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.     

Partial uncompetitive inhibition of horseradish peroxidase by a water-miscible ionic liquid [BMIM][MeSO4

The ionic liquid, 1-butyl-3-methylimidazolium methylsulfate ([BMIM][MeSO₄]), was used to investigate the catalytic mechanism of horseradish peroxidase (HRP). The ionic liquid decreased both K_m and k_cat values for the HRP-catalyzed oxidation of guaiacol (2-methoxyphenol) by H₂O₂. These studies imply that [BMIM][MeSO₄] inhibits the enzyme in an uncompetitive manner. The incorporation of substrate stabilization effects measured by a thermodynamic method into the partial uncompetitive inhibition scheme successfully describes HRP-catalysis in the presence of [BMIM][MeSO₄], which participates as the inhibitor. The inhibition constant of the ionic liquid was 0.051 M. The turn-over number of the native HRP was almost 14-times higher than that of the HRP-ionic liquid complex indicating that [BMIM][MeSO₄] does not form a dead-end complex with HRP.     

Effect of ionic liquid on the kinetics of peroxidase catalysis

The effect of a water-miscible ionic liquid, 1-butyl-3-methylimidazolium tetrafluoroborate ([BMIM][BF4]), on the horseradish peroxidase (HRP)-catalyzed oxidation of 2-methoxyphenol (guaiacol) with hydrogen peroxide (H2O2) was investigated. HRP maintains its high activity in the aqueous mixtures containing various concentrations of the ionic liquid and even in 90% (v/v) ionic liquid. In order to minimize the effect of solution viscosity on the kinetic constants of HRP catalysis, the enzymatic reactions in the subsequent kinetic study were performed in water-ionic liquid mixtures containing 25% (v/v) ionic liquid at maximum. As the concentration of [BMIM][BF4] increased for the oxidation of guaiacol by HRP, the K(m) value increased with a slight decrease in the k(cat) value: The K(m) value increased from 2.8 mM in 100% (v/v) water to 22.5 mM in 25% (v/v) ionic liquid, indicating that ionic liquid significantly weakens the binding affinity of guaiacol to HRP.     

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.     

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     

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.     

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.     

Partitioning of acidic, basic and neutral amino acids into imidazolium-based ionic liquids

In this paper, partitioning behaviors of typical neutral (Alanine), acidic (Glutamic acid) and basic (Lysine) amino acids into imidazolium-based ionic liquids [C₄mim][PF₆], [C₆mim][PF₆], [C₈mim][PF₆], [C₆mim][BF₄] and [C₈mim][BF₄] as extracting solvents were examined. [C₆mim][BF₄] showed the best efficiency for partitioning of amino acids. The partition coefficients of amino acids in ionic liquids were found to depend strongly on pH of the aqueous solution, amino acid and ionic liquid chemical structures. Different chemical forms of amino acids in aqueous solutions were pH dependent, so the pH value of the aqueous phase was a determining factor for extraction of amino acids into ionic liquid phase. Both water content of ionic liquids and charge densities of their anionic and cationic parts were important factors for partitioning of cationic and anionic forms of amino acids into ionic liquid phase. Extracted amino acids were back extracted into phosphate buffer solutions adjusted on appropriate pH values. The results showed that ionic liquids could be used as suitable modifiers on the stationary phase of an HPLC column for efficient separation of acidic, basic, and neutral amino acids.     

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.     

Hydroxynitrile Lyase Catalysis in Ionic Liquid-containing Systems

The cleavage of mandelonitrile catalysed by hydroxynitrile lyases (HNL) from Prunus amygdalus (PaHNL) and Manihot esculenta (MeHNL) proceeded more rapidly in monophasic aqueous media containing 1-propyl-3-methylimidazolium tetrafluoroborate [C₄MIm][BF₄] than in media containing acetonitrile or THF. Both HNLs were much more thermostable in [C₄MIm][BF₄] than in acetonitrile or THF. The addition of each of the four ionic liquids 1-butyl-, 1-pentyl- and 1-hexyl-3-methylimidazolium tetrafluoroborates at 2–6% (v/v in the aqueous phase) increased both the enzyme activity and the product e.e. in the PaHNL-catalysed transcyanation in an aqueous/DIPE biphasic system. However, MeHNL was inactivated by the ionic liquids, as indicated by the decreased reaction rate, substrate conversion and product e.e.     

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.     

Correlation between thermostability and stability of glycosidases in ionic liquid

The activity and stability of a β-glycosidase (Thermus thermophilus) and two α-galactosidases (Thermotoga maritima and Bacillus stearothermophilus) were studied in different hydrophilic ionic liquid (IL)/water ratios. For the ILs used, the glycosidases showed the best stability and activity in 1,3-dimethylimidazolium methyl sulfate [MMIM][MeSO₄] and 1,2,3-trimethylimidazolium methyl sulfate [TMIM][MeSO₄]. A close correlation was observed between the thermostability of the enzymes and their stability in IL media. At high IL concentration (80%), a time-dependent irreversible denaturing effect was observed on glycosidases while, at lower concentration (<30%), a reversible inactivation affecting mainly the k _cat was obtained. The results demonstrate that highly thermostable glycosidases are more suitable for biocatalytic reactions in water-miscible ILs.     

The peroxidase activity of cytochrome <Emphasis Type="Italic">b</Emphasis><Subscript>6</Subscript><Emphasis Type="Italic">f</Emphasis> complex from spinach chloroplasts

The cytochrome b ₆ f (Cyt b ₆ f) complex, which functions as a plastoquinol-plastocyanin oxidoreductase and mediates the linear electron flow between photosystem II (PSII) and photosystem I (PSI) and the cyclic electron flow around PSI, was isolated from spinach (Spinacia oleracea L.) chloroplasts using n-octyl-β-D-glucopyranoside (β-OG). The preparation was also able to catalyze the peroxidase-like reaction in the presence of hydrogen peroxide (H₂O₂) and guaiacol. The optimal conditions for peroxidase activity of the preparation included: pH 3.6, ionic strength 0.1, and temperature 35°C. The apparent Michaelis constant (K _m) values for H₂O₂ and guaiacol were 50 mM and 2 mM, respectively. The bimolecular rate constant (k _obs) was about 26 M⁻¹ s⁻¹ and the turnover number (K _cat) was about 60 min⁻¹ (20 mM guaiacol, 100 mM sodium phosphate, pH 3.6, 25°C, [H₂O₂]<100mM). These parameters were similar to those of several other heme-containing proteins, such as myoglobin and Cyt c.     

Enantioselective hydrogenation in ionic liquids: Recyclability of the [Rh(COD)(DIPAMP)]BF4 catalyst in [bmim][BF4]

Asymmetric hydrogenation of (Z)-α-acetamidocinnamic acid and methyl-(Z)-α-acetamidocinnamate by [Rh(COD)(DIPAMP)][BF₄] catalyst was studied in ionic liquid/isopropanol two-phase catalytic system. In this system 97–100% conversion was achieved and the enantioselectivity values were over 90%. Application of [bmim][BF₄] ionic liquid made it possible to recycle the catalyst in consecutive cycles. After four cycles, neither significant conversion nor enantioselectivity decrease was observed.     

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.     

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%.     

Lipase-catalyzed acylation of lily polysaccharide in ionic liquid-containing systems

A comparative study was made of immobilized Burkholderia cepacia lipase (PSL-C)-catalyzed acylation of lily polysaccharide (LP) with vinyl acetate in organic solvents, ionic liquids (ILs) and IL-containing systems. The degree of substitution (DS) of the modified LP was used to evaluate the extent of acylation and thus enzymatic activity. In this manner, an eco-friendly solvent, 2-methyltetrahydrofuran (MeTHF), was found to be the most suitable organic reaction medium. However, compared to MeTHF, enhanced enzyme activity was observed when 1-butyl-3-methylimidazolium tetrafluorobrate ([C₄MIm][BF₄]) was used as the solvent. To further enhance the DS of the modified LP product, co-solvent mixtures of [C₄MIm][BF₄] and MeTHF were investigated. Among the various MeTHF–[C₄MIm][BF₄] systems examined, 20% (v/v) MeTHF–[C₄MIm][BF₄] produced the highest DS. In this reaction medium, the optimal water activity, reaction temperature and time were 0.33, 55 °C and 18 h, respectively, producing a product DS as high as 0.67. The PSL-C enzyme exhibited a much higher stability in the IL-containing system. Additionally, PSL-C-catalyzed acylation of LP was highly regioselective, causing acylation of only C6OH.     

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.     

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.     

Laccases from Basidiomycetes: physicochemical characteristics and substrate specificity towards methoxyphenolic compounds

Laccases from the Basidiomycetes Coriolus hirsutus, Coriolus zonatus, Cerrena maxima, and Coriolisimus fulvocinerea have been isolated and purified to homogeneity and partially characterized. The kinetics of oxidation of different methoxyphenolic compounds by the fungal laccases has been studied. As laccase substrates, such methoxyphenolic compounds as 4-hydroxy-3,5-dimethoxycinnamic acid (sinapinic acid), 4-hydroxy-3-methoxycinnamic acid (ferulic acid), and 2-methoxyphenol (guaiacol) were used. The stoichiometries of the enzymatic reactions were determined: guaiacol and sinapinic acid are one-electron donors and their oxidation apparently results in the formation of dimers. It was established that k _cat/K _m, which indicates the effectiveness of catalysis, increases in the series guaiacol, ferulic acid, and sinapinic acid. This fact might be connected with the influence of substituents of the phenolic ring of the substrates. This phenomenon was established for fungal laccases with different physicochemical properties, amino acid composition, and carbohydrate content. This suggests that all fungal laccases possess the same mechanism of interaction between organic substrate electron donors and the copper-containing active site of the enzyme and that this interaction determines the observed values of the kinetic parameters.