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%.     

Adverse effect of chloride impurities on lipase-catalyzed transesterifications in ionic liquids

The adverse influence of chloride impurities on the lipase-catalyzed transesterification in ionic liquid is described. The activity of lipase from Rhizomucor miehei exponentially decreased with increasing Cl⁻ content in 1-octyl-3-methylimidazolium bis[(trifluoromethyl)sulfonyl] amide, [Omim][Tf₂N], and the activity of lipase in [Omim][Tf₂N] mixture containing 2% [Omim] [Cl] was only about 2% of the activity in pure [Omim][Tf₂N]. The activity of lipase from Candidantarctica linearly decreased at about 5% with every 1% increase in [Omim][Cl] with there being no activity in [Omim][Tf₂N] containing about 20% [Omim][Cl].     

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.     

Extracting keratin from chicken feathers by using a hydrophobic ionic liquid

Keratin was extracted from chicken feathers by using a hydrophobic ionic liquid (IL), 1-hydroxyethyl-3-methylimidazolium bis(trifluoromethanesulfonyl)amide ([HOEMIm][NTf₂]). Extracted keratin has good solubility in water while the ionic liquid is immiscible with water, and therefore the extracted keratin could be easily separated from the reaction system by water. The effects of ionic liquid, NaHSO₃, reaction temperature and time were investigated and extracting conditions were optimized. The maximum yield of keratin was up to about 21% with mass ratio of feathers to NaHSO₃1:1 and mass ratio of feathers to ionic liquid 1:40 at 80 °C for 4 h. Moreover, there was no obvious loss in the yield after ionic liquid was reused for five batches under optimized conditions. In addition, the recovery of ionic liquid was about 95% each time. The results indicated that [HOEMIm][NTf₂] was very efficient as catalyst and solvent for dissolving feathers and could be easily recovered due to its hydrophobicity.     

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.     

Asymmetric alkene epoxidation by Mn(III)salen catalyst in ionic liquids

The enantioselective epoxidation of 6-cyano-2,2-dimethylchromene (Chrom) catalysed by the Jacobsen catalyst, using sodium hypochlorite (NaOCl) as oxygen source, at room temperature, was performed in a series of 1,3-dialkylimidazolium and tetra-alkyl-dimethylguanidium based ionic liquids. All the room temperature ionic liquids (RTILs) could be used as reaction media for the enantioselective epoxidation of the alkene giving, generally, moderate to good epoxide yields and enantiomeric excesses (ee%).For the series of ionic liquids derived from the 1,3-dialkylimidazolium cation, it was observed some relationship between the RTILs physical properties and the catalytic reaction parameters, exemplified by linear correlations between (i) the ee% and the α Kamlet-Taft parameter (hydrogen bond acidity of the solvent) for CH₂Cl₂ and [C₄m_nim][BF₄] ionic liquids (n = 1 or 2), and (ii) the ee% and the β Kamlet-Taft parameter (hydrogen bond basicity of the solvent) for CH₂Cl₂ and [C₄mim][X] ionic liquids (X = PF₆, NTf₂ or BF₄).All the RTILs could be reused in further catalytic cycles, with the exception of [C₈mim][PF₆]. The reutilisation of the Jacobsen catalyst for four times generally led to a decrease in the epoxide yield and to a slight decrease in the enantioselectivity. The recycling of the catalyst could be improved by imparting an ionic character to the complex through abstraction of the axially coordinated chloride anion (Cat 2). Other oxygen sources, such as iodosylbenzene, hydrogen peroxide and urea-hydrogen peroxide adduct, were also tested coupled with Jacobsen catalyst, but the best results were achieved with NaOCl.     

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.     

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.     

Whole-cell biocatalysis: Evaluation of new hydrophobic ionic liquids for efficient asymmetric reduction of prochiral ketones

A biphasic process design is often applied in whole-cell biocatalysis if substrate and product have low water solubility, are unstable in water or toxic for the biocatalyst. Some water immiscible ionic liquids (ILs) with adequate distribution coefficients have already been applied successfully as second liquid phase, which acts as a substrate reservoir and in situ extractant for the product. In this work, 12 new ILs were evaluated with respect to their applicability in biphasic asymmetric reductions of prochiral ketones in comparison to 9 already published ILs. The ILs under study are composed of seven different cations and three different anions. Recombinant Escherichia coli was used as whole-cell biocatalyst overexpressing the genes of a Lactobacillus brevis alcohol dehydrogenase (LB-ADH) and a Candida boidinii formate dehydrogenase (CB-FDH) for cofactor regeneration. Best results were achieved if ionic liquids with [PF6]- and [NTF]-anions were applied, whereas [FAP]-ILs showed minor qualification, e.g., the use of [HMPL][NTF] as second liquid phase for asymmetric synthesis of (R)-2-octanol resulted in a space–time-yield of 180 g L⁻¹ d⁻¹, a chemical yield of 95% and an enantiomeric excess of 99.7% in a simple batch process.     

Synthesis of poly(ε-caprolactone) by an immobilized lipase coated with ionic liquids in a solvent-free condition

Polycaprolactone (PCL) was synthesized by ring-opening polymerization of ε-caprolactone through two different enzymatic processes. The lipase from Candida antarctica B, immobilized on macroporous acrylic acid beads, was employed either untreated or coated with small amounts of ionic liquids (ILs). Monocationic ionic liquids, [C _n MIm][NTf₂] (n = 2, 6, 12), as well as a dicationic ionic liquid, ([C₄(C₆Im)₂][NTf₂]₂), were used to coat the immobilized lipase and also as the reaction medium. In both methods, the polarity, anion of the ILs concentration and viscosity strongly influenced the reaction. Coating the immobilized enzyme with ILs improved catalytic activity and less ILs was required to produce PCL with a higher molecular weight and reaction yield. At 60 °C and ILs/Novozyme-435 coating ratio of 3:1 (w/w) for 48 h, the highest M _w and reaction yield of PCL were 35,600 g/mol and 62 % in the case of [C₁₂MIm][NTf₂], while the M _w and reaction yield of PCL was 20,300 g/mol and 54 % with [C₁₂MIm][NTf₂] and catalyzed by untreated lipase.     

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.     

Identification of suitable ionic liquids for application in the enzymatic hydrolysis of rutin by an automated screening

An automated method in milliliter scale was developed for the screening of process parameters concerning the hydrolysis of the flavonoid rutin catalyzed by the rhamnosidase activity of naringinase from Penicillium decumbens. Besides the effect of additives such as ionic liquids and low molecular salts, the productivity in a multiple phase system as well as the recyclability of the enzyme in repetitive batches were studied. The hydrophobic ionic liquid (IL) trihexyl(tetradecyl)phosphonium bis(trifluormethylsulfonyl)imide [P(h₃)t][Tf₂N] was identified to combine the most favorable characteristics out of 23 investigated ILs with regard to enzyme compatibility, substrate solubility and enzyme partition coefficient. Also, for the corresponding cations 1-ethyl-3-methylimidazolium [EMIM], 1-butyl-3-methylimidazolium [BMIM], 1-butyl-1-methylpyrrolidinium [BMPL] and 1-octyl-3-methylimidazolium [OMIM], the entity with the [Tf₂N] anion was best tolerated by the naringinase. With increasing IL content, higher space time yields with up to 1.5 g/(L h) for 80% (v/v) [P(h₃)t][Tf₂N] were achieved. Enhanced specific enzyme activity was observed in the presence of Ca²⁺ ions. By addition of [P(h₃)t][Tf₂N] and calcium chloride, the reactive aqueous phase was successfully used in three repetitive batches with full conversion.     

Production of chiral compound using recombinant Escherichia coli cells co-expressing reductase and glucose dehydrogenase in an ionic liquid/water two phase system

(S)-3-Chloro-1-phenyl-1-propanol ((S)-CPPO) is a useful chiral building block for the synthesis of anti-depressant drugs. The yeast reductase, YOL151W, evidences enantioselective reduction activity, converting 3-chloro-1-phenyl-1-propanone (3-CPP) into (S)-CPPO. Escherichia coli whole cells co-expressing YOL151W and Bacillus subtilis glucose dehydrogenase were employed for the synthesis of CPPO following permeabilization treatment. A reaction system employing these recombinant E. coli whole cells could convert 30 mM 3-CPP enantioselectively into (S)-CPPO. In an effort to enhance substrate solubility and to prevent substrate/product inhibition during the enzyme reaction process, a variety of ionic liquids were tested and [Bmim][NTf₂] was ultimately selected for the ionic liquid/water two phase system. Tween 40 was added to accomplish the efficient mixing of the two phases. Using these recombinant E. coli whole cells and the [Bmim][NTf₂]/water two phase system, 100 mM (S)-CPPO was generated with an enantiomeric excess of >99%.     

On the nature of ionic liquids and their effects on lipases that catalyze ester synthesis

Free and immobilized lipases from Candida antarctica (CALA and CALB), Thermomyces lanuginosus (TLL) and Rhizomucor miehei (RML) were used as catalysts in the synthesis of butyl propionate by transesterification in reaction media consisting in nine different ionic liquids. Enzyme activities were clearly dependent on the nature of the ions, the results being improving as the alkyl chain length of the imidazolium cation increased, and as a function of the type of anion ([PF₆], [BF₄] or [ethylsulphate]). The best synthetic activity (655.5 U/mg protein at 40 °C) was obtained when free CALB were assayed in the water-miscible IL cocosalkyl pentaethoxy methyl ammonium methosulfate ([CPMA][MS]), and was clearly related with the water content of the medium. The synthetic activity of free CALB in [CPMA][MS] was enhanced with the increase in temperature, while practically no effect was obtained for TLL. The ability of free CALB to synthesize aliphatic esters of different alkyl chain lengths, using different alkyl vinyl esters and 1-alkanols as substrates, was also studied in [CPMA][MS], the best results (4500 U/mg protein) being obtained for the synthesis of hexyl butyrate.     

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.     

Substrate stabilization and noncompetitive inhibition effects of a water-miscible ionic liquid [BMPy][BF<Subscript>4</Subscript>] in the catalysis of horseradish peroxidase

The inhibition mechanism of a water-miscible ionic liquid, N-butyl-3-methypyridinium tetrafluoroborate ([BMPy][BF₄]), on the catalysis of horseradish peroxidase (HRP) was investigated. The K _m value for the oxidation of guaiacol (2-methoxyphenol) with H₂O₂ catalyzed by HRP increased from 2.8 mM in 100% water to 12.6 mM in 25% (v/v) [BMPy][BF₄]. This increase of K _m by the ionic liquid was elucidated to be caused by the strong stabilization of the ground state of guaiacol by the ionic liquid. On the contrary, the k _cat value for the HRP-catalyzed reaction decreased from 13.8/sec in 100% water to 6.7/sec in 25% (v/v) [BMPy][BF₄]. Such decrease of k _cat value of HRP catalysis by the increasing content of [BMPy][BF₄] was described using the noncompetitive inhibition of the enzyme by the ionic liquid. The value of the inhibition constant of [BMPy][BF₄] was 1.48 M indicating that the ionic liquid exerts a weak noncompetitive inhibition effect on the HRP catalysis.     

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.     

Catalytic activity of α-chymotrypsin in enzymatic peptide synthesis in ionic liquids

The catalytic activity of α-chymotrypsin in the enzymatic peptide synthesis of N-acetyl-l-tryptophan ethyl ester with glycyl glycinamide was examined in ionic liquids and organic solvents. The water content in 1-ethyl-3-methylimidazolium bis(fluorosulfonyl)imide ([emim][FSI]) affected the initial rates of peptide synthesis and hydrolysis. The activity of α-chymotrypsin was influenced by a kind of anions consisting of the same cation, [emim], when an ionic liquid was used as a solvent. The initial rate of peptide synthesis was improved 16-fold by changing from an organic solvent, acetonitrile, to an ionic liquid, [emim][FSI], at 25 °C. The activity of α-chymotrypsin in the peptide synthesis in [emim][FSI] was 17 times greater than that in acetonitrile at 60 °C, although the activity of α-chymotrypsin in the peptide synthesis gradually decreased with an increase in reaction temperature in [emim][FSI], similar to organic solvents. Moreover, α-chymotrypsin exhibited activity in [emim][FSI] and [emim][PF₆] at 80 °C.     

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.     

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.