It is well known that the performance of enzymes in organic solvents as well as in ionic liquids is affected by common factors such as water activity, pH as well as the existence of excipients and impurities. Moreover, the physicochemical properties of ionic liquids such as viscosity, polarity, hydrophobicity, kosmotropicity, etc. also have strong effect on the activity and stability of enzymes in ionic liquids. The effect of these physicochemical properties of ionic liquids on enzyme activity and stability are summarized in Table 10.2.

In general, enzymes are optimized in nature to perform best in aqueous envi­ronment, at neutral pH, temperature below 40 °C and at low osmotic pressure. When enzymes are used in either pure organic solvents or ionic liquids, the

minimum amount of water, which is best described by the water activity, is of crucial importance to maintain the enzyme activity. For ionic liquids, the same methods can be used to maintain a constant water activity as those established for organic solvents [33]. However, in some ionic liquids (e. g. fluoride contained ionic liquids), the water present in the reaction system may cause hydrolysis of ionic liquids and result in reduced enzyme activity and stability. This is attributed by the change in pH of system and the inhibition effect of the hydrolyzed products. The pH strongly effects the activity and stability of enzyme in ionic liquids media as shown in work of Tavares et al. [48]. In this study, the activity of laccase was well maintained at pH 9.0 for 7 days (with activity loss about 10 %) in aqueous solution of 1-ethyl-3-methylimidazolium 2-(2-methoxyethoxy) ethylsulfate, [Emim] [MDEGSO4], while significantly reduced at pH 5.0. Moreover, the impurities present in ionic liquids are also important factor that need to be taken into account when carrying out enzymatic reactions in ionic liquids. The impurities may influ­ence the physicochemical properties of ionic liquids and hence, enzymatic reaction. For example, the activity of immobilized CALB (Novozym 435) in [Omim][Tf2N] decrease linearly with chloride content while the activity of immobilized lipase from Rhizomer miehei ( Lipozyme IM) drastically decreases in the presence of [Omim][Cl] [49].

Since ionic liquids are composed only of ions, the effect of ions on the enzyme activity and stability is also an important factor. Ions can affect the stability of proteins through the interactions between the ions and charged amino acid groups in the proteins [50]. Some enzymes require metal ions such as cobalt, manganese, zinc, etc. for their activity. If these ions are removed or interfered by interaction

m—————— Stabilizing Destabilizing———————————— ►

Anion F"> PO43′> SO42′> CH3COO’> Cl’> Br’> I’> SCN’

Cation (CH3)4N+>(CH3)NH2+> NH4+> K+> Na+> Cs+> Li+> Mg2+> Ca2+> Ba2+

Fig. 10.1 The Hofmeister series as an order of the ion effect on protein stability (Reprinted with permission from Ref. [43]. Copyright © 2013, Elsevier) with ionic liquids, enzyme might be inactivated. Several researchers have proposed Hofmeister series to explain the behavior of enzymes in aqueous solution of ionic liquids. Hofmeister series (Fig. 10.1) which indicates the kosmotropicity of individual cations and anions of ionic liquids may be a good guide for choosing ionic liquids for enzyme activity and stability in aqueous solutions [43, 44, 46]. The anion such as PO43~, SO42~, CO32~ (kosmotropic anions), and cations such as NH4+, K+, Cs+ (chaotropic cations) stabilize enzymes, while chaotropic anions and kosmotropic cations destabilize them. However, the influence of Hofmeister series is complicated when enzymes are present in nearly anhydrous ionic liquids.

Properties of ionic liquids such as viscosity, polarity, and hydrophobicity also affect the enzymes in ionic liquids. Ionic liquids are well recognized to have higher viscosity than conventional organic solvents. As a general trend, enzymes are more active in less viscous ionic liquids that can be attributed to the mass transfer limitation in high viscous media. For example, a-chymotrypsin maintained higher activity in less viscous [Emim][Tf2N] (34 mPa • s) than that in high viscous [MTOA][Tf2N] (574 mPa • s) for the synthesis of N-acetyl-L-tyrosine propyl ester [26]. Ionic liquids are considered as highly polar account on their ionic nature. The polarity of ionic liquids has been empirically determined by means of a variety of solvatochromic probe dyes [51]. For instance, betaine dye no. 30 or Reichardt’s dye has been used to characterize ionic liquid polarity by the solvent polarity parameter ET(30) and the corresponding normalized polarity scale £^. In addition, the Kamlet-Taft parameters (a, p and n*, which quantify hydrogen-bond donating ability (acidity), hydrogen-bond accepting ability (basicity) and polarity/polariz — ability, respectively), determined by set of dyes, have also been used to quantify the polarity of ionic liquids [52, 53]. Based on solvatochromic probes studies, ionic liquids have polarity close to low chain alcohol or formamide [54]. Several studies have demonstrated that activities and stabilities of enzyme in ionic liquids were increased with increasing ionic liquids polarity [24, 26] although there were some reports showing no clear effect of polarity on enzyme [21,45]. Hydrophobicity may be considered as narrow concept of polarity. However, it is practically important to separate hydrophobicity to polarity since the former is often related to the misci­bility with water [55]. Depending on the structure of cation and anions, ionic liquids can be hydrophilic or hydrophobic. As a general rule, the structure of anions has strong influence on the hydrophobicity of ionic liquids than the contribution of cations. Hydrophilic anions such as halides, carboxyl groups, having high hydrogen bonding capability, strongly interact with enzyme resulting in the conformational change and deactivation of enzymes. On the other hand, the hydrophobicity of ionic
liquids increases as the alkyl chain length in cations increases. In some reports, ionic liquids with long alkyl chain in cations may behave as surfactants in aqueous solution and have strong stabilizing impact on enzyme [56, 57]. However, in some cases, the long alkyl chain might have negative effect on the activity and stability of enzyme due to the resulted higher viscosity [58]. In practice, the log P (logarithm of partition coefficient between octanol and water) scale can be used to quantify the hydrophobicity of ionic liquids, and in some reports this scale can be used to optimize enzyme activity and stability in ionic liquids [21, 46, 59, 60].