Modeling stability and flexibility of α-Chymotrypsin in room temperature ionic liquids

We investigate the structure and dynamics of α-Chymotrypsin in five room temperature ionic liquids (RTILs) sharing a common cation, hydrated with different water percentages (w/w) (weight of water over protein). Results from molecular dynamics simulations are correlated with experimental evidences from studies on the activity of enzymes in RTILs. α-Chymotrypsin protein structure is closer to its native crystallographic structure in RTILs than in aqueous environment. We show that the structural properties of α-Chymotrypsin were affected by the water concentration assayed in a typical bell-shaped profile, which is also frequently reported for organic solvents. The protein structure was more native like at 10–20% of water (w/w) for all RTILs except for [BMIM][Cl]. We found that the fluctuations of the main chain in [BMIM][BF₄] and [BMIM][TfO] were not significantly affected by the increasing amount of water. However, we were able to show that the flexible regions were the ones more hydrated, indicating that water is responsible for the flexibility of the protein. The solvation of the enzyme in water-immiscible RTILs, such as [BMIM][PF₆] and [BMIM][Tf₂N] lead to higher enzyme flexibility at increased water content. Enzyme solvation by [BMIM][Cl] resulted in ion penetration in the core enzyme structure, causing incremented flexibility and destabilization at low water percentages. All RTILs stripped water molecules from the protein surface, following a similar behavior also found in organic solvents. Anions formed structured arrangements around the protein, which allowed non-stripped water molecules to localize on the protein 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.     

Lipase‐catalyzed enantioselective transesterification of prochiral 1‐((1,3‐dihydroxypropan‐2‐yloxy)methyl)‐5,6,7,8‐tetrahydroquinazoline‐2,4(1H,3H)‐dione in ionic liquids

The application of ionic liquids as solvents for transesterification of prochiral pirymidine acyclonucleoside using lipase (EC 3.1.1.3) Amano PS from Burkholderia cepacia (BCL) is reported. The effect of using medium reaction, acyl group donor, and temperature on the activity and enantioselectivity of BCL was studied. From the investigated ionic solvents, the hydrophobic ionic liquid [BMIM]PF₆] was the preferred medium for enzymatic reactions. However, the best result was obtained in the mixture [BMIM][PF₆]:TBME (1:1 v/v) at 50°C. Enzyme activity and selectivity in [BMIM][PF₆]:TBME (1:1 v/v) was slightly higher in than in conventional organic solvents (for example, TBME), and in this condition, good activity and enantioselectivity were associated with unique properties of ionic liquid such as hydrophobicity and high polarity. Independently of solvents, monester of (R)‐configuration was obtained in excess. Under optimal conditions, desymmetrization of the prochiral compound using different acyl donors was performed. If vinyl butyrate was used as the acylating agent, BCL completely selectively acylated enantiotopic hydroxyl groups.     

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].     

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.     

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.     

Pseudomonas cepacia lipase catalyzed esterification and transesterification of 3-(furan-2-yl) propanoic acid/ethyl ester: A comparison in ionic liquids vs hexane

A comparison of the Pseudomonas cepacia lipase (lipase PS) catalyzed esterification of 3-(furan-2-yl) propanoic acid and transesterification of ethyl 3-(furan-2-yl) propanoate with six straight chain alcohols (propanol to octanol) in ionic liquids and hexane was carried out. The ionic liquids selected, [Bmim]BF₄, [Bmim]PF₆, and [Bmim]Tf₂N, consisted of an identical cation and different anions. This is the first report on the biocatalyzed synthesis of these esters. In all the media, lipase PS catalyzed esterification of 3-(furan-2-yl) propanoic acid resulted in high yields of the esters compared to the transesterification of ethyl 3-(furan-2-yl) propanoate. [Bmim]Tf₂N proved to be the best; yielding 98–67% of the product by lipase PS catalyzed esterification. The lipase PS–[Bmim]Tf₂N and lipase PS–[Bmim]PF₆ mixture was recycled five times without any decrease in the yields of the products and was found to be operationally stable up to 10 months at room temperature.     

Alpha-chymotrypsin catalysis in imidazolium-based ionic liquids

The transesterification reaction of N-acetyl-L-phenylalanine ethyl ester with 1-propanol catalyzed by alpha-chymotrypsin was examined in the ionic liquids 1-butyl-3-methylimidazolium hexafluorophosphate ([bmim][PF(6)]) and 1-octyl-3-methylimidazolium hexafluorophosphate ([omim][PF(6)]), and in combination with supercritical carbon dioxide (SC-CO(2)). The activity of alpha-chymotrypsin was studied to determine whether trends in solvent polarity, water activity, and enzyme support properties, observed with this enzyme in conventional organic solvents, hold for the novel environment provided by ionic liquids. alpha-Chymotrypsin freeze-dried with K(2)HPO(4), KCl, or poly(ethylene glycol) demonstrated no activity in [bmim][PF(6)] or [omim][PF(6)] at very low water concentrations, but moderate transesterification rates were observed with the ionic liquids containing 0.25% water (v/v) and higher. However, the physical complexation of the enzyme with poly(ethylene glycol) or KCl did not substantially stimulate activity in the ionic liquids, unlike that observed in hexane or isooctane. Activities were considerably higher in [omim][PF(6)] than [bmim][PF(6)]. Added water was not necessary for enzyme activity when ionic liquids were combined with SC-CO(2). These results indicate that [bmim][PF(6)] and [omim][PF(6)] provide a relatively polar environment, which can be modified with nonpolar SC-CO(2) to optimize enzyme activity.     

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     

Properties of an ionic liquid-tolerant <Emphasis Type="Italic">Bacillus</Emphasis><Emphasis Type="Italic">amyloliquefaciens</Emphasis> CMW1 and its extracellular protease

An ionic liquid-tolerant bacterium, Bacillus amyloliquefaciens CMW1, was isolated from a Japanese fermented soybean paste. Strain CMW1 grew in the presence of 10 % (v/v) 1-butyl-3-methylimidazolium chloride ([BMIM]Cl), a commonly used ionic liquid. Additionally, strain CMW1 grew adequately in the presence of the hydrophilic ionic liquids 10 % (v/v) 1-ethyl-3-methylimidazolium trifluoromethanesulfonate ([EMIM]CF₃SO₃) or 2.5 % (v/v) 1-butyl-3-methylimidazolium trifluoromethanesulfonate ([BMIM]CF₃SO₃). Strain CMW1 produced an extracellular protease (BapIL) in the culture medium. BapIL was stable in the presence of 80 % (v/v) ionic liquids, [EMIM]CF₃SO₃, [BMIM]Cl, [BMIM]CF₃SO₃, 1-butyl-3-methylimidazolium tetrafluoroborate, 1-butyl-3-methylimidazolium hexafluorophosphate, and 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide, and functioned in 10 % (v/v) these ionic liquids. BapIL was stable at pH 4.0–12.6 or in 4004 mM NaCl solution, and exhibited activity in the presence of 50 % (v/v) hydrophilic or hydrophobic organic solvents. BapIL was completely inhibited by 1 mM PMSF and partially by 5 mM EDTA. BapIL belongs to the true subtilisins according to analysis of the deduced amino acid sequence. We showed that BapIL from the ionic liquid-tolerant B. amyloliquefaciens CMW1 exhibited tolerance to ionic liquid and halo, alkaline, and organic solvents.     

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

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

High transesterification activities of immobilized proteases in new ether-functionalized ionic liquids

Three new ether-functionalized hydrophobic ionic liquids (ILs) were synthesized. Two proteases (subtilisin and α-chymotrypsin both covalently immobilized on chitosan) exhibited high synthetic activity (1–3 μmol/min·g) and selectivity (>97–99%, esterification over hydrolysis) in these ILs containing 10–15% (v/v) water during the transesterification of N-acetyl-l-phenylalanine ethyl ester with 1-propanol. However, the same reaction in t-butanol or [BMIM][Tf₂N], with water contents higher than 2% (v/v) yielded low synthetic activities (0.2–1 μmol/min g in t-butanol) and/or poor selectivity (<40%). The high synthetic activities of proteases in ether-functionalized ILs at high water contents are explained by two reasons: (1) the hydrogen-bond donors in ILs (R₁–O–R₂) and chitosan (–OH) controlling the thermodynamic water activity of the reaction system, and (2) the protective role of the ether chain in reducing the cation–protein interaction.     

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

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

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.     

Environmentally friendly, efficient resolution of racemic secondary alcohols by lipase-catalyzed enantioselective transesterification in ionic liquids in the presence of organic bases

The lipase-catalyzed enantioselective transesterification of racemic secondary alcohols was studied using vinyl acetate as acyl donor in two imidazolium-based ionic liquids vs. hexane (Scheme), both in the absence and presence of catalytic amounts of organic bases such as triethylamine (Et(3)N) or pyridine. The organic bases generally enhanced both the rate and enantioselectivity of the reaction. Further, the system 1-butyl-3-methyl-1H-imidazolium hexafluorophosphate/Candida antarctica lipase B ([bmim][PF(6)]/CALB) could be readily recycled four times without significant loss in activity or enantioselectivity.     

Crystallisation of inorganic salts containing 18-crown-6 from ionic liquids

Reaction of the zwitterionic imidazolium salt [(CH₂COOH)(CH₂COO)im] with K₂CO₃ or BaO in the presence of 18-crown-6 affords the salts [(CH₂COO)₂im][K(18-crown-6)] and [(CH₂COO)₂im]₂[Ba(18-crown-6)], respectively. Recrystallisation of these crown complexes from the ionic liquid 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)amide, [emim][Tf₂N], at a water interface, results in the formation of new salts in which the original anion is replaced by Tf₂N⁻. Single crystal X-ray diffraction has been performed on two of the salts. Notably, the potassium structure containing 18-crown-6 and Tf₂N⁻ forms a linear chain coordination polymer that can be regarded as metal organic frameworks (MOFs). Moreover, this study provides insights into the separation of group I and II metal ions using crown ethers in combination with ionic liquids.     

Enzymatic selective acylation of glycosides in ionic liquids: significantly enhanced reactivity and regioselectivity

The enzymatic selective acylations of carbohydrates in ionic liquids were explored in both organic solvents and ionic liquids to see any significant differences in terms of reactivity and regioselectivity between two different classes of reaction media. Monoprotected glycosides (methyl-6-O-trityl-glucosides and galactosides) were chosen as the substrates with Candida rugosa lipase as an acylation enzyme. Two organic solvents, THF and chloroform, and two ionic liquids, [BMIM]⁺PF₆⁻ ([BMIM]⁺ = 1-butyl-3-methylimidazolium) and [MOEMIM]⁺PF₆⁻ ([MOEMIM]⁺ = 1-methoxyethyl-3-methylimidazolium), were employed as reaction media. The enzymatic reactions were performed in the presence of vinyl acetate at room temperature. It was observed that the reactions in ionic liquids took place more rapidly and more selectively than those in conventional organic solvents.     

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.     

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

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

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.