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%.     

Evaluation of ionic liquids on phototrophic microbes and their use in biofuel extraction and isolation

Multiple ionic liquids (ILs) were assessed for their ability to extract branched, unsaturated hydrocarbons from an aqueous medium. In addition, IL cytotoxicity studies were performed on two phototrophic microbes, Synechocystis sp. PCC6803 and Botryococcus braunii var Showa. The optimum IL for use in an isoprenoid hydrocarbon extraction may vary based on the biological source of the isoprenoids. Our results suggest that ionic liquids have the potential to serve as novel biocompatible milking agents for extracting high-value chemicals from the microbes, with toxicity to both species minimized by considerations of ionic liquid structure and hydrophobicity.     

Effect of ions and other compatible solutes on enzyme activity, and its implication for biocatalysis using ionic liquids

The effect of ions on enzyme activity and stability usually follows the Hofmeister series (or the kosmotropicity order): kosmotropic anions and chaotropic cations stabilize enzymes while chaotropic anions and kosmotropic cations destabilize them. The effect of ionic liquids (ILs) on the enzyme activity/stability/enantioselectivity is complicated especially when there is no or little water presence in the IL media. However, when aqueous solutions of hydrophilic ILs are employed as reaction media, the enzyme seems to follow the Hofmeister series since ILs dissociate into individual ions in water.     

Enhanced enzyme-catalyzed synthesis of l-methionine with ionic liquid additives

The low solubility of l-methionine and low activity of enzyme are the major hurdles during l-methionine production by the enzymatic conversion approach. In this study, we investigated various ionic liquids (ILs) as additives for the enzyme-catalyzed production of l-methionine from O-acetyl L-homoserine and methyl mercaptan. Among the ILs evaluated, we found that tetraalkylammonium hydroxide ILs enhanced the solubility of l-methionine as well as the activity of the enzyme. Methionine solubility decreased with increasing alkyl chain length but increased with increasing IL concentration. l-methionine could be dissolved up to 232 g/L in 10% tetramethylammonium hydroxide solution. The enzyme O-acetylhomoserine aminocarboxypropyltransferase reached its maximum activity when the IL concentration was 2.5% (3 times higher than that without ILs) and significantly decreased with increasing IL concentration. The stability of the enzyme also decreased rapidly after 2 h of incubation regardless of the presence or absence of ILs. Nevertheless, 74 g/L of l-methionine could be produced in a reaction media containing 2.5% tetraethylammonium hydroxide compared to 35 g/L of l-methionine obtained in a reaction system without ILs.     

Hydrolysis of cellulose in SO₃H-functionalized ionic liquids

Influence of acidity and structure of ionic liquids on microcrystalline cellulose (MCC) hydrolysis was investigated. MnCl₂-containing ionic liquids (ILs) were efficient catalysts and achieved MCC conversion rates of 91.2% and selectivities for 5-hydroxymethyl furfural (HMF), furfural and levulinic acid (LA) of 45.7%, 26.2% and 10.5%, respectively. X-ray diffractometry indicated that catalytic hydrolysis of MCC in ionic liquids resulted in the changes to MCC crystallinity and transformation of cellulose I into cellulose II. SO₃H-functionalized ionic liquids showed higher activities than non-functionalized ILs. The simplicity of the chemical transformation of cellulose provides a new approach for the use this polymer as raw material for renewable energy and chemical industries.     

The effect of ionic liquid media on activity,selectivity and stability of Candida antarctica lipase B in transesterification reactions

Nineteen different 1,3-dialkylimidazolium-based ionic liquids (ILs) were used as reaction media for the synthesis of butyl butyrate by transesterification from vinyl butyrate and 1-butanol catalyzed by Candida antarctica lipase B (CaLB). The reaction was also carried out in hexane as a reference solvent. In all the water-immiscible ILs assayed, the enzymatic activity and selectivity were higher than that obtained in hexane. However, in water-miscible ILs, the activity was lower than in the reference solvent, although they showed >99.99% selectivity. Two solvent properties, hydrophobicity and nucleophilicity, were considered key parameters for analyzing the behavior of CaLB in ILs. In the case of ILs based on the same anion, the synthetic activity was gradually enhanced by increasing cation hydrophobicity. Furthermore, the activity of CaLB was greater in ILs containing anions of lower nucleophilicity. Stability studies indicate that CaLB exhibited greater stability in water-immiscible ILs than in water-miscible ILs.     

Kinetic study on the enzymatic resolution of homophenylalanine ester using ionic liquids

Two ionic liquids (ILs) were investigated as novel media for the enzymatic resolution of amino acid ester to obtain enantiomeric amino acid homophenylalanine. The effects of solvent nature, polarity, and concentration on the kinetic resolution were investigated. With change in solvent concentration, a systematic study shows that an improved enzyme activity can be obtained by adjusting these solvent parameters.     

Ionization basis for activation of enzymes soluble in ionic liquids

BackgroundThe complex interactions between electrolytes and proteins have been studied for more than a century. However, understanding is not yet complete and does not provide a basis for predicting the activity of enzymes in ionic media. The use of ionic liquids (ILs) as reaction medium has opened up new opportunities for better understanding of the mechanism of enzymatic catalysis. Although a number of properties of ILs have been correlated with enzyme function, these relationships are not completely understood at a molecular level.MethodsWe propose that ILs must be able to promote ionization of protein ionizable groups in order to dissolve active enzymes. The biocompatible IL need to possess a functional group with large donor number and acceptor number in both cationic and anionic units, each of which is based on a high dielectric constant lead structure. We designed and synthesized two series of ILs and determined their ionizing–dissociating abilities and activities of lipases soluble in these new ILs.ResultsThe results showed that the ionizing–dissociating abilities of ILs paralleled the catalytic activity trend of lipases dissolved in the ILs. The activities of lipases soluble in the newly designed ILs were comparable to those in water.ConclusionsWe can conclude that ionizing–dissociating abilities of an IL can be used as a basis for predicting the activity of enzymes soluble in the IL.General significanceIonization basis for activation of enzymes gives a deeper understanding of the behavior of enzymes in non-aqueous media at a molecular level.     

Solubility and stability of cytochrome c in hydrated ionic liquids: effect of oxo acid residues and kosmotropicity

Hydrated ionic liquids (ILs) were prepared by adding appropriate amounts of water to hydrophilic ILs. Some hydrated ILs show excellent solubilizing ability for proteins, keeping the basic properties of ILs. The solubility of cytochrome c (cyt c) depended on the structure of the component ions. When component anions have oxo acid residues, the resulting hydrated ILs solubilize cyt c quite well. In such hydrated ILs, the structure and activity of cyt c is influenced by the kosmotropicity of the component ions. We synthesized ILs from various ions having different kosmotropicity, including dihydrogen phosphate (dhp), dibutylphosphate, acetate, lactate, and methanesulfonate as anions. The activity of the dissolved cyt c depends on the permutations of kosmotropicity of the component ions. cyt c shows no structural change and retains its activity when dissolved in the hydrated choline dhp, which is an excellent combination of chaotropic cation and kosmotropic anion. Furthermore, cyt c dissolved in the hydrated choline dhp remained in a native state and was active after 18 months of storage at room temperature.     

Cover Picture: Biotechnology Journal 8/2016

This regular issue of BTJ includes articles on biocatalysis, biochemical engineering, and bioprocess engineering. This cover page highlights the applications of biomolecules (e.g., proteins, enzymes, and DNA) in ionic liquids (ILs). The technological utility of biomolecules can be enhanced significantly by combining them with ILs. Image is provided by Magaret Sivapragasam, Muhammad Moniruzzaman, and Masahiro Goto authors of ”Recent advances in exploiting ionic liquids for biomolecules: Solubility, stability and applications“ ( http://dx.doi.org/10.1002/biot.201500603 ).     

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.     

Ionic liquids promote amyloid formation from α-synuclein

The slow process required for α-synuclein to form amyloid fibrils is a major obstacle in the development of therapeutic compounds for α-synuclein-related neurodegenerative diseases such as Parkinson’s disease (PD). Here we have developed an efficient method by which amyloid fibrils can be formed from α-synuclein using ionic liquids (ILs). This report indicates that ILs could potentially be used as a stimulator for the amyloid formation of α-synuclein.     

Recent advances in exploiting ionic liquids for biomolecules: Solubility,stability and applications

The technological utility of biomolecules (e.g. proteins, enzymes and DNA) can be significantly enhanced by combining them with ionic liquids (ILs) – potentially attractive ”green“ and ”designer“ solvents – rather than using in conventional organic solvents or water. In recent years, ILs have been used as solvents, cosolvents, and reagents for biocatalysis, biotransformation, protein preservation and stabilization, DNA solubilization and stabilization, and other biomolecule‐based applications. Using ILs can dramatically enhance the structural and chemical stability of proteins, DNA, and enzymes. This article reviews the recent technological developments of ILs in protein‐, enzyme‐, and DNA‐based applications. We discuss the different routes to increase biomolecule stability and activity in ILs, and the design of biomolecule‐friendly ILs that can dissolve biomolecules with minimum alteration to their structure. This information will be helpful to design IL‐based processes in biotechnology and the biological sciences that can serve as novel and selective processes for enzymatic reactions, protein and DNA stability, and other biomolecule‐based applications.     

Enhanced production of fructose palmitate by lipase-catalyzed esterification in ionic liquids

For the enhancement of enzyme activity, application of ultrasound irradiation on lipase-catalyzed esterification of fructose with palmitic acid in ionic liquids (ILs) mixture containing supersaturated fructose solution was investigated. In the mixture of [Bmim][TfO] and [Omim][Tf₂N] (1:1, v/v), 1.44 times higher enzyme activity (29.2 μmoL/min/g) was achieved under ultrasound irradiation. Besides, ultrasound irradiation enhanced enzyme stability in viscous ILs mixture. After 5 times reuse of Novozym 435 and ILs mixture, 84.4% of initial enzyme activity was remained under ultrasound irradiation, while the residual activity using magnetic stirring only method was 76.2%. These results show that enzymatic reaction in viscous ILs mixture under ultrasound irradiation is an effective method for enzyme activity, as well as, enzyme stability resulting in economic competitiveness of green process.     

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.     

Aqueous ionic liquid pretreatment of straw

Pretreatment is the key to unlock the recalcitrance of lignocellulose for cellulosic biofuels production. Increasing attention has been drawn to ionic liquids (ILs) for pretreatment of lignocellulosic biomass, because this approach has several advantages over conventional methods. However, cost and energy-intensive recycling of the solvents are major constraints preventing ILs from commercial viability. In this work, a mixture of ionic liquid 1-ethyl-3-methylimidazolium acetate and water was demonstrated to be effective for pretreatment of lignocellulosic biomass, evidenced by the removal of lignin and a reduction in cellulose crystallinity. A higher fermentable sugar yield (81%) was obtained than for pure ionic liquid pretreatment under the same conditions (67%). Aqueous ionic liquid pretreatment has the advantages of less usage and easier recycling of ILs, and reduced viscosity.     

A theoretical investigation of the interactions between hydroxyl-functionalized ionic liquid and water/methanol/dimethyl sulfoxide

Density functional calculations have been used to investigate the interactions of 1-(2-hydroxyethyl)-3-methylimidazolium ([C₂OHmim]⁺)-based ionic liquids (hydroxyl ILs) with water (H₂O), methanol (CH₃OH), and dimethyl sulfoxide (DMSO). It was found that the cosolvent molecules interact with the anion and cation of each ionic liquid through different atoms, i.e., H and O atoms, respectively. The interactions between the cosolvent molecules and 1-ethyl-3-methylimizolium ([C₂mim]⁺)-based ionic liquids (nonhydroxyl ILs) were also studied for comparison. In the cosolvent–[nonhydroxyl ILs] systems, a furcated H-bond was formed between the O atom of the cosolvent molecule and the C2-H and C6-H, while there were always H-bonds involving the OH group of the cation in the cosolvent–[hydroxyl ILs] systems. Introducing an OH group on the ethyl side of the imidazolium ring may change the order of solubility of the molecular liquids.     

The study of factors affecting the enzymatic hydrolysis of cellulose after ionic liquid pretreatment

Cellulose resource has got much attention as a promising replacement of fossil fuel. The hydrolysis of cellulose is the key step to chemical product and liquid transportation fuel. In this paper a serials of chloride, acetate, and formate based ionic liquids were used as solvents to dissolve cellulose. The cellulose regenerated from ILs was characterized by FTIR and X-ray powder diffraction. From the characterization and analysis, it was found that the original close and compact structure has changed a lot. After enzymatic hydrolysis, different kinds of ionic liquids (ILs) have different yields of the reducing sugar (TRS). They are 100%, 90.72%, and 88.92% from 1-butyl-3-methylimidazolium chloride ([BMIM]Cl), 1-ethyl-3-methylimidazolium acetate ([EMIM][OAc]), 1-butyl-3-methylimidazolium formate ([BMIM][HCOO]) respectively after enzymatic hydrolysis at 50 °C for 5 h. The results indicated that the yields and the hydrolysis rates were improved apparently after ILs pretreatment comparing with the untreated substrates.     

Spectroscopic insight into the interaction of bovine serum albumin with imidazolium‐based ionic liquids in aqueous solution

The study of protein–ionic liquid interactions is very important because of the widespread use of ionic liquids as protein stabilizer in the recent years. In this work, the interaction of bovine serum albumin (BSA) with different imidazolium‐based ionic liquids (ILs) such as [1‐ethyl‐3‐methyl‐imidazolium ethyl sulfate (EmimESO₄), 1‐ethyl‐3‐methyl‐imidazolium chloride (EmimCl) and 1‐butyl‐3‐methyl‐imidazolium chloride (BmimCl)] has been investigated using different spectroscopic techniques. The intrinsic fluorescence of BSA is quenched by ILs by the dynamic mechanism. The thermodynamic analysis demonstrates that very weak interactions exist between BSA and ILs. 8‐Anilino‐1‐naphthalenesulfonic acid (ANS) fluorescence and lifetime measurements reveal the formation of the compact structure of BSA in IL medium. The conformational changes of BSA were monitored by CD analysis. Temperature‐dependent ultraviolet (UV) measurements were done to study the thermal stability of BSA. The thermal stability of BSA in the presence of ILs follows the trend EmimESO₄ > EmimCl > BmimCl and in the presence of more hydrophobic IL, destabilization increases rapidly as a function of concentration.     

A profile of the in vitro anti-tumor activity of imidazolium-based ionic liquids

The anti-cancer activity and cytotoxicity of imidazolium-based ionic liquids has been determined for the first time via NCI’s in vitro 60 human tumor cell lines. The preliminary SAR showed that the chain length of alkyl substitution at N-3 position of imidazole ring plays crucial role towards anti-tumor activity and cytotoxicity of these ionic liquids. The ionic liquids with alkyl substitution of C-12 chain length were found to be effective against all 60 tumor cell lines and show very low cytotoxicity in most of the cases. Further increase in chain length resulted in enhanced growth inhibition of tumor cell lines as well as high cytotoxicity. Interestingly, active compounds 1-dodecyl-3-methylimidazolium chloride (8), 1-dodecyl-3-methylimidazolium tetrafluoroborate (9), 1-hexadecyl-3-methylimidazoium chloride (10), 1-octadecyl-3-methylimidazolium chloride (11), 1-octadecyl-3-methylimidazolium hexafluorophosphate (12), 1-octadecyl-3-methylimidazolium bis(triflic)imide (13) and 1-octadecyl-3-methylimidazolium tris(pentafluoroethyl)trifluorophosphate (14) were highly active against leukemia cell lines, especially compounds 13 and 14 where the cytotoxicity was also very low as given by LC₅₀ >100 μM in all six leukemia cell lines.