Combination use of ultrasound irradiation and ionic liquid in enzymatic isomerization of glucose to fructose

The synthetic effect of the combination use of ultrasound irradiation (UI) and ionic liquid (IL) on improving enzyme activity was studied when they were employed in isomerization of glucose to fructose by immobilized glucose isomerase (IGI, produced from streptomyces murinus and immobilized on silica). Both of UI and IL [EMIM][Cl], which was screened as the best medium for this reaction, were found to increase the enzyme activity in isomerization reaction. And a further increase of enzyme activity was observed by combination use of UI and IL. A systematic screening and optimization of the reaction parameters in ILs under UI on the IGI activity were performed. Under the optimum reaction conditions, 45.3% yield of fructose was achieved in 10 h under UI in [EMIM][Cl], compared to only 41.5% yield under stirring in [EMIM][Cl], 44.2% under UI without [EMIM][Cl] and 38.9% under stirring without [EMIM][Cl] in 12 h, respectively. High thermal stability and reusability of IGI was also observed under UI in [EMIM][Cl]. These results indicated that the combination use of UI and IL might be a fast and efficient method for enzymatic isomerization of glucose to fructose.     

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

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

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.     

Enhanced activity and stability of ionic liquid-pretreated lipase

The activity and stability of Mucor javanicus lipase pretreated with various ionic liquids (ILs) were investigated. The results show that the activity and stability of lipase pretreated with ILs were higher than those of untreated lipase for the hydrolysis reaction in an aqueous medium. The activities of lipase pretreated with ILs such as [Bmim][PF₆], [Emim][Tf₂N], [Bmim][BF₄] and [Emim][BF₄] were 1.81, 1.66, 1.56 and 1.60 times higher than that of untreated lipase, respectively. Furthermore, activities of lipase in ILs were well maintained even after 7 days of incubation in ILs at 60 °C, while untreated lipase in phosphate buffer was fully inactivated only after 12 h of incubation at the same temperature. These results suggest that pretreatment of lipase with ILs might form IL-coated lipase which causes the structural change of lipase, and thus, enhances the activity and stability of lipase in aqueous solution.     

Peroxidase biocatalysis in water-soluble ionic liquids: activity,kinetic and thermal stability

Abstract

The activity and stability of commercial peroxidase was investigated in the presence of five 1-alkyl-3-methylimidazolium-based ionic liquids (ILs) with either bromide or chloride anions: [C_xmim][X]. The peroxidase activity and stability were better for the shorter alkyl chain lengths of the ILs and peroxidase was more stable in the presence of the bromide anion, rather than chloride. The thermal inactivation profile was studied from 45 to 60 °C in [C₄mim][Cl] and [C₄mim][Br]. The activation energy was also determined. Kinetic analysis of the enzyme in the presence of the [C₄mim][Br] or control (buffer solution) showed that the KM value increased 5-fold and Vm decreased 13-fold in the presence of the IL. The increase in KM indicates that this IL can reduce the binding affinity between substrate and enzyme.     

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.     

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.     

Compatible ionic liquid-cellulases system for hydrolysis of lignocellulosic biomass

Ionic liquids (ILs) have been increasingly recognized as novel solvents for dissolution and pretreatment of cellulose. However, cellulases are inactivated in the presence of ILs, even when present at low concentrations. To more fully exploit the benefits of ILs it is critical to develop a compatible IL‐cellulases system in which the IL is able to effectively solubilize and activate the lignocellulosic biomass, and the cellulases possess high stability and activity. In this study, we investigated the stability and activity of a commercially available cellulases mixture in the presence of different concentrations of 1‐ethyl‐3‐methylimidazolium acetate ([Emim][OAc]). A mixture of cellulases and β‐glucosidase (Celluclast1.5L, from Trichoderma reesei, and Novozyme188, from Aspergillus niger, respectively) retained 77% and 65% of its original activity after being pre‐incubated in 15% and 20% (w/v) IL solutions, respectively, at 50°C for 3 h. The cellulases mixture also retained high activity in 15% [Emim][OAc] to hydrolyze Avicel, a model substrate for cellulose analysis, with conversion efficiency of approximately 91%. Notably, the presence of different amounts of yellow poplar lignin did not interfere significantly with the enzymatic hydrolysis of Avicel. Using this IL‐cellulase system (15% [Emim][OAc]), the saccharification of yellow poplar biomass was also significantly improved (33%) compared to the untreated control (3%) during the first hour of enzymatic hydrolysis. Together, these findings provide compelling evidence that [Emim][OAc] was compatible with the cellulase mixture, and this compatible IL‐cellulases system is promising for efficient activation and hydrolysis of native biomass to produce biofuels and co‐products from the individual biomass components. Bioeng. 2011; 108:1042–1048. © 2010 Wiley Periodicals, Inc.     

Efficient pretreatment of lignocellulose in ionic liquids/co-solvent for enzymatic hydrolysis enhancement into fermentable sugars

Ionic liquids (ILs) have been widely used as alternative solvents for biomass pretreatment, however, efficient methods that enable economically use of ILs at large scale have not been established. In this study, a new method in which ILs and polar organic solvents (ILs/co-solvent systems) was proposed for efficient pretreatment of lignocellulosic materials. The combination use of appropriate ILs and organic co-solvents can significantly enhance the solubility of lignocellulose due to the lower viscosity of ILs/co-solvent mixture as compared to those of pure ILs while the hydrogen bond basicity was maintained. In addition, the solubility of lignocellulosic materials in ILs/co-solvent system was found to be correlated with the Kamlet-Taft solvent parameters. Moreover, the use of microwave heating also enhances the efficiency of lignocellulose pretreatment. For example, the microwave-assisted [Emim][OAc]-DMSO (1:1 volume ratio) treated-rice straw could be hydrolyzed at least 22 times faster than that of untreated-rice straw by cellulase from Trichoderma reesei. This enhancement was attributed by several factors including more efficient lignin extraction, less crystalline cellulose and lower residual ILs in treated-rice straw. The produced sugars can be effectively fermented by Pichia stipitis for ethanol production. Moreover, [Emim][OAc]-DMSO mixture could be reused at least 5 times without significantly decrease in effectiveness demonstrated that the use of ILs/co-solvent was potential alternative method for large-scale biomass pretreatment.     

Ionic liquids as alternative co-solvents for laccase: study of enzyme activity and stability

The activity and stability of commercial laccase (DeniLite base) in three different water soluble ionic liquids (ILs) (1-ethyl-3-methylimidazolium 2-(2-methoxyethoxy) ethylsulfate, [emim][[MDEGSO4], 1-ethyl-3-methylimidazolium ethylsulfate, [emim][EtSO4], and 1-ethyl-3-methylimidazolium methanesulfonate, [emim][MeSO3]) have been studied and compared to that in two organic solvents (acetonitrile and dimethyl sulfoxide). Initial enzyme activities were similar among the ILs if the same conditions were used. A high reduction on initial enzyme activity was found with acidic pH (5.0). The effect of pH and solvent concentration on enzyme stability were investigated in more detail for 1 week. The enzyme maintained a high stability at pH 9.0 for all ILs tested. [emim][MDEGSO4] was the most promising IL for laccase with an activity loss of about 10% after 7 days of incubation. The kinetic studies in the presence of ABTS as substrate allowed to calculate the Michaelis- Menten parameters. Good agreement was found between experimental data and calculated values using the Michaelis-Menten mechanism, with a total average relative deviation of 2.1%.     

α-Rhamnosidase and β-glucosidase expressed by naringinase immobilized on new ionic liquid sol-gel matrices: Activity and stability studies

Novel ionic liquid (IL) sol-gel materials development, for enzyme immobilization, was the goal of this work. The deglycosylation of natural glycosides were performed with α-l-rhamnosidase and β-d-glucosidase activities expressed by naringinase. To attain that goal ILs with different structures were incorporated in TMOS/Glycerol sol-gel matrices and used on naringinase immobilization.The most striking feature of ILs incorporation on TMOS/Glycerol matrices was the positive impact on the enzyme activity and stability, which were evaluated in fifty consecutive runs. The efficiency of α-rhamnosidase expressed by naringinase TMOS/Glycerol@ILs matrices increased with cation hydrophobicity as follows: [OMIM] > [BMIM] > [EMIM] > [C₂OHMIM] > [BIM] and [OMIM] ≈ [E₂-MPy] ? [E₃-MPy]. Regarding the imidazolium family, the hydrophobic nature of the cation resulted in higher α-rhamnosidase efficiencies: [BMIM]BF₄ ? [C₂OHMIM]BF₄ ? [BIM]BF₄. Small differences in the IL cation structure resulted in important differences in the enzyme activity and stability, namely [E₃-MPy] and [E₂-MPy] allowed an impressive difference in the α-rhamnosidase activity and stability of almost 150%. The hydrophobic nature of the anion influenced positively α-rhamnosidase activity and stability. In the BMIM series the more hydrophobic anions (PF₆⁻, BF₄⁻ and Tf₂N⁻) led to higher activities than TFA. SEM analysis showed that the matrices are shaped lens with a film structure which varies within the lens, depending on the presence and the nature of the IL.The kinetics parameters, using naringin and prunin as substrates, were evaluated with free and naringinase encapsulated, respectively on TMOS/Glycerol@[OMIM][Tf₂N] and TMOS/Glycerol@[C₂OHMIM][PF₆] and on TMOS/Glycerol. An improved stability and efficiency of α-l-rhamnosidase and β-glucosidase expressed by encapsulated naringinase on TMOS/Glycerol@[OMIM][Tf₂N] and TMOS/Glycerol@[C₂OHMIM][PF₆] were achieved. In addition to these advantageous, with ILs as sol-gel templates, environmental friendly processes can be implemented.     

Influence of ionic liquids as additives on sol–gel immobilized lipase

The immobilization of lipase from Candida rugosa, using ionic liquids as additives to protect the inactivation of lipase by released alcohol and shrinking of gel during sol–gel process, was investigated. The influence of various factors, such as structure of ionic liquids, content of ionic liquids and types of precursor in the sol–gel process on the activity and stability of immobilized lipase was also studied. The highest hydrolytic activity of immobilized lipase was obtained when the hydrophilic ionic liquid, [C₂mim][BF₄], was used as an additive, while the highest stability of immobilized lipase was obtained by using hydrophobic ionic liquid, [C₁₆mim][Tf₂N]. Therefore, the binary mixtures of these ionic liquids as additives were used to obtain the optimal immobilized lipase, which shows both high activity and stability. The hydrolysis and esterification activities of lipase co-immobilized with the mixture of 1:1 at molar ratio of [C₂mim][BF₄] and [C₁₆mim][Tf₂N] were 10-fold and 14-fold greater than in silica gel without ionic liquids (ILs), respectively. After 5 days incubation of this immobilized lipase in n-hexane at 50 °C, 84% of initial activity was remained, while the residual activity of the lipase immobilized without ILs was 28%.     

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.     

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     

The Effect of Hydrophilic Ionic Liquids 1-Ethyl-3-Methylimidazolium Lactate and Choline Lactate on Lipid Vesicle Fusion

Ionic liquids (ILs) are room-temperature molten salts that have applications in both physical sciences and more recently in the purification of proteins and lipids, gene transfection and sample preparation for electron microscopy (EM) studies. Transfection of genes into cells requires membrane fusion between the cell membrane and the transfection reagent, thus, ILs may be induce a membrane fusion event. To clarify the behavior of ILs with cell membranes the effect of ILs on model membranes, i.e., liposomes, were investigated. We used two standard ILs, 1-ethyl-3-methylimidazolium lactate ([EMI][Lac]) and choline lactate ([Ch][Lac]), and focused on whether these ILs can induce lipid vesicle fusion. Fluorescence resonance energy transfer and dynamic light scattering were employed to determine whether the ILs induced vesicle fusion. Vesicle solutions at low IL concentrations showed negligible fusion when compared with the controls in the absence of ILs. At concentrations of 30% (v/v), both types of ILs induced vesicle fusion up to 1.3 and 1.6 times the fluorescence intensity of the control in the presence of [Ch][Lac] and [EMI][Lac], respectively. This is the first demonstration that [EMI][Lac] and [Ch][Lac] induce vesicle fusion at high IL concentrations and this observation should have a significant influence on basic biophysical studies. Conversely, the ability to avoid vesicle fusion at low IL concentrations is clearly advantageous for EM studies of lipid samples and cells. This new information describing IL-lipid membrane interactions should impact EM observations examining cell morphology.     

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.     

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.     

Improving the catalytic performance of porcine pancreatic lipase in the presence of [MMIm][MeSO4] with the modification of functional ionic liquids

Porcine pancreatic lipase (PPL) was chemically modified with various functional ionic liquids (ILs) to increase its catalytic performance in water-miscible IL. Catalytic activity and thermostability were tested with a p-nitrophenyl palmitate (pNPP) hydrolysis reaction. The native enzyme lost 18% of its initial activity in 0.4 M [MMIm][MeSO₄], whereas the activities of all the modified enzymes increased. The [HOOCBMIm][Cl] modification led to a 2-fold increase in activity in 0.3 M [MMIm][MeSO₄] than in aqueous. All the modified enzymes exhibited higher thermostability compared with the native enzyme at high temperature. In particular, the [HOOCBMIm][Cl] modification led to a 6-fold increase in thermostability at 60 °C. Conformational changes were confirmed by fluorescence spectroscopy and circular dichroism spectroscopy to elucidate the mechanism of catalytic performance alteration.     

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 ionic liquids activation on laccase from Trametes versicolor: Enzymatic stability and activity

The stability and activity of laccase from Trametes versicolor in two water‐soluble ionic liquids (ILs), namely 1‐butyl‐3‐methylimidazolium methyl sulfate, [bmim][MeSO₄] and 1,3‐dimethylimidazolium methyl sulfate, [mmim][MeSO₄], were investigated in this study. Thermal inactivation of laccase was characterized in the presence of these both ILs and as expected first‐order kinetics was followed. Inactivation rate constant (k), half‐life time (t_1/2), and energy of activation (Ea) were determined. Kinetics of 2,2′‐azino‐bis(3‐ethylbenzthiazoline‐6‐sulfonic acid) oxidation by laccase in the presence of these ILs was studied and Michaelis–Menten parameters were calculated. There is no enzymatic inactivation since the maximum reaction rate remained constant for IL concentrations up to 25%, and surprisingly, it was found that laccase was activated for concentrations of 35% of ILs, since the reaction rate increased 1.7 times.