Increase in Thermal Stability of Proteins by Aprotic Ionic Liquids

Proteins are biomolecules of great importance in the biochemical processes such as the med‐ ical, pharmaceutical, and food fields, since they exhibit their outstanding biological activities under mild condition. However, most of proteins dissolved in an aqueous solution are im‐ mediately denatured and inactivated at high temperatures due to the disruption of weak in‐ teractions including ionic interactions, hydrogen bonds, and hydrophobic interactions, which are prime determinants of protein tertiary structures [5-3]. In particular, protein ag‐ gregation easily occurs upon the exposure of the hydrophobic parts of proteins, which are usually located in the inside of native proteins, and this phenomenon becomes the major problem because of the fast irreversible inactivation. Thermal denaturation of proteins is a serious problem not only in the separation and storage of proteins but also in the processes of biotransformation, biosensing, drug production, and food manufacturing. Several strat‐ egies have so far been proposed in order to prevent thermal denaturation of proteins [4-14]. They include chemical modification, immobilization, genetic modification, and addition of stabilizing agents. The addition of stabilizing agents is one of the most convenient methods for minimizing thermal denaturation, compared to other methods. It has been reported that inorganic salts, polyols, sugars, amino acids, amino acid derivatives, chaotropic reagents, and water-miscible organic solvents are available for improving protein stability. However, these additives do not sufficiently prevent irreversible protein aggregation or some of them are no longer stable at high temperatures.

ents of ionic liquids. It has recently been reported that protic ionic liquids such as alkylammonium salts keep the stability of proteins in an aqueous solution at high temperatures [17,18], and amyloid fibrils of proteins are dissolved in protic ionic liquids and are refolded by dilution with an aqueous solution [19]. On the other hand, biotransformation in ionic liquids has increasingly been studied . Aprotic ionic liquids such as immidazolium salts have mainly been employed as reaction media, since the high activity of enzymes is exhibited as usual. We have found that the activity of protease is highly maintained not only in waterimmiscible aprotic ionic liquids but also in water-miscible aprotic ionic liquids [23,24].     However, despite such potential capability of aprotic ionic liquids, there have not been any works on the thermal stability of proteins in aqueous solutions containing water-miscible aprotic ionic liquids.
In the chapter, the effect of water-miscible aprotic ionic liquids consisting of 1-ethyl-3-methylimidazolium cations and several kinds of anions on thermal stability of proteins in aqueous solutions is mainly discussed [25].

Dependence of the remaining activity of lysozyme on the concentration of ionic liquids after the incubation at 25 o C
As a model protein, hen egg white lysozyme has been employed as shown in Fig. 1, since it has been well investigated regarding its structure, properties, and functions [26]. Lysozyme is a compact protein of 129 amino acids which folds into a compact globular structure. The molecular weight of lysozyme is 14,300, and the structure of lysozyme includes α-helices, β sheets, random coils, β turns, and disulfide bonds, which are typical structures of proteins. Lysozyme attacks peptidoglycans in the cell walls of Gram-positive bacteria, and catalyzes hydrolysis of 1,4-beta-linkages between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in a peptidoglycan. Accordingly, lysozyme has been used as an anti-inflammatory agent, a preservative, a freshness-keeping agent, an antibacterial agent, a disinfectant, and so on.
Room temperature ionic liquids of alkyl imidazolium cations are widely used, and are commercially available. Figure 2 shows structures and properties of ionic liquids introduced in this chapter. It has been well known that ions and other compatible solutes affect enzyme activity [22]. Figure 3 shows the plot of the remaining activity of lysozyme against the concentration of ionic liquids after the incubation at 25

Thermal inactivation of lysozyme
When proteins dissolved in an aqueous solution are placed at high temperatures, most of proteins are immediately unfolded due to the disruption of weak interactions including ionic effects, hydrogen bonds, and hydrophobic interactions, which are prime determinants of protein tertiary structures. In addition, the intermolecular aggregation among unfolded proteins and the chemical deterioration reactions in unfolded proteins proceed as shown in Fig.  4 [2,29,30]. In particular, protein aggregation easily occurs upon the exposure of the hydrophobic surfaces of a protein, and this phenomenon becomes the major problem because of the fast irreversible inactivation. On the other hand, when a heated solution of denatured proteins without protein aggregation is slowly cooled back to its normal biological temperature, the reverse process, which is renaturation with restoration of protein function, tends to occur. Accordingly, if stabilizing agents can sufficiently prevent irreversible aggregation of unfolded proteins, it is expected that unfolded proteins are refolded by cooling treatment, and the high remaining activity is obtained.   ately became turbid due to the aggregation of thermally-denatured proteins, as soon as heat treatment was carried out, as shown in Fig. 6(b). It has been reported that the precipitation due to protein aggregation at high temperatures is observed above 10 μM lysozyme [31]. As lysozyme concentration in the present work was 100 μM (1.4 mg/mL) which was ten times higher than that, the formation of protein aggregation was dramatically accelerated. Ammonium sulfate, which was an inorganic salt, glucose and glycerol, which were polyols, and urea, which was a chemical denaturant, inhibited the formation of protein aggregation, and exhibited thermal stabilization to some extent. β-Cyclodextrin, which was an inclusion compound, pectin, which was a thickener, and Triton-X, which was a nonionic surfactant, could not maintain the stability of lysozyme at high temperatures, although they were widely used as a stabilizer.  Fig. 6(a). When lysozyme solution in the presence of protic ionic liquids such as alkylammonium formates is heated at 90 ℃, protein aggregation is prevented, and any cloudy appearance is absent [18]. The hydrophobic core of lysozyme unfolded by heat interacts with the cation of ionic liquids, and cation adsorption results in acquisition of a net positive charge preventing aggregation via electrostatic repulsion [17].

Refolding of lysozyme by ionic liquids
When the formation of protein aggregation is inhibited at high temperatures by ionic liquids, and thermally-denatured proteins are individually dispersed in an aqueous solution, it is probably that denatured proteins are gradually refolded under cooling conditions. Figure  7 shows [Tf] rapidly increased with incubation time at 25 ℃, and reached a plateau around 2 and 7 min, respectively. In sufficiently low concentration of proteins, where protein aggregation is not formed, when the hydrophobic core of proteins is exposed, but the disulfide bonds keep intact, denatured proteins gradually tend to refold to their native structures on cooling after thermal denaturation [32][33][34][35][36]. The refolding of thermally-denatured proteins is enhanced in the presence of protic ionic liquids such as alkylammonium nitrate and alkylammonium formates [32,21]. Moreover, N'-alkyl and N'-(ωhydroxyalkyl) N-methylimidazolium chlorides refold denatured proteins such as hen egg white lysozyme and the single-chain antibody fragment ScFvOx [37].

Dependence of the remaining activity of lysozyme on the concentration of ionic liquids via heat treatment
The stability of proteins depends upon the kind and concentration of electrolytes [27,28]. Figure 8 shows the plot of the remaining activity of lysozyme against the concentration of ionic liquids after the heat treatment at 90 ℃ for 30 min. reported that after heat treatment the remaining activity of lysozyme increases with an increase in the concentration of ethylammonium formate and 2-methoxyethylammonium formate, while the remaining activity increases at low concentration of propylammonium formate, but at higher concentrations of propylammonium formate the protein spontaneously denatures [18]. Thus, the dependence of concentration of ionic liquids on the remaining activity of proteins changes by switching from one ionic liquid to another. Figure 9. Thermal denaturation curves of lysozyme with or without ionic liquids. The aqueous solution of 100 μM lysozyme with or without ionic liquids was incubated in a silicone oil bath thermostated at requisite temperature for 30 min.

Dependence of the remaining activity of lysozyme on the temperature of heat treatment
The thermal inactivation of proteins more rapidly proceeds by higher temperatures. Figure 9 shows the relationship between the temperature of heat treatment and the remaining activity of lysozyme in aqueous solutions containing water-miscible ionic liquids after the heat treatment for 30 min. As seen in the figure, the remaining activity of lysozyme without ionic liquids gradually decreased with an increase in temperature below 70 ℃, accompanied with the formation of precipitation due to protein aggregation, drastically dropped in the range from 70 to 80 ℃, and was then lost at temperatures of 80 ℃ or higher. The transition temperature was exhibited around 75 ℃, similar to the case measured by differential scanning calorimetry [17]. On the other hand, the remaining activity of lysozyme with 1.

Time course of remaining activity of lysozyme via heat treatment with or without ionic liquids
Heating time directly enhances the thermal inactivation of proteins. Figure 10 shows time course of remaining activity of lysozyme with or without ionic liquids through the heat treatment at 90 ℃. The remaining activity of lysozyme without ionic liquids dramatically decreased with an increase in time, accompanied with the formation of protein aggregation, and was almost lost at 10 min. It has been reported that the remaining activity in the thermal denaturation process accompanied with the formation of protein aggregation follows firstorder kinetics [31]. As seen in the figure, the relationship of the remaining activity of proteins in the absence of ionic liquids with the time of heat treatment could be correlated by first-order kinetics. On the other hand,  4. The plots of remaining activity versus heat treatment time on thermal inactivation of lysozyme in the presence of ionic liquids followed first-order kinetics on linearity. It has been reported that the thermal inactivation of lysozyme obeyed first-order kinetics when it irreversibly proceeded by the covalent change without the formation of protein aggregation [39]. Table 1 represents rate constants and half lives of inactivation of lysozyme with or without ionic liquids calculated from the fitting curves in Fig. 10

Conclusion
In this chapter the effect of water-miscible aprotic ionic liquids on thermal stability of lysozyme has been described. Aprotic ionic liquids could sufficiently prevent thermally-denatured proteins from aggregating. The activity of lysozyme in the presence of aprotic ionic liquids was kept to some extent, just after heat treatment at high temperatures. Moreover, thermally-denatured lysozyme was effectively refolded by cooling. Consequently, the high remaining activity of lysozyme was obtained. These results indicate that aprotic ionic liquids act not only as an inhibitor of protein aggregation, but also as a protective agent in the native structure of protein and an accelerator in the refolding of thermally-denatured pro-

Author details
Hidetaka Noritomi 1 1 Tokyo Metropolitan University Japan