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Biotechnol. J. 2016, 11, 1000–1013 DOI 10.1002/biot.201500603

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BiotechnologyJournal

Review

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

Magaret Sivapragasam1, Muhammad Moniruzzaman1 and Masahiro Goto2,3

1 Centre of Research in Ionic Liquids (CORIL), Chemical Engineering Department, Universiti Teknologi PETRONAS, Bandar Seri Iskandar, Malaysia

2 Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, Fukuoka, Japan3 Center for Future Chemistry, Kyushu University, Fukuoka, Japan

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 “design-er” 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 pres-ervation 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 biomole-cules 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 appli-cations.

Keywords: DNA solubility · Enzyme activity · Ionic liquid · Microorganism · Protein stability

Correspondence: Prof. Masahiro Goto, Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, 744 Moto-oka, Fukuo-ka 819-0395, Japan E-mail: [email protected]

Abbreviations: BCL, Burkholderia cepacia lipase; [Bmpyrro][DHP], N-butyl-N-methylpyrrolidinium dihydrogen phosphate; [Bmpyrro][NTf2], N-butyl-N-methylpyrrolidinium imide; [Bzmim][Cl], 1-methyl-3-m-methoxylbenzylimi-dazolium chloride; [Bzmim][TSA], 1-methyl-3-m-methoxylbenzylimidazolium toluenesulonate; [Bzmim][BF4], 1-methyl-3-m-methoxylbenzylimidazolium tetraflouroborate; [C1mim][DMP], 1,3-dimethylimidazolium dimethylphos-phate; [(C10)2NMDG-Br], N,N-didecyl-N-methyl-D-glucaminium bromide; [C16POHIM-Br], 1-(1,2-dihydroxypropyl)-3-hexadecylimidazolium bromide; [C2mim][NTf2], 1-ethyl-3-methylimizadolium bis(trifluoromethylsulfonyl)amide; [C4mpy][N(CN)2], 1-butyl-3-methylpyridinium dicyanamide; [C6MIM][Cl], 1-hexyl-3-methylimidazolium chloride; [Ch][Gly], choline glycolate; [Ch][DHCit], cholinium dihydrogen citrate; [Ch][Lac], cholinium lactate; [Ch][Pyr], choline pyruvate; [Ch][TACl], cholinium (2-hydroxyethyl)(trimethylam-

Received 29 OCT 2015Revised 30 MAR 2016Accepted 17 MAY 2016

monium chloride); CRL, Candida rugosa lipase; (DEAA), diethylammonium acetate; (DEAS), diethylammonium hydrogen sulfate; (DEAP), diethyl-ammonium dihydrogen phosphate; GTL, Geobacillus thermocatenolatus lipase; [HOOCMMIm][Cl], 3-(2-carboxymethyl)-1-methylimidazolium chlo-ride; [HOOCEMIm][Cl], 3-(2-carboxymethyl)-1-ethylimidazolium chloride; ([Me(OEt)3-Et-Im][NTf2], 1-ethyl-3-(2-(2-ethoxyethoxy)ethoxy)ethylimidazo-lium bis(trifluoromethylsulfonyl) imide; [Me-(OEt)3-Me-Et-Im][NTf2], 1-ethyl-3-(2-(2-methoxyethoxy)ethoxyethyl-2-methylimidazolium bis(trifluo-romethylsulfonyl)imide; [Me(OEt)3-Et3N][NTf2], triethyl(2-(2-methoxyeth-oxy)ethoxy)-ethylammonium bis(trifluoromethylsulfonyl)imide; [Mor1,2][Br], N-ethyl-N-methyl-morpholinium bromide; [Pmim][TSA], 1-(2-propoxy-ethyl)-3-methylimidazolium p-toluenesulfonic acid; PPL, porcine pancreatic lipase; RDL, Rhizopus delemar lipase; [Tdim][Br], 1-tetradecyl-3-methylimida-zolium bromide; (TEAA), trimethyl ammonium acetate; (TMAP), trimethyl-ammonium dihydrogen phosphate; (TMAS), trimethylammonium hydro-gen sulfate; [Toma][TFA], trioctylmethylammonium trifluoroacetate; TLL, Thermomyces lanuginosus lipase

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Biotechnol. J. 2016, 11, 1000–1013

1 Introduction

Ionic liquids (ILs) have emerged as an attractive “green” recyclable alternative to toxic, hazardous, flammable, and highly volatile organic compounds (VOCs) [1–6]. ILs are entirely composed of ions. They generally consist of organic cations, namely, derivatives of imidazolium, pyri-dinium, pyrrolidinium, cholinium, ammonium, morpho-linium and phosphonium, and either organic or inorganic anions (Fig. 1). ILs are liquids at or far below ambient temperature. Their low melting point originates from the asymmetry of at least one of the ions and weak intermo-lecular attractions. ILs possess many unique and design-able physicochemical properties that make them excel-lent candidates for VOC replacements. These properties include negligible vapor pressure, multiple solvation interactions with organic and inorganic compounds, excellent chemical and thermal stability, high ionic con-ductivity and a large electrochemical window [1, 2]. The physicochemical properties of ILs, such as their viscosity, hydrophobicity, density, and solubility, can be tuned by

simply selecting different combinations of cations and anions, or changing the attached substituents, leading to the use of the terms “designer” and “task-specific” ILs [7].ILs are extensively used in chemistry (e.g. organic synthe-sis and nanomaterial synthesis) [8, 9], chemical engineer-ing (e.g. separation, extraction, and catalytic reactions) [10–12], biotechnology (e.g. biocatalysis and biofuel pro-duction) [13–18], and pharmaceutics (e.g. drug formula-tion, drug delivery, and formulation of active pharmaceu-tical ingredients) [19–24]. ILs are of particular interest as new and highly efficient solvents/co-solvents/agents for protein- and enzyme-based applications, and they have many advantages over their conventional counterparts (see Box 1). The technological utility of biomolecules, particularly enzymes, can be significantly enhanced by using ILs rather than organic solvents or aqueous reaction media because of the unique solvent characteristics of ILs. Several reviews have been published focusing on biocatalysis and biotransformations in ILs [25–29], protein extraction and their stability in ILs [30], and DNA solubil-ity and stability in ILs [31]. ILs are being increasingly used

Figure 1. Some common cations and anions used for the synthesis of ILs


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Biotechnol. J. 2016, 11, 1000–1013

for biomolecules because of the discovery of new and unique biocompatible ILs, as well as the development of useful techniques in which biomolecules exhibit more pronounced solubility, activity, and stability in ILs. Here, we review some recent technological developments of ILs in protein-, enzyme-, and DNA-based applications. This review is not intended to be comprehensive, but rather to present a general overview of the research using ILs for biomolecule-based applications from early 2010.

2 Ionic liquids and their biocompatibility characteristics

Although ILs have emerged as very promising solvents for biomolecules, biocompatibility is the main challenge for their biological applications. To use ILs for biomolecules, they should preferably be nontoxic. Recently, the cytotox-icity, environmental toxicity and microbial toxicity of many ILs have been investigated, and it has been found that nontoxic ILs can be generated by changing their anion/cation combination [32–35]. Nontoxic ILs can be produced by selecting biocompatible organic cations and inorganic anions. It is widely accepted that the head group of the cation has a deciding role in the toxicity [35] and longer side chains have a more severe effect on living cells. Generally, the toxicity can be decreased by either reducing the hydrophobicity of the side chain by shorten-ing long alkyl chains (C < 4) or by introducing polar func-

tional groups (i.e. ether, hydroxyl, or nitrile groups) into the IL cations [36]. In addition to the strong side-chain effect, the chemical structure of the cationic group also has a significant effect on the toxicity [37]. In general, imidazolium, pyridinium and quinolinium head groups have strong toxicological effects even when they are linked to short polar side chains. The morpholinium head group has been found to be the least toxic, and it can be used to design inherently safe ILs, especially when com-bined with short polar side chains and nontoxic anions [38]. Regarding the anions, most of the tested ILs show no significant toxicological effects. However, hydrophobic and most fluorinated species are not suitable for nontoxic ILs [36]. Very recently, it was found that oleate-, hex-anoate-, and geranate-based ILs are nontoxic and can disrupt the skin without irritation [33]. However, the bio-compatibility of ILs should be defined in the context of their specific target applications and discussed within regulatory and environmental requirements.

3 Application of ILs to biomolecules

3.1 Protein solubility and stability in ILs

Studies of protein solubility and stability are typically car-ried out in aqueous solution. However, in many cases, applications of proteins in water are limited by their low stability, especially their thermal stability during storage and their reaction processes. To overcome this problem, several approaches have been proposed, including chem-ical modification, immobilization and addition of stabiliz-ing agents. However, most of these methods do not pre-vent irreversible thermal denaturation of proteins in water. Recently, ILs have been extensively used as either co-solvents with water in biphasic systems [39–41] or as “neat” ILs to maintain protein stability. A number of stud-ies have shown that ILs are effective for maintaining or even increasing protein solubility and stability (Table 1). The unique physicochemical properties of ILs make them excellent solvents to improve the separation efficiency of important biomolecules, such as amino acids, proteins, carbohydrates, lactic acid, antibiotics and alkaloids, from different media [30, 42]. To assess the functionality of a particular protein in ILs, structural information and the function of biomolecules in ILs are crucial for understand-ing their metabolic role. Protein stability during extraction and purification procedures is essential for their applica-tions because changes in the protein environment can alter their native state [43].

Separation of a target protein from its complexed mix-tures accounts for about 50–80% of the total production cost [44]. Traditionally, proteins are purified by methods such as ionic precipitation, chromatography, electropho-resis, and liquid–liquid extraction. However, these meth-ods have certain disadvantages, including high costs,

Box 1

Advantages of using biomolecules (enzymes, protein and DNA) in ILs

• Scope to design particular bioprocesses because of the tailor-made and inimitable physical/chemi-cal/biological properties of ILs

• In many cases, biomolecules have enhanced solu-bilities, stabilities (both operational and thermal), and lifetime in ILs

• Ease of substrate, product and ILs recovery after biocatalysis and biotransformation in ILs

• Ease of biomolecule recovery by filtration or cen-trifugation

• Prevents self-aggregation of biomolecules during solubilization

• ILs are excellent solvents for protein refolding and crystallization

• Enhanced solubility of insoluble/sparingly soluble substrates in ILs, which is beneficial for biomole-cule-based reactions

• Ease of immobilization of biomolecules in highly viscous ILs for biochemical processes




Biotechnol. J. 2016, 11, 1000–1013

time consumption, loss of biological activity, and lack of robustness [44]. Aqueous two-phase systems (ATPSs) overcome these disadvantages because of their robust-ness, short processing time, low energy consumption, easy scale up, and biocompatibility. ATPSs typically con-sist of two aqueous-rich phases of polymer/polymer, salt/salt, or polymer/salt. ATPSs offer the advantage of selec-tive partitioning of a target molecule, which depends on its affinity for each aqueous-rich phase, as well as other factors such as temperature, pH, and system composition [45]. Rogers and co-workers [46] pioneered the introduc-tion of IL into ATPSs. This was successful in overcoming the disadvantages of polymer-based ATPSs, such as their high viscosity that hinders mass transfer leading to slow phase separation [45]. IL-based aqueous phase extraction is the preferred choice of protein separation because of its mild nature, low viscosity, rapid phase separation, high extraction efficiency, and gentle biocompatible environ-

ment [42, 44, 45, 47]. With the introduction of ILs into ATPSs, the narrow range of polarity differences between the coexisting phases of polymer-based systems can be extended [48]. Considering the abovementioned facts, IL-based ATPSs can be considered as novel liquid parti-tioning systems [49]. As a liquid–liquid extractive tech-nique, ATPSs are simple, low cost, and relatively reliable in scale-up, and they show great potential for separation applications. IL/inorganic salt separation systems are highly ionic media, and therefore might not be compati-ble with the extracted product because many biomole-cules have narrow tolerance limits of ionic strength [50]. Using an IL with ATPS extraction methods is performed by replacing the polymer component of the conventional polymer–salt two-phase system with the IL. Imidazolium-based ILs are usually used, although some studies have been performed with the phosphonium cation [45, 51]. To successfully use ILs as extraction solvents or correctly

Table 1. ILs in protein applications

Entry ILs used Protein Remarks Ref.

1 [Pmim][TSA], [Bzmim][TSA] BSA BSA stable and 1-(2-alkoxy-ethyl)-3-methylimidazolium chloride showed the greatest potential to separate protein from saccha-ride solutions

[42]

2 [C2mim][Cl], [C2mim][NTf2] Zein Protein soluble at up to 15 wt% in both ILs at 80°C [56]

3 [Ch][DHP] Cyt c, peroxidase, ascorbate oxidase, azurin, pseudoazurin, fructose dehydrogenase

All proteins soluble in IL at concentrations above 1 mMAll proteins retained enzymatic activity with significant thermal stability

[57]

4 (DEAA), (DEAS),(DEAP), (TEAA),(TMAS),(TMAA), (TMAP)

CT Tertiary structure (rearrangement of β-sheets) disrupted with an increase of temperatureTertiary structure retained at 80°C and 85°C in TMAS and TMAP, respectively

[59]

5 [C2mim][Cl] Human urine Direct extraction of urine by IL without any chemical interactions between proteins and IL moiety, and no protein alteration

[88]

6 [Ch][Sac], [Bmpyrro][NTf2] [Ch][DHP], [Bmpyrro][DHP] [Ch][(CH2)2 PO4, [C2mim][X], where X = [CDP], [Ac], or [MeSO3]

cyt c Cyt c structure, thermal stability, and long-term stability main-tained in [Ch][DHP] even after 18 months storage

[89]

7 [Ch][X], where X = [TACl], [DHCit], [Bit], [Ac], [DHP], [Prop], [Gly], [But], or [Lac]

BSA BSA completely extracted (92–100%) in a single step while maintaining its activity and stability using the ILs [Ch][DHCit], [Ch][But], [Ch][Prop], [Ch][Ac], and [Ch][Gly]

[107]

8 Iolilyte 221 PG, Cyphos 108 Rubisco (ribulose-1,5-biphosphate car-boxylase oxygenase)

Decrease in enzyme activity and protein aggregation with increasing IL concentrationRubisco structurally and functionally stable in 10% v/v ILs

[108]

9 [Tdim][Br] BSA IL stabilizes BSA below its critical micelle concentration (CMC) and destabilizes it above its CMC

[109]

10 [Ch][X], where X = [TES], [tricine], [HEPES], or [Cl]

BSA Helicity in the order of [Ch][TES] > [Ch][tricine] > [Ch][HEPES] > sucrose > TES > [Ch][Cl] > HEPES > tricineProtein stable and maintained α-helical structure in all IL sys-tems

[110]




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design new ILs to meet the requirements of any particular task, it is crucial to effectively manipulate the experimen-tal parameters that influence the partitioning of biomole-cules between the IL and the aqueous medium, as well as to have a deep understanding of the interactions involved [52]. For example, the performance of IL-based ATPSs is highly dependent on the IL side chain [53]. As the number of carbon atoms in the alkyl chain increases (C > 6), the ability for formation of a biphasic system changes because it no longer follows the hydrophobicity of the IL (possibly because of micellar formation in aqueous solution). This is more pronounced in anions than cations [54].

ILs are also used as solvents for chemical modification of proteins. Solubilization of proteins in ILs prevents aggregation and thus improves their stability and activity [55]. It has been found that starch and zein can com-pletely dissolve in 5–10% choline-based ILs [56]. Fujita and Ohno [57] investigated the effectiveness of Hy[Ch][DP] as a novel solvent for metalloproteins. The IL dis-solved some metalloproteins, although the mechanism is not yet clear. Proteins dissolved in Hy[Ch][DP] were shown to retain the same surroundings of the active site and secondary structure as in a buffer solution. Tomé et al. [52] concluded that the IL extraction ability for trypto-phan decreases with increasing length of the alkyl side chain and increasing number of substitutions in the IL cation. They also reported that pyrrolidinium-based ILs are much better extraction phases than ILs composed of imidazolium and pyridinium rings. Another widely stud-ied protein is the green fluorescent protein (GFP). Heller et al. [55] found that GFP is soluble in [C4mim][Cl] and the thermal stability of GFP is lower in [C4mim][Cl] than in aqueous solution, increasing its sensitivity to thermal unfolding [57]. They suggested that [C4mim][Cl]–water mixtures are poor candidate solvents for biocatalysis because of the strong denaturing character of the solvent. Wolski et al. [58] found that GFP retained its ability to fluorescence in [Mmim][DMP], emim ethylsulfate, and 2-hydroxyethyl-trimethylammonium lactate at 75–85% v/v. However, other water-miscible ILs can provide better protein solubility in biocatalytic processes. The [C4OC-

2mim][anion] IL shows potential to separate proteins from saccharide solutions, and therefore ether-functionalized IL-ATPSs show potential to separate biomolecules in complex systems [47]. Attri and Venkatesu [59] reported that ILs with more hydrophobic ammonium cations con-taining relatively long alkyl chains (diethyl ammonium or triethyl ammonium) are weak stabilizers for α-chymotrypsin (CT), while those containing small alkyl chains (trimethyl ammonium) are strong stabilizers and therefore more biocompatible for the CT structure. They then concluded that ILs show potential as new and promising co-solvent media for the CT structure. Baker and Heller [60] demon-strated that aqueous mixtures of [C4mim]Cl IL influence the solution structures of cytochrome c (cyt c) and human serum albumin (HAS) in a unique and concentration

dependent way. The [C4mim]Cl IL exhibited control over the oligomerization state of these proteins in a way that was dependent on the specific protein under considera-tion. It is clear that ILs increase the solubility and stability of proteins in various situations (Table 1). This can be enhanced by tuning the physicochemical properties of ILs, including their viscosity, hydrophobicity, kosmotro-picity, and polarity.

3.2 Enzyme activity and stability in ILs

As discussed in Section 1, the use of ILs for enzymes has advantages over conventional organic solvents and water, including high conversion rates [61, 62] and enantioselec-tivity [63], better enzyme stability [64–69], recoverability, and recyclability [70–72]. Furthermore, ILs can be used to perform biotransformations with polar or hydrophilic sub-strates that are insoluble or sparingly soluble in most organic solvents, such as amino acids and carbohydrates [73]. However, in some cases, the use of enzymes in ILs is limited by their low solubility, activity, and stability [20, 25]. Attempts have been made to address these limita-tions, including developing enzyme compatible ILs [74, 75], enzyme immobilization on supports [76], chemical modification with activated stabilizing agents [77, 78], and combining enzymes with suitable surfactants to form microemulsions [79–82]. Many reviews focusing on enzyme-catalyzed reactions in ILs have been published, indicating the advances of the entire field [20, 25] or spe-cific research groups [26]. This is why this review is not intended to be comprehensive, but rather to present the recent (2010 until present) research in activation and sta-bilization of enzymes in IL media.

A wide range of enzymatic reactions have been car-ried out in ILs, including esterification, transesterification, alcoholysis, aminolysis, hydrolysis, and polymerization. We summarize the recently studied enzyme-catalyzed reactions in ILs in Tables 2 and 3. All of the examples included here indicate that the reaction rates and yields are comparable with or higher than those obtained in organic systems.

After the first successful report of biocatalysis in ILs by Russell et al. in 2000 [83], the activity and stability of a large number of enzymes in ILs have been evaluated, such as lipases [66, 68, 71, 84], proteases [80, 85], alcohol dehydrogenases [86], and oxidoreductases [80]. Com-pared with other enzymes, lipases have been extensively used in ILs and show higher stability in ILs than tradi-tional solvents (Table 2). The behavior of oxidases in ILs is less clear. However, hydrolytic enzymes, such as lipases, proteases, and glycosidases, have been widely studied in IL reaction systems [87]. Many factors affect enzyme performance in ILs, including the polarity, cation, anion, alkyl chain length, hydrophobicity, ion specific effects and viscosity [88, 89]. The anion in the IL has been found to have a profound effect on the stability and activ-




Biotechnol. J. 2016, 11, 1000–1013

Table 2. Lipase-catalyzed (free, immobilized and modified lipase) reactions using ILs

Entry ILs used Lipase Remarks Ref.

1 [C2mim][Cl], [C8mim][Cl], [C4mim][X], where X = [CH3SO3],[CF3SO3], [CH3COO], [Cl],[Br], [CF3COO], [HSO4], or [N(CN)2]

CAL (free) Hydrophilic ILs have a deleterious influence on the enzy-matic activity

[54]

2 [C10mim][Cl] CAL (free) Relative enzyme activities (ActIL/ActBf ) for IL molar con-centrations ranging from 0.000–0.150 mol L−1 show an increase of up to six-fold

[54]

3 [CnC1im][Sac] [CnC1im][Ac] (n = 4, 6, 8, or 10)

CRL and RDL(free)

Coating of lipase from Rhizopus delemar with 0.1 M [C8C1im][Sac] or [C8C1im][Ac] resulted in an up to 1.5-fold increase in the initial enzyme activity. Addition of 0.02 M [C4C1im][Sac] or [C4C1im][Ac] stabilized the lipase and its activity was preserved for more than 25 days at room tem-perature

[66]

4 (ChAA) (AA = alaninate, glycinate, or lysinate)

TLL (free) Stable enzyme activity and none of the ILs had a deleteri-ous effect for concentrations lower than 2 M

[67]

5 [C2OHmim]-CRL-[H2PO4], [C2OHmim]-CRL-[Cl], [C2OHmim]-CRL-[BF4],[HOOCEPEG350Im]-CRL-[H2PO4], [HOOCEPEG350Im]-CRL-[Cl], [HOOCEPEG350Im]-CRL-[BF4]

CRL (modified with polyethylene glycol functional ILs)

Lipase modified by [HOOCEPEG350Im][H2PO4] increased the catalytic activity by 1.7-fold and the thermostability by five-fold at 50°C

[68]

6 [C2mim][X], where X = [BF4], [PF6], [NO3], [MeOSO3], or [C2mim][BF4], [bdmim][BF4], [bdmim][PF6]

GTL, CRL (immo-bilized onto Octyl Sepharose)

Activity of GTL increased seven-fold using [Emim][PF6] and five-fold using [Emim][MeSO3]Regioselectivity of Candida rugosa lipase in the hydrolysis of peracetylated thymidine improved (from 72 to 81% product yield) in the presence of [Emim][PF6]

[69]

7 [C2mim][PF6], [C2mim][BF4] Lipase (Lip1) from an Antarctic ther-mophilic bacterium (free)

Maximum reaction rate of Lip1 improved in the presence of both ILs. [C2mim][PF6 ] in the reaction mixture resulted in higher hydrolytic activity and catalytic efficiency of the enzyme

[84]

8 [C2mim][X], where X = [BF4], [Cl], [PF6] or [SCN]

BCL (immobilized onto starch film, agar gel and loofa sponge)

Degree of conversion to products using organic solvent/IL mixtures was considerably higher compared to the pure organic solvents

[111]

9 [C2mim][X], where X= [PF6], [NO3], [CF3SO3], or [BF4]

CRL (free) Activity and stability of lipase depended on the type and content of the IL complex

[112]

10 [HOOCMMIm][Cl], [HOOCEMIm][Cl], [HOOCC2mim][Cl], [HOOCMMIm][PF6], [Ch][NO3], [Ch][H2PO4]

PPL (modified with various functional ILs)

[HOOCB-MIm][Cl] modification led to a two-fold increase in the activity in 0.3 M [MMIm][MeSO4] compared with the aqueous formAll of the modified enzymes exhibited higher thermostabil-ity compared with the native enzyme at high temperature[HOOCC2mim][Cl] modification led to a six-fold increase in the thermostability at 60°C

[113]

11 [HOOCMMIm][Cl], [HOOCEMIm][Cl] [HOOCC2mim][Cl], [HOOCC2mim][H2PO4], [HOOCMMIm][PF6], [Ch][NO3], [Ch][H2PO4]

PPL (modified with various functional ILs)

Hydrolytic activity enhanced by ILs with chaotropic cations and kosmotropic anionsMore than four-fold increase with [Ch][H2PO4] modifica-tionWith [HOOCC2mim][Cl], there was 12-fold thermostability increase at 60°C and more than seven-fold enantioselectiv-ity enhancement compared with the native enzyme

[114]




Biotechnol. J. 2016, 11, 1000–1013

ity of enzymes. Anions that have high hydrogen-bond-forming capabilities strongly interact with enzymes and cause conformational changes. For example, Noritomi et al. [90] compared the effect of various anions on the activ-ity of lysozyme and found that lysozyme had lower activ-ity in [C2mim][Cl] than in [C2mim][BF4] and [C2mim][NTf2]. In contrast, cations interact with enzymes by van der Waals forces. The stability of the enzyme increases with elongation of the alkyl chain. However, the cation effect is closely linked to the viscosity of the IL, and the viscosity increases with increasing alkyl chain length. Machado and Saraiva [91] investigated the effect of the

cation chain length of alkyl-imidazolium based ILs (ethyl-, butyl-, and hexyl-imidazolium based ILs) on the enzyme activity of horseradish peroxidase. They found that 1-hex-yl-3-methylimidazolium chloride lowered the peroxidase enzyme activity. Attri et al. [59] found that less hydropho-bic ILs enhance enzyme refolding. They found that less hydrophobic ILs such as [TEA][Ac] and [TEA][PO4] stabi-lized CT while more hydrophobic ILs such as [Bzmim][Cl] and [Bzmim][BF4] acted as destabilizers [59].

ILs are more viscous than many conventional solvents, and it has been reported that the viscosity of ILs influ-ences enzyme stability and activity [86]. Bihari et al. [86]

Table 2. Lipase-catalyzed (free, immobilized and modified lipase) reactions using ILs (continue)

Entry ILs used Lipase Remarks Ref.

12 [C2mim][X], where X = [C2SO3], [C4SO3], [C6SO3], [C2SO4], [C4SO4], or [C6SO4]

CAL (free) Extraction with [C2mim][C4SO4] and ammonium sulfate resulted in enzyme recovery of 99%

[115]

13 [C2mim][NTf2], [Me(OEt)3–Et3N][NTf2], Me(OEt)3–Et–Im][NTf2], ([Me–(OEt)3–Me–Et–Im][NTf2], [Ch][Ac]

Novozyme 435 (free)

Maintained 92% of its activity after 48 h pre-incubation in choline acetate/glycerol (1:1.5), and 50% after 168 h pre-incubation

[116]

14 [Cnmim]Cl (n= 2, 4, 6, 8), [C4mim] [N(CN)2], [C8pyr] [N(CN)2], [C4mpip][Cl], [C4mpyr][Cl], [C4mpyrr][Cl], [C4mim][CF3SO3], [C4mim][CH3SO3]

CAL (free) Highest purification parameters observed for [C8mim][Cl], with a purification factor of 2.6 ± 0.1 and enzyme recovery of 95.9 ± 0.2%

[117]

15 [HOOCMMIm][Cl], [HOOCEMIm][Cl][HOOCC2mim][Cl], [HOOCC2mim][H2PO4], [HOOCMMIm][PF6]

CAL (modified with imidazolium functional ILs)

After modification, lipase was activated and achieved a high catalytic efficiency in the aqueous phase[HOOCMMIm][Cl] modification resulted in a 1.5-fold increase in the catalytic efficiency

[118]

Table 3. Enzyme (non-lipase) catalyzed reactions in ILs

Entry ILs used Enzyme Remarks Ref.

1 [C2mim][X], where X = [Cl], [BF4], or [NTF2]

Lysozyme Higher enzyme activity in [C2mim][BF4] and [C2mim][NTF2] [85]

2 [C2mim][EtSO4] Cyt c Secondary structure remained largely intact and tertiary structure significantly changed upon dissolution. Trends for cyt c in this IL resemble those for acid-denatured cyt c

[87]

3 [Ch][AA] Phosphatidylserine IL acted as reaction media via enzymatic transphosphatidylationMore than 70% of its original activity maintained in the IL

[119]

4 [Pmim][Br] Human serum albumin

[pmim][Br] acted as a denaturant when the protein was in the native state

[120]

5 [C2mim][Cl], [C6mim][Cl] α-Amylases from Bacillus amyloliquefaciens and Bacillus lichiniformis

Loss of activity and stability of both mesophilic (Bacillus amyloliquefaciens) and thermophilic (Bacillus lichiniformis) α-amylases in the presence of [C2mim][Cl] and [C6mim][Cl]

[121]

6 [C6mim][Cl], [Toma][TFA], [C2mim][OctSO4], [C2mim][MDEGSO4], [C2mim][CF3SO3], [C2mpy]·[C4F9SO3], [C4mpy][N(CN)2]

Flavonoids (rutin, esculin)

Overall, ILs containing NTf2−, PF6

−, and BF4− anions were most

successful as reaction media while ILs containing anions with stronger solvating properties resulted in decreased yields

[122]




Biotechnol. J. 2016, 11, 1000–1013

investigated dissolution of cyt c in [C2mim][EtSO4]. They found that the secondary structure of the enzyme remains intact but the tertiary structure significantly changes. Binding of 1-tetradecyl-3-methylimidazolium bromide to BSA was investigated by Geng et al. [88]. They concluded that ILs reduced burial of the tryptophan residue at low concentrations but denatured BSA at high concentra-tions. When enzyme formate dehydrogenase was dis-solved in [C1mim][DMP], [C4mim][CH3COO], and [C1mim][CH3H2PO2OCH3] ILs, the enzyme strongly denatured but each denaturation occurred with a different mechanism. Thus, ILs not only provide a novel and efficient reaction medium but they also act as an efficient participant in various chemical/biological reactions [92].

Enzymes such as lipases, proteases, glycosidases, and oxidoreductases retain their activity in the presence of ILs even with low water content. For example, lipase main-tains its activity in anhydrous hydrophobic IL media, and its selectivity and operational stability are better than in traditional volatile solvents [89]. Enzymes are most active in ILs that have large hydrophobic noncoordinating ani-ons, such as [BF4] and [PF6]. The most deactivating ILs contain anions that are capable of breaking hydrogen bonds, such as [Cl]. Lahtinen et al. [93] found that pro-longed incubation of Melanocarpus albomyces laccase in the presence of an IL caused an additional inactivating effect [93]. However, without understanding how and with which enzyme structures the IL ions interact at the molecular level, it was not possible to explain these obser-vations. This is because the effect of ILs on the stability and activity of immobilized laccase has not been com-pletely investigated [90].

In the past few years, a large number of enzymatic reac-tions have been carried out in systems containing ILs, including resolution of 1-phenylethanol, alcoholysis, perhy-drolysis, and ammoniolysis [20, 25]. Munoz et al. [87] reported that even though a wide range of enzymatic reac-tions have been tested using a variety of ILs, most of the studies focused on measuring the enzyme activity without considering the kinetic catalysis behavior in the ILs. A suit-able IL should maximize the solubility of the substrate while minimizing the negative effect on the enzyme activ-ity. Interestingly, Gao et al. [26] suggested that the activity of an enzyme in a particular IL is solely dependent on the enzyme–substrate relationship. Thus, it is necessary to thoroughly understand the IL chemistry before it is select-ed as a solvent/media for an enzymatic reaction.

3.3 DNA solubility and stability in ILs

Extraction of DNA from biological samples is typically performed by the phenol/chloroform method [31]. This is based on the underlying principle that proteins dissolve in the organic phenol/chloroform mixture while DNA remains in the aqueous solution. However, the depend-ence of these protocols on organic solvents and time-

consuming steps has resulted in development of more environmentally friendly techniques that are capable of high sample throughput. To find a medium in which DNA is both soluble with long term stability is a bottleneck in DNA technology. Recently, a number of studies have reported the use of ILs in life sciences for separation and extraction of nucleic acids, especially DNA [94–96]. As environmentally benign solvents, ILs are used for DNA storage because of their many advantages, including the enhanced solubility and excellent stability of DNA in ILs [31]. Interestingly, optimization of the IL properties seems to enhance the extraction efficiency of DNA. Li et al. [97] investigated the effect of IL alkyl chain length in the pres-ence of hydroxyl groups and obtained the highest extrac-tion efficiencies of DNA using C16POHIM-Br and [(C10)2NMDG-Br]. [C4mim][PF6] is the most extensively studied IL for extraction of DNA because hydrophobic interactions between the alkyl chain of this IL and DNA enhances DNA/IL binding [97].

Several studies have investigated the stability of DNA in various solvents. It has been discovered that the factors that affect the helical structure and denaturation of DNA in ILs include the ionic strength, pH, temperature, and solvent strength. Table 4 summarizes the various applica-tions of ILs for DNA extraction, preservation, and stabili-zation. Jumbri et al. [98] found that DNA is not stable and loses its native β-helical structure when dissolved in for-mamide, methanol or dimethyl sulfoxide. Vijayaraghavan et al. [99] investigated the stability of DNA in a series of hydrated choline-based ILs. They found that the chemical and structural stability of DNA are preserved up to a year using ILs. Ding et al. [100] investigated the binding char-acteristics and molecular mechanisms of the interaction between [C4mim][Cl] (a typical IL) and DNA. They con-cluded that even though the results provide information about the interactions between ILs and DNA, the molecu-lar mechanism of the interactions are still unclear.

Taking advantage of the benefits of ILs, Clark et al. [101] used magnetic ILs as a solvent for DNA extraction. They obtained 57% DNA recovery with an unmodified DNA sequence. According to the study of Tateishi-Kari-mata et al. [102], [Ch][DHP] profoundly stabilizes DNA triplex formation compared with a conventional aqueous buffer at neutral pH. Furthermore, high concentrations of ILs and deep eutectic solvents have a profound effect on DNA stability and do not induce extensive structural changes in the double-helix structure [103].

Nishimura et al. [104] prepared an IL with adenine and cytosine, and attempted to make an IL domain inside the double strands of DNA using nucleic acid bases as the starting units of the cation structures. However, a high-density IL columnar domain could not be obtained because the double-stranded structure collapsed. They then used IL-robed DNA with the phosphate group out-side the DNA chain, and the double-stranded structure did not collapse.




Biotechnol. J. 2016, 11, 1000–1013

Long-term preservation of DNA under ambient condi-tions is a major challenge. DNA is susceptible to slow hydrolytic reactions, such as depurination and deamina-tion, which causes serious damage to the DNA structure. Chandran et al. [105] investigated the binding character-istics and molecular mechanisms of interactions between IL molecules and DNA. They combined molecular dynam-ics simulations, circular dichroism spectroscopy, UV-vis spectroscopy, and fluorescent dye displacement assays to

determine the important factors that stabilize DNA in hydrated ILs. Although numerous methods are used for purification and preconcentration of DNA, nucleic acid extraction remains a formidable bottleneck.

Because of the success of DNA extraction with ILs, considerable effort has been devoted to understand the mechanism of DNA and its interactions with ILs and to develop new bio-ILs for dissolution of DNA [100, 106]. In the future, with careful engineering of IL structures, they

Table 4. ILs in DNA applications

Entry ILs DNA/RNA origin Remarks Ref.

1 [C2mim][Cl], [C10mim][Br], [C16mim][Br], [C10pohim][Br], [C16POHIM][Br], [(C10)2NMDG[Br]

DNA sodium salt from salmon testes

Highest extraction efficiencies of DNA obtained using [C16POHIM][Br] and [(C10)2NMDG][Br]Extraction efficiencies higher than 97%

[75]

2 [C4bim][Br] Calf thymus DNA Conformation was closer to its native structure in ILs

[98]

3 [C4mim][X], where X = [Cl], [NO3], or [lactate], [Ch][NO3], [Ch][lactate]

Dickerson–Drew dodecamer (DD, 5´-d-CGC-GAATTCGCG)2-3´Calf thymus DNA

DNA retained its native β-conformation in all systems[C4mim]+ bound to the minor groove more effec-tively because of its planar geometry and better charge delocalization

[99]

4 [Ch][DHP] DNA triplexes (Ts1, Ts2 and Ts3

Triplex formation significantly stabilized in ILs compared with an aqueous buffer at neutral pH

[102]

5 [Ch][DP] Small interfering RNA Prolonged biological stability of siRNA [104]

6 [C2emim][Br] DNA isolated from salmon milt

IL-robed DNA maintained double-helix structure [105]

7 [Ch][Pyr], [Ch][Gly] DNA (Salmon testes) Solubilized DNA maintained its chemical and structural stability for up to one year when stored at room temperature (25°C)

[106]

8 [b–4C–im][Br] Streptomyces coelicolor genomic DNA

Effective for promoting the polymerase chain reaction

[123]

9 [Mor1,2][Br] Calf-thymus DNA Hydrodynamic radius of DNA remained more or less constant in the presence of [Mor1,2][Br], suggesting that the structure of DNA is retained in the presence of the IL

[124]

10 [C2mim][Ac], [C2mim][Cl] Maize DNA DNA extraction from maize using pure ILsPure ILs for genomic DNA were not suitable with reproducibility but IL–aqueous-buffer system notably increased amount of extracted DNA

[125]

11 [Ch][DHP] DNA source not mentioned

DNA chemically stabilized in the IL [126]

12 [C4C1pyrr][NTf2], [P6,6,6,14][NTf2], [(OH)2C2(OH)2C2 C4NH][NTf2], [(OH)2 C2 C1 C1NH][NTf2], [C6 C1im][FAP], [P6,6,6,14][FAP], [(OH)2 C2 C1 C1NH][C2CO2], [N1,8,8,8] cation combined with [IO3]

−

RNA from feline calcivirus

[C1C1im][C1PO2OH] disintegrated the feline DNA with promising recovery rates and purity

[127]

13 [(C8)3BnN+][FeCl3Br−], [C16BnIM]2C12

2+[NTf2−,FeCl3Br−]

DNA from salmon testes

High extraction efficiencies obtained for short oligonucleotides with [(C8)3BnN+][FeCl3Br−] while [C16BnIM)2C12

2+[NTf2−,FeCl3Br−] gave high extrac-

tion efficiencies for longer stDNA

[128]




Biotechnol. J. 2016, 11, 1000–1013

could be used to produce innovative DNA extraction sys-tems, ion-conductive DNA films, and DNA preservation media.

4 Concluding remarks

ILs have been established as potential solvents/reagents for biomolecules and related techniques. In fact, ILs pro-vide a new and powerful platform for proteins, enzymes, and DNA, in which they show enhanced solubility, activ-ity, and stability. However, more research is needed to obtain quantitative and physically robust data to design ILs and IL-based technology for biomolecule enhance-ment. ILs have many molecular action modes through their interactions with phospholipids, proteins, and DNA. The main challenges in using ILs for biomolecules are the significant uncertainty in IL toxicity (both ecotoxicity and cytotoxicity), biodegradability, and the potential effect of ILs on biomolecules. Recently, significant steps have been made to understand and create cleaner routes for preparation of biocompatible and biodegradable ILs. We strongly believe that biocompatible ILs will be available in the near future, and that this will increase the use of ILs in biomolecular applications.

MS thanks the Centre of Research in Ionic Liquids (CORIL), Universiti Teknologi PETRONAS for financial support (Postdoctoral Fellowship)

The authors declare no financial or commercial conflict of interest

5 References [1] Rogers, R. D., Seddon, K. R., Ionic liquids: Industrial applications for

green chemistry. ACS Symposium Series, Vol. 818, American Chemical Society, Washington, DC, 2002.

[2] Welton, T., Room-temperature ionic liquids. Solvents for synthesis and catalysis. Chem. Rev. 1999, 99, 2071–2084.

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[4] Zolfigol, M. A., Khazaei, A., Moosavi-Zare, A. R., Zare, A. et al., Rapid synthesis of 1-amidoalkyl-2-naphthols over sulphonic acid functionalized imidazolium salts. Appl. Catal., A 2011, 400, 70–81.

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Magaret Sivapragasam is a postdoctor-

al research scientist at the Centre of

Research in Ionic Liquids, (CORIL),

Universiti Teknologi PETRONAS. Her

current projects include that of ecotoxi-

cology impact of Ionic Liquids (IL) on

various environmental flora and micro-

bial adaptability to ILs. Magaret has a

vast research background in the field of

biotechnology which includes recombinant protein downstream pro-

cessing and a six year experience in dealing with chromatographic sep-

arations. She obtained her Ph.D from Universiti Putra Malaysia where

she majored in Bioprocess Engineering.

Muhammad Moniruzzaman received

his B.Sc. in Chemical Engineering from

Bangladesh University of Engineering

and Technology (BUET), Bangladesh.

He then moved to Japan where he got

his M.Sc. (2004) and Ph.D (2007) in

biochemical engineering from Kanaz-

awa University. He then worked at

Kyushu University, Japan and Okayama

University, Japan. Currently, he is a faculty at Universiti Teknologi PET-

RONAS, Malaysia. His current research interests focus on the applica-

tion of ionic liquids as alternative “green” solvents in biocatalysis/

catalysis, biomass degradation, bioactive compound separation, and

pharmaceutics including drug delivery and formulations.

Masahiro Goto is a professor in the

department of Applied Chemistry at the

Graduate School of Engineering,

Kyushu University (Japan), and also the

director of center for Transdermal Drug

Delivery Systems (TDDS) at the same

university. His current research interest

is surfactant-based technology, and he

has been working on pharmaceutical

application of ionic liquids, transdermal vaccine, and drug delivery sys-

tems using surfactant molecules. He has published 14 books, 107

review articles, and more than 450 papers in scientific journals. He is

now deputy editor of Biochemical Engineering Journal.




Biotechnol. J. 2016, 11, 1000–1013

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© 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.biotechnology-journal.com

Meeting reportMetabolic Engineering Summit BeijingXiangbin Chen

http://dx.doi.org/10.1002/biot.201500664

CommentaryToward improved host cell protein impurity assessmentJong Youn Baik, Kelvin H. Lee


ReviewRecent advances in exploiting ionic liquids for biomolecules: Solubility, stability and applicationsMagaret Sivapragasam, Muhammad Moniruzzaman and Masahiro Goto


Research ArticleQuantitative definition and monitoring of the host cell protein proteome using iTRAQ – a study of an industrial mAb producing CHO-S cell lineLesley M. Chiverton, Caroline Evans, Jagroop Pandhal, Andrew R. Landels, Byron J. Rees, Peter R. Levison, Phillip C. Wright and C. Mark Smales


Research ArticleGeneric HPLC platform for automated enzyme reaction monitoring: Advancing the assay toolbox for transaminases and other PLP-dependent enzymesTim Börner, Carl Grey and Patrick Adlercreutz


Research ArticleHyperosmotic stimulus study discloses benefits in ATP supply and reveals miRNA/mRNA targets to improve recombinant protein production of CHO cellsJennifer Pfizenmaier, Lisa Junghans, Attila Teleki and Ralf Takors


Research Article Stirred tank bioreactor culture combined with serum-/xenogeneic-free culture medium enables an efficient expansion of umbilical cord-derived mesenchymal stem/stromal cellsAmanda Mizukami, Ana Fernandes-Platzgummer, Joana G. Carmelo, Kamilla Swiech, Dimas T. Covas, Joaquim M. S. Cabral and Cláudia L. da Silva


Research ArticleMono- and dichromatic LED illumination leads to enhanced growth and energy conversion for high-efficiency cultivation of microalgae for application in spaceInes Wagner, Christian Steinweg and Clemens Posten


Research ArticleImproving carbohydrate production of Chlorella sorokiniana NIES-2168 through semi-continuous process coupled with mixotrophic cultivationYue Wang, Sheng-Yi Chiu, Shih-Hsin Ho, Zhuo Liu, Tomohisa Hasunuma, Ting-Ting Chang, Kuan-Fu Chang, Jo-Shu Chang, Nan-Qi Ren and Akihiko Kondo


Biotechnology Journal – list of articles published in the August 2016 issue.

Cover illustrationThis 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 Moni ruzzaman, 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).

Research ArticleA dual enzyme system composed of a polyester hydrolase and a carboxylesterase enhances the biocatalytic degradation of polyethylene terephthalate films Markus Barth, Annett Honak, Thorsten Oeser, Ren Wei, Matheus R. Belisário-Ferrari, Johannes Then, Juliane Schmidt and Wolfgang Zimmermann


Research ArticleDisulfide-bridging PEGylation during refolding for the more efficient production of modified proteinsClaire Ginn, Ji-won Choi and Steve Brocchini


Research ArticleIdentifying and retargeting transcriptional hot spots in the human genomeJoseph K. Cheng, Amanda M. Lewis, Do Soon Kim, Timothy Dyess and Hal S. Alper


Biotech MethodEasyClone-MarkerFree: A vector toolkit for marker-less integration of genes into Saccharomyces cerevisiae via CRISPR-Cas9Mathew M. Jessop-Fabre, Tadas Jakociunas, Vratislav Stovicek, Zongjie Dai, Michael K. Jensen, Jay D. Keasling and Irina Borodina


© 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.biotechnology-journal.com

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