of [bmim][PF₆] at 313.15 K, using an equimolar ratio of vinyl acetate/(R,S)-1-phenylethanol after 3 h of bioconversion, the highest possible conversion (50%) was reached with enantiomeric excess for substrate higher than 99%.     

Effect of mobile phase additives on resolution of some nucleic compounds in high performance liquid chromatography

Here we investigate the chromatographic behavior, with reversed-phase high performance liquid chromatography (RP-HPLC) of nucleic compounds (nucleobases, nucleosides, and nucleotides) on a C₁₈ column in several different mobile phase additives, including1-butyl-3-methylimidazolium tetrafuloroborate ([BMIm][BF₄]), 1-ethyl-3-methylimidazolium methylsulfate ([EMIm][MS]) ionic liquids, ammonium formate, and potassium phosphate. The effect of the alkyl group length, the imidazolium ring, and the ionic liquid's counterions on retention and resolution of the samples were tested. The results show the potential application of a used buffer system, ion pairing system, and ionic liquid as mobile phase additives in liquid chromatography resolution of nucleic compounds.     

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.     

Optimization of lipase-catalyzed glucose fatty acid ester synthesis in a two-phase system containing ionic liquids and t-BuOH

Selective lipase-catalyzed synthesis of glucose fatty acid esters in two-phase systems consisting of an ionic liquid (1-butyl-3-methyl imidazolium tetrafluoroborate [BMIM][BF₄] or 1-butyl-3-methyl imidazolium hexafluorophosphate [BMIM][PF₆]) and t-butanol as organic solvent was investigated. The best enzyme was commercially available lipase B from Candida antarctica (CAL-B), but also lipase from Thermomyces lanuginosa (TLL) gave good conversion. After thorough optimization of several reaction conditions (chain-length and type of acyl donor, temperature, reaction time, percentage of co-solvent) conversions up to 60% could be achieved using fatty acid vinyl ester as acyl donors in [BMIM][PF₆] in the presence of 40% t-BuOH with CAL-B at 60 °C.     

Lipase-catalyzed transesterification in several reaction systems: An application of room temperature lonic liquids for bi-phasic production ofn-butyl acetate

Organic solvents are widely used in biotransformation systems. There are many efforts to reduce the consumption of organic solvents because of their toxicity to the environment and human health. In recent years, several groups have started to explore novel organic solvents called room temperature ionic liquids in order to substitute conventional organic solvents. In this work, lipase-catalyzed transesterification in several uni-and bi-phasic systems was studied. Two representative hydrophobic ionic liquids based on 1-butyl-3-methylimidazolum coupled with hexafluorophosphate ([BMIM][PF₆]) and bis[(trifluoromethylsulfonyl) imide] ([BMIM] [Tf₂N]) were employed as reaction media for the transesterification ofn-butanol. The commercial lipase, Novozym 435, was used for the transesterification reaction with vinyl acetate as an acyl donor, The conversion yield was increased around 10% in a water/[BMIM][Tf₂N], bi-phasic system compared with that in a water/hexane system. A higher distribution of substrates into the water phase is believed to enhance the conversion yield in a water/[BMIM][Tf₂N] system. Partion coefficients of the substrates in the water/[BMIM][Tf₂N] bi-phasic system were higher than three times that found in the water/hexane system, while n-butyl acetate showed a similar distribution in both systems. Thus, RTILs appear to be a promising substitute of organic solvents in some biotransformation systems.     

Strong substrate-stabilizing effect of a water-miscible ionic liquid [BMIM][BF<Subscript>4</Subscript>] in the catalysis of horseradish peroxidase

The effects of a water-miscible ionic liquid, 1-butyl-3-methylimidazolium tetrafluoroborate ([BMIM][BF₄]), on both thermodynamics and kinetic mechanism of the horseradish peroxidase (HRP)-catalyzed oxidation of guaiacol (2-methoxyphenol) by H₂O₂ were investigated. The ionic liquid stabilized the ground state of guaiacol by causing an 8-fold increase of K_m from 3 to 23 mM upon the addition of 25% (v/v) [BMIM][BF₄]. In addition, the effect of [BMIM][BF₄] in decreasing the k_cat value of HRP catalysis was described by a non-competitive inhibition mechanism. The value of the inhibition constant of [BMIM][BF₄] was 2.9 M indicating that the ionic liquid plays the role of a weak non-competitive inhibitor for HRP catalysis.     

Enzyme Catalysed Synthesis in Ambient Temperature Ionic Liquids

The activity of three different lipases, a glycosidase and a protease in ionic liquids has been studied. Ambient temperature ionic liquids are a new class of solvents that are nonvolatile and nonflammable and thus an interesting alternative to classical organic solvents. Monitoring the synthesis of a simple ester, all lipases were found to exhibit both excellent activity and stability in the non-polar ionic liquid 1-butyl-3-methylimidazohum hexaflurophosphate ([bmin][PF₆], 1). Furthermore, β-galactosidase from E. coli and the Subtilisin protease Savinase^TM were both found to exhibit a hydrolytic activity in a 50% aqueous solution of the water-miscible ionic liquid 1-butyl-3-methyhmidazoUum tetra-fluoroborate ([bmin][BF₄], 2) comparable to the activity observed in 50% aqueous solutions of ethanol and acetonitrile.     

Thermal Stability and Activity Regain of Horseradish Peroxidase in Aqueous Mixtures of Imidazolium-Based Ionic Liquids

Thermal deactivation kinetics of horseradish peroxidase (HRP) were studied from 45 to 90 °C in phosphate buffer and 5–25% (v,w/v) 1-butyl-3-methylimidazolium tetrafluoroborate [BMIM][BF₄] and 1-butyl-3-methylimidazolium chloride [BMIM][Cl]. HRP activity at 25 °C was not affected by the presence of ionic liquids up to 20% (v,w/v). Increasing the ionic liquids concentration up to 25% (v,w/v) changed the biphasic character of deactivation kinetics to an apparent single first-order step. The presence of 5–10% (v/v) [BMIM][BF₄] significantly improved HRP thermal stability with lower activation energies for the deactivation second phase (83–87 kJ mol⁻¹). After deactivation, enhanced activity regain of the enzyme, up to 70–80% of the initial activity, was found in 25% (v/v) [BMIM][BF₄] and 10% (w/v) [BMIM][Cl] and correlated to prevalence of the deactivation first phase.     

Effect of different reaction parameters on the lipase-catalyzed selective acylation of polyhydroxylated natural compounds in ionic liquids

The effect of various reaction parameters on the enzymatic acylation of plant polyhydroxylated compounds, including phenolic and flavonoid glucosides (salicin, helicin, esculin and naringin), was investigated in imidazolium-based ionic liquids (1-butyl-3-methylimidazolium tetrafluoroborate [bmim]BF₄ and 1-butyl-3-methylimidazolium hexafluorophosphate [bmim]PF₆), using immobilized lipase B from Candida antarctica. The conversion yield, the regioselectivity and the reaction rate of the biocatalytic process strongly depended on the ionic liquid used, their water content, the incubation temperature, as well as the solubility and the concentration of substrates. For most glucosides tested, one major product (monoacylated derivative) was detected as a result of the acylation of the primary hydroxyl group of glucose moiety. The acylation rate and the regioselectivity of the process are higher in [bmim]BF₄, where the solubility of all glucosides is significantly higher than in [bmim]PF₆ or acetone. Response surface methodology (RSM) based on a five level-three variable central composite circumscribed design, was employed to evaluate the interactive effect of the molar ratio of substrates (MR), the initial concentration of glucoside (N) and the reaction time (RT), as well as for their optimization in [bmim]BF₄. At the optimal reaction conditions the maximum acylation yield was 87%. The amount of monoacylated derivatives produced in a single-step biocatalytic process reached values up to 31.6 g/l which is considerably higher than those reported for organic media.     

Effect of ionic liquid [BMIM][PF<Subscript>6</Subscript>] on asymmetric reduction of ethyl 2-oxo-4-phenylbutyrate by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The effect of ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM][PF₆]) on the asymmetric reduction of ethyl 2-oxo-4-phenylbutyrate (EOPB) to synthesize optical active ethyl 2-hydroxy-4-phenylbutyrate (EHPB) catalyzed by Saccharomyces cerevisiae was investigated. (R)-EHPB [70.4%, e.e.(R)] is obtained using ethyl ether or benzene as the solvent. The main product is (S)-EHPB [27.7%, e.e.(S)] in [BMIM][PF₆]. However, in ionic liquid-water (10:1, v/v) biphasic system, the enantioselectivity of the reduction is shifted towards (R)-side, and e.e.(R) is increased from 6.6 to 82.5% with the addition of ethanol (1%, v/v). The effect of the use of [BMIM][PF₆] as an additive in relatively small amounts on the reduction was also studied. We find that there is a decline in the enantioselectivity of the reduction in benzene. In addition, a decrease in the conversion of EOPB and the yield of EHPB with increasing [BMIM][PF₆] concentrations occurs in either organic solvent–water biphasic systems or benzene.     

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.     

A Monte Carlo simulation study to predict the solubility of H2S in ionic liquids with 1-butyl-3-methylimidazolium ([C4mim+]) cation and tetrafluoroborate ([BF4−]), hexaflorophosphate ([PF6−]) and bis(trifluoromethanesulfonyl)amide ([Tf2N−]) anions

The solubilities of H₂S in ionic liquids, 1-butyl-3-methylimidazolium tetrafluoroborate ([C₄mim⁺][BF₄^?]), 1-butyl-3-methylimidazolium hexaflorophosphate ([C₄mim⁺][PF₆^?]) and -butyl-3-methylimidazolium tetrafluoroborate bis(trifluoromethanesulphonyl)amide ([C₄mim⁺][Tf₂N^?]) are predicted using isothermal–isobaric Gibbs ensemble Monte Carlo method (NPT-GEMC) at temperatures ranging from 333 to 453 K and pressure up to 20 bar. The low pressure points (up to 3 bar) of the absorption isotherms are fitted to a straight line to get a rough estimation of the Henry’s law constants. The van’t Hoff relationship is used to estimate the partial molar enthalpy of the absorption. The obtained results are in a good agreement with Jou and Mather [18], and Jalili et al. [13]. When comparing the solubility in ILs, it is found that H₂S solubility is highest for [C₄mim⁺][Tf₂N^?], followed by [C₄mim⁺][PF₆^?]. The lowest solubility is observed in [C₄mim⁺][BF₄^?]. The highest solubility in [C₄mim⁺][Tf₂N^?] is consistent with Jalili et al. [13]. However, their results indicate slightly higher solubility in [C₄mim⁺][BF₄^?] than in [C₄mim⁺][PF₆^?], and do not agree with the predictions. Upon absorption, the molar volumes of the mixtures decrease linearly, showing only small changes in volume. The effect of H₂S absorption on ILs is further studied by calculating the radial distribution functions between the ions. The results indicate that the solute molecules accommodate themselves in the cavities without significantly disturbing the ionic arrangement of the ions, similar to CO₂ absorption in ILs. The spatial distribution functions show similar spatial distribution for H₂S around cation in all of the studied ILs, whereas the distribution around anion depends on the shape and flexibility of the anion. The mechanism of H₂S absorption is studied by computing the van der Waals (VDW) and electrostatic (ELEC) energies. It is observed that the solubility of H₂S in the studied ILs is primarily controlled by VDW interaction. When comparing the interaction of H₂S with the ions, it is found that solute molecules interact with cations mainly due to VDW interaction. Both VDW and ELEC energies contribute in the interaction between H₂S and anions.     

Candida antarctica lipase B catalyzed enantioselective acylation of pyrimidine acyclonucleoside

Abstract

The influence of solvent and acyl group donor on selectivity of the transesterification reaction of 1-[1′,3′-dihydroxy-2′-propoxymethyl]-5-methyluracil, a structural analogue of ganciclovir was examined. Lipase (EC 3.1.1.3) B from Candida antarctica (CALB) enabled desymmetrization of prochiral hydroxyl groups when 1-butyl-3-methylimidazolium hexafluorophosphate ([Bmim][PF₆]) was used as a reaction medium. It was observed that CALB was up to 2.7–4 times more enantioselective in the ionic liquid [Bmim][PF₆] than in conventional organic solvents.     

H, F-HOESY and PGSE diffusion studies on ionic liquids: The effect of co-solvent on structure

¹H, ¹⁹F-HOESY studies on the ionic liquid based on 1-butyl-3-methylimidazolium tetrafluoroborate, [C₄C₁im]BF₄, [C₄C₁im][N(OTf)₂], and, partially, [C₄C₁im]PF₆, are reported. The addition of methanol separates the ions; however, in dichloromethane solution the anions and cations show strong HOESY contacts even after dilution and taken together with the PGSE diffusion measurements, the data suggest strong ion pairing in this solvent.