The effect of imidazolium cations on the structure and activity of the Candida antarctica Lipase B enzyme in ionic liquids.

In order to understand how cations affect the structural changes and enzyme activity of Lipase B from Candida antarctica, we performed all-atom molecular dynamics simulations of CALB in four types of ionic liquids (ILs) with varying sizes of imidazolium cations and correlated these results with the experimentally determined CALB activity. The imidazolium cations under study differ in the alkyl tail length in the following order: [Emim](+) < [Bmim](+) < [Hmim](+) < [Omim](+). We observed that the best enzyme activity and structural stability of CALB are obtained in [Bmim][TfO] and [Hmim][TfO]. In contrast, in [Emim][TfO], bonding of [TfO](-) to LYS-290 disrupts the interactions between LYS-290 and ILE-285, which leads to a closed catalytic gate conformation with low accessibility of substrates to the catalytic triad. In [Omim][TfO], strong hydrophobic interactions between [Omim](+) and LEU-278 result in a significant loss of the secondary structure of the α-10 helix and cause the exposure of the catalytic triad to ILs, which affects the stability of the catalytic triad and consequently deteriorates the enzyme activity. Overall, our study indicates that a high ion coordination number ([Emim][TfO]) or the presence of a long hydrophobic tail ([Omim][TfO]) can facilitate ion-protein interactions that cause structural distortions and a decrease in CALB enzyme activity in ILs.

The relationship between enhanced enzyme activity and structural dynamics in ionic liquids: a combined computational and experimental study.

Candida antarctica lipase B (CALB) is an efficient biocatalyst for hydrolysis, esterification, and polymerization reactions. In order to understand how to control enzyme activity and stability we performed a combined experimental and molecular dynamics simulation study of CALB in organic solvents and ionic liquids (ILs). Our results demonstrate that the conformational changes of the active site cavity are directly related to enzyme activity and decrease in the following order: [Bmim][TfO] > tert-butanol > [Bmim][Cl]. The entrance to the cavity is modulated by two isoleucines, ILE-189 and ILE-285, one of which is located on the α-10 helix. The α-10 helix can substantially change its conformation due to specific interactions with solvent molecules. This change is acutely evident in [Bmim][Cl] where interactions of LYS-290 with chlorine anions caused a conformational switch between α-helix and turn. Disruption of the α-10 helix structure results in a narrow cavity entrance and, thus, reduced the activity of CALB in [Bmim][Cl]. Finally, our results show that the electrostatic energy between solvents in this study and CALB is correlated with the structural changes leading to differences in enzyme activity.

Optimization of lipase-catalyzed synthesis of caffeic acid phenethyl ester in ionic liquids by response surface methodology.

Lipase-catalyzed caffeic acid phenethyl ester (CAPE) synthesis in ionic liquid, 1-ethyl-3-methylimidazolium bis[(trifluoromethyl)sulfonyl]imide ([Emim][Tf(2)N]), was investigated in this study. The effects of several reaction conditions, including reaction time, reaction temperature, substrate molar ratio of phenethyl alcohol to caffeic acid (CA), and weight ratio of enzyme to CA, on CAPE yield were examined. In a single parameter study, the highest CAPE yield in [Emim][Tf(2)N] was obtained at 70 °C with a substrate molar ratio of 30:1 and weight ratio of enzyme to CA of 15:1. Based on these results, response surface methodology (RSM) with a 3-level-4-factor central composite rotatable design (CCRD) was adopted to evaluate enzymatic synthesis of CAPE in [Emim][Tf(2)N]. The four major factors were reaction time (36-60 h), reaction temperature (65-75 °C), substrate molar ratio of phenethyl alcohol to CA (20:1-40:1), and weight ratio of enzyme to CA (10:1-20:1). A quadratic equation model was used to analyze the experimental data at a 95 % confidence level (p < 0.05). A maximum conversion yield of 99.8 % was obtained under the optimized reaction conditions [60 h, 73.7 °C, substrate molar ratio of phenethyl alcohol to CA (27.1:1), and weight ratio of enzyme to CA (17.8:1)] established by our statistical method, whereas the experimental conversion yield was 96.6 ± 2 %.

Effect of ionic liquids on enzymatic synthesis of caffeic acid phenethyl ester.

Although caffeic acid phenethyl ester (CAPE), an active flavonoid, plays an important role in the antioxidant activity of honeybee propolis, the isolation of CAPE from honeybee propolis is time-consuming due to wide variety of impurities present. Therefore, biochemical method to synthesize CAPE was investigated in this study. Since ionic liquids (ILs) possess some unique characteristics as appreciated alternatives to conventional solvents for certain biotransformation, the effect of ILs as reaction media for enzymatic synthesis of CAPE was assessed. Several factors including substrate molar ratio, and reaction temperature affecting the conversion yield of lipase-catalyzed CAPE synthesis were also investigated. Reaction yields were significantly higher in hydrophobic ILs than in hydrophilic ILs (almost zero). Among nine hydrophobic ILs tested, the highest conversion of synthetic reaction was obtained in 1-ethyl-3-methylimidazolium bis[(trifluoromethyl)sulfonyl]imide ([Emim][Tf(2)N]). A reaction temperature of 70 °C was found to give high conversion. In addition, optimal substrate molar ratio between phenethyl alcohol and caffeic acid (CA) was decreased significantly from 92:1 to 30:1 when ILs were used instead of isooctane.

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][Tf(2)N]) 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][Tf(2)N] 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.

Beta-glucosidase coating on polymer nanofibers for improved cellulosic ethanol production.

Beta-glucosidase (betaG) can relieve the product inhibition of cellobiose in the cellulosic ethanol production by converting cellobiose into glucose. For the potential recycled uses, betaG was immobilized and stabilized in the form of enzyme coating on polymer nanofibers. The betaG coating (EC-betaG) was fabricated by crosslinking additional betaG molecules onto covalently attached betaG molecules (CA-betaG) via glutaraldehyde treatment. The initial activity of EC-betaG was 36 times higher than that of CA-betaG. After 20 days of incubation under shaking, CA-betaG and EC-betaG retained 33 and 91% of each initial activity, respectively. Magnetic nanofibers were also used for easy recovery and recycled uses of betaG coating. It is anticipated that the recycled uses of highly active and stable betaG coating can improve the economics of cellulosic ethanol production so long as economical materials are employed as a host of enzyme immobilization.

Highly stable trypsin-aggregate coatings on polymer nanofibers for repeated protein digestion.

A stable and robust trypsin-based biocatalytic system was developed and demonstrated for proteomic applications. The system utilizes polymer nanofibers coated with trypsin aggregates for immobilized protease digestions. After covalently attaching an initial layer of trypsin to the polymer nanofibers, highly concentrated trypsin molecules are crosslinked to the layered trypsin by way of a glutaraldehyde treatment. This process produced a 300-fold increase in trypsin activity compared with a conventional method for covalent trypsin immobilization, and proved to be robust in that it still maintained a high level of activity after a year of repeated recycling. This highly stable form of immobilized trypsin was resistant to autolysis, enabling repeated digestions of BSA over 40 days and successful peptide identification by LC-MS/MS. This active and stable form of immobilized trypsin was successfully employed in the digestion of yeast proteome extract with high reproducibility and within shorter time than conventional protein digestion using solution phase trypsin. Finally, the immobilized trypsin was resistant to proteolysis when exposed to other enzymes (i.e., chymotrypsin), which makes it suitable for use in "real-world" proteomic applications. Overall, the biocatalytic nanofibers with trypsin aggregate coatings proved to be an effective approach for repeated and automated protein digestion in proteomic analyses.

Separation of D-psicose and D-fructose using simulated moving bed chromatography.

Simulated moving bed (SMB) processes have been widely used in the sugar industries with ion-exchange resin as a stationary phase. D-psicose, a rare monosaccharide known as a valuable pharmaceutical substrate, was synthesized by the enzymatic conversion from D-fructose. The SMB process was adopted to separate D-psicose from D-fructose. Before the SMB experiment, the reaction mixture including D-psicose and D-fructose was treated by a deashing process to remove contaminants, such as buffers, proteins, and other organic materials. Four columns packed with Dowex 50WX4-Ca2+ (200-400 mesh) ion-exchange resins were used in the four-zone SMB. Single-step frontal analysis was performed to estimate the isotherm parameters of each monosaccharide. The operating conditions of the SMB process were determined based on the Equilibrium Theory. According to the simulation of the SMB process, the purity and yield of extract product (D-psicose) achieved were 99.04 and 97.46%, respectively and those of raffinate product (D-fructose) were 99.06 and 99.53%, respectively. Under the optimized operating condition, complete separation (extract purity = 99.36%, raffinate purity = 99.67%) was achieved experimentally.

Whole-Cell Biocatalysis in Ionic Liquids.

The use of whole-cell biocatalysis in ionic liquid (IL)-containing systems has attracted increasing attention in recent years. Compared to bioreactions catalyzed by isolated enzymes, the major advantage of using whole cells in biocatalytic processes is that the cells provide a natural intracellular environment for the enzymes to function with in situ cofactor regeneration. To date, the applications of whole-cell biocatalysis in IL-containing systems have focused on the production of valuable compounds, mainly through reduction, oxidation, hydrolysis, and transesterification reactions. The interaction mechanisms between the ILs and biocatalysts in whole-cell biocatalysis offer the possibility to effectively integrate ILs with biotransformation. This chapter discusses these interaction mechanisms between ILs and whole-cell catalysts. In addition, examples of whole-cell catalyzed reactions with ILs will also be discussed. Graphical Abstract.

Separation characteristics of hydrophilic ionic liquids from ionic liquids-water solution by ultrasonic atomization.

Ionic liquids (ILs) have attracted much attention as promising alternatives for volatile organic solvents. Although the applications of ILs have been found in a diverse range of fields, there are a limited number of methods for the recovery of ILs so far. As an efficient separation method, therefore, ultrasonic atomization has been attempted to recover hydrophilic ILs, [Bmim][BF4], from ILs-water solution. In order to examine the separation characteristics of hydrophilic ILs-water solution, ultrasonic atomization of hydrophilic ILs-water solution was performed under various operating conditions such as initial ILs concentration, ultrasonic electric power, carrier gas flow rate, and operating temperature. The result showed that hydrophilic ILs recovery yield increased with a decrease in ultrasonic electric power, gas velocity, and temperature. As an increase in initial ILs concentration, however, higher ILs recovery yield was obtained. After 6 h of ultrasonic atomization of 50% (v/v) [Bmim][BF4]-water solution, 93.4% of initial ILs amount was recovered without any changes in their structure at ultrasonic power of 10 W, carrier gas flow of 5 L/min and temperature of 20 °C. It demonstrated that ultrasonic atomization could be used for the recovery of ILs from ILs-aqueous solution.

Enhanced lipase-catalyzed synthesis of sugar fatty acid esters using supersaturated sugar solution in ionic liquids.

A solvent-mediated method (SMM) was used to prepare supersaturated sugar solutions in hydrophobic and mixture of hydrophilic/hydrophobic ionic liquids (ILs), namely, [Bmim][Tf2N] and [Bmim][TfO]/[Bmim][Tf2N], respectively. In this method, sugars were first solubilized in a mixture of organic solvent and water (i.e. methanol:water, 1:1 v/v), and then added to [Bmim][Tf2N] and/or [Bmim][TfO]/[Bmim][Tf2N] mixture. Supersaturated sugar solution in ILs were obtained by removing organic solvents and water under vacuum evaporation. Sugar solubilities in ILs, especially in hydrophobic IL ([Bmim][Tf2N]) and in [Bmim][TfO]/[Bmim][Tf2N] mixture prepared by SMM were greater than in ILs prepared using water-mediated method (WMM), which suggested methanol aided sugar solvation in hydrophobic media. In addition, interactions between glucose molecules and between glucose and methanol, water, and IL were investigated by all-atom molecular dynamics (MD) simulation. The MD simulation results showed that initial water and water/methanol molecules around glucose were gradually replaced by IL anions. Notably, SMM resulted in stronger interaction between IL anions and glucose than WMM, which was attributed to greater solubility of sugar in ILs prepared by SMM. Resultantly, the productivity of lipase-catalyzed production of glucose laurate using supersaturated glucose solution in [Bmim][TfO]/[Bmim][Tf2N] mixture prepared by SMM was at least 1.76-fold greater than that obtained in IL mixture prepared by WMM.

Simple synthesis of functionalized superparamagnetic magnetite/silica core/shell nanoparticles and their application as magnetically separable high-performance biocatalysts.

Uniformly sized silica-coated magnetic nanoparticles (magnetite@silica) are synthesized in a simple one-pot process using reverse micelles as nanoreactors. The core diameter of the magnetic nanoparticles is easily controlled by adjusting the w value ([polar solvent]/[surfactant]) in the reverse-micelle solution, and the thickness of the silica shell is easily controlled by varying the amount of tetraethyl orthosilicate added after the synthesis of the magnetite cores. Several grams of monodisperse magnetite@silica nanoparticles can be synthesized without going through any size-selection process. When crosslinked enzyme molecules form clusters on the surfaces of the magnetite@silica nanoparticles, the resulting hybrid composites are magnetically separable, highly active, and stable under harsh shaking conditions for more than 15 days. Conversely, covalently attached enzymes on the surface of the magnetite@silica nanoparticles are deactivated under the same conditions.

Lipase-catalyzed synthesis of fatty acid sugar ester using extremely supersaturated sugar solution in ionic liquids.

The low solubility of sugars has hampered the lipase-catalyzed synthesis of fatty acid sugar esters in organic solvents and ionic liquids (ILs), because several solvents that are able to effectively dissolve sugars are detrimental to enzymes. In this work, in order to prepare a high concentration of sugars in ILs, we have developed a new procedure that entails mixing an aqueous sugar solution into ILs followed by removal of the water from the solution. The glucose concentrations in the supersaturated [Emim][TfO] and [Bmim][TfO] were 19 and 10 times higher, respectively, than the solubilities (6.1 and 4.8 g/L) of glucose in the ILs at 25 degrees C. Furthermore, the supersaturated glucose solutions in ILs were maintained over a long period of time without any significant loss of glucose. In ILs that were extremely supersaturated with glucose, lipase-catalyzed esterifications of glucose with vinyl laurate, and lauric acid were successfully carried out. The conversion increased from 8% to 96% at 1 day of reaction by using supersaturated solution in [Bmim][TfO] which had dissolved glucose concentration of 400% higher than its solubility, compared with the reaction using saturated glucose solution. By making the glucose concentration in ILs much higher than the solubility through our novel and simple method, the initial rate and conversion of the lipase-catalyzed reaction were significantly improved.

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.

Aspergillus niger whole-cell catalyzed synthesis of caffeic acid phenethyl ester in ionic liquids.

Synthesis of caffeic acid ester essentially requires an efficient esterification process to produce various kinds of medicinally important ester derivatives. In the present study, a comprehensive and comparative analysis of whole-cell catalyzed caffeic acid esters production in ionic liquids (ILs) media was performed. Olive oil induced mycelial mass of halotolerant Aspergillus niger (A.niger) EXF 4321 was freeze dried and used as a catalyst. To ensure maximum solubilization of caffeic acid for highest substrate loading several ILs were screened and 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide ([Emim][Tf2N]) was found to have the maximum solubility and favoured for enzymatic activity of freeze dried mycelia. The whole-cell catalyzed synthesis of caffeic acid phenethyl ester (CAPE) conditions were optimized and bioconversion up to 84% was achieved at a substrate molar ratio of 1:20 (caffeic acid:2-phenyl ethanol), 30°C for 12h. Results obtained during this study were encouraging and helpful to design a bioreactor system to produce caffeic acid derived esters.

Affinity separation by protein conjugated IgG in aqueous two-phase systems using horseradish peroxidase as a ligand carrier.

A novel affinity separation method in an aqueous two-phase system (ATPS) is suggested, using protein conjugated IgG as a ligand. For verification of the proposed approach, horseradish peroxidase (HRP) and human IgG was used as a ligand carrier and affinity ligand, respectively. The partition of the affinity ligand, human IgG, was controlled by the conjugation of HRP. Two ATPSs, one consisting of potassium phosphate (15%, w/w) and polyethylene glycol (PEG, M.W. 1450, 10%, w/w) and the other of dextran T500 (5%, w/w) and PEG (M.W. 8000, 5%, w/w), were used. The conjugated human IgG-HRP favored a PEG-rich top phase, whereas human IgG, rabbit anti-human IgG and goat anti-mouse IgG preferred a salt or dextran-rich bottom phase. Using the conjugated human IgG-HRP, rabbit anti-human IgG was successfully separated into a PEG-rich top phase from the mixture with goat anti-mouse IgG. The appropriate molar ratio between human IgG-HRP and rabbit anti-human IgG was around 3:1 and 1:1 for the salt and dextran-based ATPS, respectively. The dextran-based ATPS showed a better recovery yield and purity than the salt-based ATPS for the range of test conditions employed in this experiment. The yield and purity of the recovered rabbit anti-human IgG were 90.8 and 87.7%, respectively, in the dextran-based ATPS, while those in the salt-based ATPS were 78.2 and 73.2%.

Heterologous expression and optimized one-step separation of levansucrase via elastin-like polypeptides tagging system.

Elastin-like polypeptides (ELPs) undergo a reversible inverse phase transition upon a change in temperature. This thermally triggered phase transition allows for a simple and rapid means of purifying a fusion protein. Recovery of ELPs-tagged fusion protein was easily achieved by aggregation, triggered either by raising temperature or by adding salt. In this study, levansucrase has been used as a model enzyme in the development of a simple one-step purification method using ELPs. The levansucrase gene cloned from Pseudomonas aurantiaca S-4380 was tagged with various sizes of ELPs to functionally express and optimize the purification of levansucrase. One of two ELPs, ELP[V-20] or ELP[V-40], was fused at the C-terminus of the levansucrase gene. A levansucrase-ELP fusion protein was expressed in Escherichia coli DH5alpha at 37 degrees C for 18 h. The molecular masses of levansucrase-ELP[V-20] and levansucrase-ELP[V-40] were determined as 56 kDa and 65 kDa, respectively. The phase transition of levansucrase-ELP[V-20] occurred at 20 degrees C in 50 mM Tris-Cl (pH 8) buffer with 3 M NaCl added, whereas the phase transition temperature (Tt) of levansucrase-ELP[V-40] was 17 degrees C with 2 M NaCl. Levansucrase was successfully purified using the phase transition characteristics of ELPs, with a recovery yield of higher than 80%, as verified by SDS-PAGE. The specific activity was measured spectrophotometrically to be 173 U/mg and 171 U/mg for levansucrase-ELP[V-20] and levansucrase-ELP[V-40], respectively, implying that the ELP-tagging system provides an efficient one-step separation method for protein purification.

Reliable fabrication method of transferable micron scale metal pattern for poly(dimethylsiloxane) metallization.

We have developed a reliable fabrication method of forming micron scale metal patterns on poly(dimethylsiloxane) (PDMS) using a pattern transfer process. A metal stack layer consisting of Au-Ti-Au layers, providing a weak but reliable adhesion, was deposited on a silicon wafer. The metal stack layer was then transferred to a PDMS substrate using serial and selective etching. We demonstrate that features as small as 2 microm were reliably transferred on to the PDMS substrate for use as interconnects and electrodes for biosensors and flexible electronics application.

Refolding of horseradish peroxidase is enhanced in presence of metal cofactors and ionic liquids.

The effects of various refolding additives, including metal cofactors, organic co-solvents, and ionic liquids, on the refolding of horseradish peroxidase (HRP), a well-known hemoprotein containing four disulfide bonds and two different types of metal centers, a ferrous ion-containing heme group and two calcium atoms, which provide a stabilizing effect on protein structure and function, were investigated. Both metal cofactors (Ca(2+) and hemin) and ionic liquids have positive impact on the refolding of HRP. For instance, the HRP refolding yield remarkably increased by over 3-fold upon addition of hemin and calcium chloride to the refolding buffer as compared to that in the conventional urea-containing refolding buffer. Moreover, the addition of ionic liquids [EMIM][Cl] to the hemin and calcium cofactor-containing refolding buffer further enhanced the HRP refolding yield up to 80% as compared to 12% in conventional refolding buffer at relatively high initial protein concentration (5 mg/ml). These results indicated that refolding method utilizing metal cofactors and ionic liquids could enhance the yield and efficiency for metalloprotein.