Another property of ILs is hydrophobicity, which should not be confused with solvent polarity. The terms hydrophilic/hydrophobic ions are often used synony­mously with water miscibility based on the miscibility of ILs with water, so that ionic liquids can be divided into two categories: hydrophobic (water-immiscible) and hydrophilic (water-miscible). The hydrophobicity of ILs is usually quantified by the log P scale, a concept derived from the partition coefficient of ILs between 1-octanol and water. Russell’s group measured the log P values (< —2.0) of several ILs, and suggested that they are very hydrophilic in nature based on Laane’s scale [31]. They also observed that free lipase (Candida rugosa) was only active in hydrophobic [BMIM][PF6] (log P = —2.39), but inactive in other hydrophilic ILs, including [BMIM][Ac] (log P = —2.77), [BMIM][NO3] (log P = —2.90) and [BMIM][TfA] [15]. Through a systematic investigation of lipase-catalyzed transesterification in over 20 ILs, it was observed that lipase activity increased with the log P value to reach a maximum, and then decreased as log P further increased (a bell-shaped dependence). These examples implied that the high hydro — phobicity (high log P) of ILs could be beneficial for enzyme stabilization [34]. How­ever, many exceptions to this rule have been reported, and ionic liquids have not been treated according to this log P concept to relate them to enzyme activity.

The water miscibility of ILs generally depends on the anions they contain, and the solubility of water an ionic liquid can be varied by changing the anion from [Cl] to [PF6]. However, this behaviour varies widely and sometimes unpredictably; for example, [BMIM][BF4], does not dissolve simple sugars to an appreciable degree and [BMIM][Cl], in contrast, dissolves massive amounts of cellulose. And yet, these ionic liquids are of similar polarity on the Reichardt’s scale. It was demonstrated that the ability of ionic liquids to dissolve complex compounds, such as sugars and proteins, mainly depends on the H-bond accepting properties of the anion. A recent measurement of the H-bond accepting properties of such ILs revealed that [BF4] or [MeSO4] were better H-bond acceptors (в = 0.61 and 0.75, respectively) than [PF6] (в = 0.50), which can be considered a reasonable expla­nation for the difference in water miscibility [85]. The high hydrogen-bonding basicity and overall hydrophilic nature of water-miscible ILs enable to dissolve enzymes (to a greater or lesser degree) while enzymes are barely soluble in hydrophobic ILs [86]. The Sheldon group [87] maintains that hydrogen bonding could be the key to understanding the interactions of proteins and ionic liquids. Water is a powerful hydrogen bonding medium and an ionic liquid must mimic water in this respect to dissolve proteins, in particular as regards the hydrogen bond — accepting properties of the anion. The interaction should not be too strong, how­ever, because, otherwise the hydrogen bonds that maintain the structural integrity of the a-helices and в-sheets will dissociate, causing the protein to unfold. So, to maintain the activity of ionic liquid-dissolved enzymes, a balance of mild hydrogen bond-accepting and donating properties is required. In contrast, in ionic liquids that do not dissolve enzymes, the enzyme preserves its native structure in this ionic liquid just as it preserves its catalytic activity. Besides, enzyme-compatible anions exhibit lower hydrogen bond basicity, which minimizes interference with the internal hydrogen bonds of an enzyme [84]. They also exhibit lower nucleophilicity and thus a lower tendency to change the enzyme’s conformation by interacting with the positively charged sites in the enzyme structure [87].

The approximate ionic association strength in aprotic solvents is listed below in increasing order [88]:

[NTf2] < [PF6] < [СЮ4] < [SCN] < [BF4] < [TfO] < [Br] < [NO3] < [TFA] < [Cl]

This order represents the strength of an anion in its interaction with solvated cations through ionic attraction, or may even represent the strength of interactions between the anion and the changed surface of macromolecules (such as proteins). Dupont [10] suggested the strength of hydrogen-bond basicity in the similarly increasing order of:

[BPh4] < [PF6] < [BF4] < [TFA]

These sequences confirm that enzyme activity is probably related with the hydrogen-bond acceptor strength of the anion: anions with low hydrogen-bond basicity are enzyme stabilizing.

In regards to IL cations, these are usually accepted to show a lower dominant effect than anions of the same charge density, because anions are more polarizable and hydrate more strongly [43]. So, cations seem to interact indirectly via interac­tion with anions, depending on the degree of coordination and the length of the alkyl chain of the cations.

Other authors suggest that the stability of the enzyme depends on the nucleophi- licity of the anion. Kaar et al. [15] observed that free Candida rugosa lipase was only active in hydrophobic [BMIM][PF6], but inactive in all hydrophilic ILs based on [NO3], [Ac] and [TFA] during the transesterification of methylmethacrylate with 2-ethyl-1-hexanol. They indicated that the last three anions are more nucleophilic than [PF6], and thus could interact with the enzyme causing protein conformational changes. However, in another study, a contradictory result was reported. Irimescu and Kato [89] carried out the CALB-catalyzed enantioselective acylation of 1-pheny- lethylamine with 4-pentenoic acid, and found that the reaction rate relied on the type of IL anions (reaction rate in a decreasing order of [TfO] > [BF4] > [PF6], keeping the cation unchanged). This suggests higher anion nucleophilicity, correlating with higher enzymatic activity. On the other hand, Lee et al. [90] measured the initial transesterification rates of three lipases (Novozym 435, Rhizomucor miehei lipase, and Candida rugosa lipase) in different ILs with the same water activity, and observed that the anion effect on the initial rates followed a decreasing order: [NTf2] > [PF6] > [TFO] > [SbF6] — [BF4]. They suggested that [TFO] and [BF4] are more nucleophilic than [PF6], although these results could be better explained by the anion hydrophobicity of IL.

An interesting phenomenon observed by Zhao et al. [34] during the transester­ification of ethyl butyrate with 1-butanol was that when the solvents (dichloromethane or ionic liquids) and substrates were dried but the lipase was not totally dried (-3 % wt water) higher reaction rates were observed with microwaves than in a water-bath. However, when the enzyme was also dried, the differences in reaction rates became insignificant. This interesting behaviour has actually been reported in a number of papers, the authors of which maintain that in a fairly dry hydrophobic solvent and substrate environment, the enzyme particle is surrounded by (at least) one layer of water molecules. The solvent is hydrophobic so it does not strip off the water layer. In this microenvironment, the water layer near the enzyme surface has a much higher relative dielectric constant (er = 80.1 at 20 °C) than the surrounding IL. Therefore, under microwave irradiation, the enzyme surface is likely to have a higher temperature than the bulk solvent due to the superheating of the water layer.

For bioethanol production, hydrophilic ILs are effective in dissolving cellulose, but the activity of cellulases decrease significantly in their presence. Therefore, IL residues should be entirely removed after cellulose regeneration [83].

When dealing with ILs in nearly anhydrous conditions, at low water content (<2 % v/v), all the assayed water-immiscible ILs (i. e. [BMIM][PF6], [BTMA] [NTf2], etc.) were shown to be suitable reaction media for biocatalytic reactions, because water-immiscible ionic liquids are nevertheless hygroscopic, as noted above, and readily absorb a small percentage of water [91], but sufficient to maintain the active conformation of the enzyme. For example, in the case of the lipase B (Novozym 435 from Novo) catalyzed transesterification of vegetable oil for biodiesel production, the best enzyme activity and a biodiesel yield of 97-98 % was obtained with [EMIM][PF6] and [BMIM][PF6], while hydrophilic ILs were poor solvents for this methanolysis for two reasons: oil insolubility (heterogeneous system) and enzyme deactivation [22, 32]. Besides, ILs with long alkyl chains (e. g., [C16MIM][NTf2] and [C18MIM][NTf2]) have been used in a homogeneous one-phase system for lipase-catalyzed biodiesel production which this avoids direct interactions between the enzyme and pure methanol and allows for the stable reuse of lipase in the ILs. These long chain and lipophilic ILs create a non-aqueous system for oil transesterification, and at the end of the reaction, a triphasic system was formed as a result of lowering the temperature, which facilitates biodiesel extraction [27, 28].

The remarkable results obtained for enzymatic reactions in water-immiscible ILs in nearly anhydrous conditions underline the suitability of these solvents as a clear alternative for biodiesel production. In this strategy, enzymes (immobilized or not) are simply suspended in the ILs, and the resulting a mixture can be used for biocatalytic reactions. Enzymes coated with ionic liquids can also be used. For example, for citronellyl ester synthesis, immobilized lipase from C. antarctica coated with ionic liquids at a very high temperature (95 °C) in hexane and solvent-free conditions was used [11].