ILs form a strong ionic matrix and the added enzyme molecules could be considered as being included rather than dissolved in the media, meaning that ILs should be regarded as liquid enzyme immobilization supports, rather than reaction media, since they enable the enzyme-IL system to be reused in consecutive operation cycles

[96] . Finally, after an enzymatic transformation process in ILs, products can usually be recovered by liquid-liquid extraction, although the organic solvents used in this step represent a clear breakdown point for the integral green design of any chemical process.

To understand the biocompatibility of ionic liquids with enzymes it is necessary to consider the structure-function relationships of enzymes in water immiscible ILs and to discern how water is partitioned between the enzyme surface and the bulk IL solution. Complementary spectroscopy measurements (e. g. fluorescence, circular dichroism, FTIR) have classically been used to investigate changes in the secondary structure of enzymes in an attempt to explain the stabilization or denaturation phenomena associated with their molecular environment. Such spectroscopic methods have been used to correlate changes in the secondary structure of monellin

[97] , CALB [98, 99] or a-chymotrypsin [100] with enzyme stability in ILs. Iborra’s group were pioneers in carrying out structural studies that revealed that the syn­thetic activity and stability exhibited by CALB in ILs was much higher than that observed in hexane, and was related with the associated conformational changes that take place in the native structure of CALB, as demonstrated by fluorescence and CD spectroscopic techniques [98]. The stabilization of CALB by hydrophobic ILs seems to be related with the observed evolution of a-helix to в-sheet secondary structures of the enzyme, resulting in a more compact enzyme conformation, that is able to exhibit high catalytic activity, suggesting that the stability of enzyme in this medium was improved by the formation of a compact, but flexible, native-like conformation of the enzyme. Turner et al. [16] described how the deactivation of the enzyme cellulase produced by water-miscible ILs (e. g., [BMIM][Cl]) is accom­panied by a fall in the fluorescence intensity maxima of the Trp parameter with respect to the native conformation in water as a result of the enhancing exposure of Trp residues to the bulk solvent and enzyme denaturation. Fluorescence spectros­copy demonstrated that monellin in a low water content (2 % v/v) in [BMpy][NTf2] resisted thermal unfolding. Fujita et al. [101] elucidated the power of hydrated [Choline][H2PO4] to maintain the activity of cytochrome c after 18 months of storage in the dissolved form at room temperature because of its ability to maintain its native secondary structure and conformation, as monitored by ATR-FTIR (attenuated total reflection Fourier transform infrared) and resonance Raman spec­troscopies. A later study found that CALB aggregates can deactivate in 1-ethyl-3- methylimidazolium-based ILs in an anion-dependent manner [42]. Studies of papain in 15 % (v/v) aqueous solutions of 1-alkyl-3-methylimidazolium-based ILs using ATR-FTIR demonstrated that the choice of anion has a significant impact

on the structure, specificity and stability of the enzyme [102]. Again, the в-sheet content in the secondary structure increased, while the a-helical content decreased.

Micaeio and Soares [103] presented a molecular dynamics simulation study of the serine protease, cutinase, in two different ILs, [BMIM][PF6] and [BMIM] [NO3]. Their work showed that the enzyme is preferentially stabilized in [BMIM] [PF6], which allows a suitable degree of hydration to be maintained at the enzyme surface and hence renders a more native-like enzyme structure, while [BMIM] [NO3] tended to be more destabilizing. These findings are in accordance with previous experimental observations [15, 86] which attributed these results to the difference in the hydrophobicity of the two ILs: [BMIM][PF6] is more hydrophobic than [BMIM][NO3] and hence is less likely to dissociate into ions to destabilize the enzyme.

A study of human serum albumin (HSA) and equine heart cytochrome c (cyt c) by CD spectroscopy and small-angle neutron scattering (SANS) demonstrated that the IL 1-butyl-3-methylimidazolium chloride ([BMIM][Cl]) not only caused sig­nificant unfolding of the a-helical proteins when present as a cosolvent with water, but [BMIM][Cl] also changed the aggregation state of HSA, suggesting that the interaction depends on the protein sequence [104].

The secondary structure can also be analyzed with FTIR spectroscopy since proteins absorb infrared wavelengths due to peptide bond vibrations. Liu et al. [32] reported that a significant decrease in the a-helix content of lipase from Burkholderia cepacia probably affects the lipase active site: the lower the a — helix, the higher the “open” conformation of the active site, allowing easier access to the substrate.

More recently, Fan et al. [105] suggested that ILs could quench the intrinsic fluorescence of papain, probably by means of a static quenching mechanism. The calculated binding constants were very small compared with that of volatile organic solvents, indicating that only very weak interaction between ILs and papain existed. The Gibbs free energy change (AG), enthalpy change (AH), and entropy change (AS) during the interaction of papain and ILs were estimated. The negative values of these parameters obtained, indicated that the interaction between ILs and papain was a spontaneous process, also implying that hydrogen bonding and van der Waals forces played important roles in the interaction processes.

The impact of water-miscible ILs on proteins was characterized by structural changes of green fluorescent protein (GFP) in aqueous solutions containing 25 and 50 % (v/v) of [BMIM][Cl]. The SANS and spectroscopic results indicated that GFP is a great deal less compact in 50 % (v/v) [BMIM][Cl] than in neat water, suggesting unfolding from the native structure. The oligomerization state of the protein in IL-containing aqueous solution changes from a dimer to a monomer in response to the IL, but does not change as a function of temperature of the IL solution. The SANS and spectroscopic results also demonstrate that the addition of this hydrophilic ionic liquid to the solution lowers the thermal stability of GFP, allowing the protein to unfold at lower temperatures than in aqueous solution [106].

An aqueous solution of free-enzyme molecules added to the hydrophobic IL phase could be regarded as being included, but not dissolved, in the medium, the

essential water shell around the protein being preserved, and providing an adequate microenvironment for the catalytic action [14]. Usually, enzymes fold by placing the non-polar residues in a hydrophobic core, while polar residues are located on the hydrated surface. A “memory” phenomenon is observed when an enzyme is placed in a dry hydrophobic system, because the biocatalyst is trapped in the native state as a consequence of the low dielectric constant of the medium. This intensifies intramolecular electrostatic interactions and enables the catalytic activity to be maintained [103, 107, 108]. The extremely ordered supramolecular structure of ILs in solid and liquid phase has been described as an extended network of cations and anions connected by hydrogen bonds [109]. This network might be able to act as a mould, maintaining an active three-dimensional structure of the enzyme in non-aqueous environments, and avoiding classical thermal unfolding. Therefore the incorporation of molecules and macromolecules in the ionic liquid network causes changes to the physico-chemical properties of these materials and can cause, in some cases, the formation of polar and non-polar regions [109]. So, enzymes in water immiscible ILs should also be considered as being included in the hydrophilic gaps of the network, where the observed enzyme stability could be attributed to the maintenance of this strong net around the protein. ILs can clearly be considered as both solvents and liquid immobilization supports because multipoint enzyme-IL interactions (ionic, hydrogen bonds, van der Waals, etc.) may occur, resulting in a supramolecular net able to maintain an active protein conformation [98] (Fig. 11.6).

A theoretical basis for predicting the compatibility of enzymes and anhydrous ionic liquids has not yet been developed, although a number of possibly contributing factors have been discussed, such as the cation H-bond donating capability, log P, formation of hydrogen-bonded nanostructures, and solvent viscosity [8]. With regard to the compatibility of enzymes and hydrophobic ionic liquids, hydrogen bonding could be the key to understanding. It is well known that the thermal stability of enzymes is enhanced in both aqueous and anhydrous media containing polyols as a consequence of an increase in hydrogen bond interactions. Thus, both the solvophobic interactions essential for maintaining the native structure and the water shell around the protein molecule are preserved by the “inclusion” of the aqueous solution of free enzyme in the IL network, resulting in a clear enhancement of enzyme stability (Fig. 11.7).

Yang [110] maintains that an ion may affect enzyme performance by playing the role of substrate, cofactor, or even inhibitor. But more generally, the effect of specific ions could be better understood by considering an ion’s ability to alter the bulk water structure, to affect the protein-water interaction, and to directly interact with the enzyme molecules. So, the effect of ions on enzyme activity and stability has usually been linked to the Hofmeister series (or the kosmotropicity order): kosmotropic anions and chaotropic cations stabilize enzymes, while chaotropic anions and kosmotropic cations destabilize them. The influence of hydrophilic ILs on the protein activity and stability usually follows the Hofmeister series when ILs dissociate into individual ions in water [111] but, unfortunately, there are many cases in which this series is not followed, especially when there is little or no water present in the IL media, and, furthermore, some authors associate

Hofmeister effects only with anions [112]. Micaelo and Soares [103] presented a molecular simulation study of an enzyme in two ionic liquids, [BMIM][PF6] and [BMIM][NO3], observing that the enzyme structure is highly dependent on the amount of water present in the IL media and that [BMIM][PF6] significantly increases protein thermostability at high temperatures, especially at low hydration values. These ILs “strip” most of the water from the enzyme surface in a degree similar to that found in the case of polar organic solvents, while the remaining water molecules at the enzyme surface are organized in many small clusters. [BMIM] [PF6] seems to retain similar amounts of water at the enzyme surface, as acetoni­trile, and supports the evidence of the polar nature of this IL. This IL [BMIM] [NO3], in contrast, replaces almost all the water at the enzyme surface, which may be the reason for its destabilizing effect on the enzyme. A more detailed analysis of enzyme solvation by the two ILs shows that the anion species dominates the

non-bonded interactions with the enzyme, as judged by the number of hydrogen bonds observed between the enzyme and the cation and anion species of each IL. The ability of ILs to dissolve molecules depends mainly on the hydrogen bond- accepting properties of the anion, as stated by Anderson et al. [113]. Moreover, Zhao et al. [83] observed that the dissolution of lipase in most hydrophilic ILs is an indication of strong interactions between the enzyme and solvent molecules. If such interactions disturb the active sites and/or are strong enough to disrupt the protein structures, the enzyme activity is lost. However, if such interactions are not too strong but allow the enzyme’s structures to be maintained, these hydrophilic ILs do not inactivate the enzyme (such as [Et3MeN][MeSO4], or AMMOENG series ILs).

Weingartner et al. [112] observed the importance of “microheterogeneity” in ILs. The charged ionic groups and non-polar residues of cations and anions give rise to the nanoscale structural heterogeneity of ILs, which is not encountered in simple molecular solvents. The resulting hydrophilic and hydrophobic patches of the IL structure have intriguing consequences for solvation because they enable dual solvent behaviour: an IL can incorporate a non-polar solute in non-polar domains, while hydrophilic domains solvate polar solutes.

Finally, the type of reaction medium used is conditioned by the type of biotrans­formation; for example, water-immiscible ILs were found to be the most effective for the production of fermentable sugars from cellulose at low water content or in nearly anhydrous conditions. Moreover, immobilized CALB was the most efficient biocatalyst for the transesterification (alcoholysis) of vegetable oils (or animal fats). Both the IL and CALB can be recycled for at least four successive reactions without any loss of activity. Furthermore, aqueous solutions of hydrophilic ILs were necessary to produce bioethanol because the presence of water is necessary for cellulose hydrolysis. In this type reaction medium, the IL and the assayed IL-to — water concentration ratio are the key criteria.

Other strategies proposed to improve the efficiency of bioethanol and biodiesel transformations include biphasic systems based on IL and scCO2 (supercritical carbon dioxide), the addition of cosolvents and a IL coating of immobilized enzyme particles. The enormous potential of immobilized multi-enzymatic or cross-linked enzyme aggregates for bioethanol synthesis in ionic liquid media has only just been realized.

11.3 Conclusions

There is, as yet, no theoretical basis for predicting the compatibility of ionic liquids with enzymes, although key parameters for this relationship depend on the type of reaction system.

(a) Water-immiscible ILs.

The enzyme structure is highly dependent on the amount of water present in the IL medium. So, the hydrophobicity of a water-immiscible ionic liquid may be considered as a constrainer of polarity, because hydrophobicity is related to miscibility with water, and the water shell around the protein molecule is essential for maintaining the activity/stability of the enzyme. Water-immiscible ionic liquids are nevertheless hygroscopic, as noted above, and readily absorb a low percentage of water. Besides, some local hydrophobic ion-enzyme macro­molecule interactions are also important for enzyme stability. Thus, for the compatibility of enzymes and anhydrous ionic liquids, a hydrophobic effect could be the key.

(b) Water-miscible ILs.

These ILs are used as aqueous ionic liquid mixtures and the ratio of the ionic liquid-to-water used is crucial to the effect it has on the enzyme. To maintain the activity of hydrophilic ionic liquid-dissolved enzymes, a balance of mild hydro­gen bond-accepting and donating properties is required, so cation and anion size and the ability to form hydrogen bonds are important for these systems because stabilization primarily results from hydrophobic forces and hydrogen-bond. The hydrogen-bond donating ability is usually a property of the cation, while the anions act as hydrogen-bond acceptors, and it has been demonstrated that the ability of ionic liquids to dissolve complex compounds, such as sugars and proteins, mainly depends on the hydrogen-bond accepting properties of the anion. So, with regard to the compatibility of enzymes and hydrophilic ionic liquids, hydrogen bonding could be the key. Ionic liquids, in particular their anions which form strong hydrogen bonds, may dissociate the hydrogen bonds that maintain the structural integrity of the a-helices and в-sheets, causing the protein to unfold wholly or partially. As discussed above, another key property for these systems is viscosity, which is strongly influenced by cation chain length.