# Ionic Liquids for Development of Heterogeneous Catalysts Based on Nanomaterials for Biocatalysis
## 2 Stabilization of Enzymes via Ionic Liquids  
Ionic liquids, known as low-temperature molten salts, are compounds consisting of organic cations and organic or inorganic anions that are classified to green chemistry substances [34]. The lack of volatility, non-flammability, and thermal stability, provide a good alternative as green solvents, which can reduce hazardous organic wastes. The significant characteristic of ionic liquids is the possibility of designing the structure and, as a result, the creation of the unique physicochemical properties [34; 35]. Accordingly, ILs have a wide range of industrial applications. Due to their modulated polarity, acidity, and viscosity, ILs are interesting alternatives for conventional organic solvents [36], hazardous catalysts [37], and absorbent and extraction agents [38; 39]. Ionic liquids have also been reported as effective enzyme stabilizers [31; 40]. In respect to the literature, many methods of enzyme stabilization via ILs were found, which can be classified in two groups. The first group of methods includes techniques where proteins are modified [30]. Enzyme immobilization is the most common method of enzyme stabilization, it includes sol-gel encapsulation, the cross-linking enzyme aggregates (CLEAs), and the protein attachment to the solid support [31; 41]. Sol-gel encapsulation is an irreversible method, where enzymes, via noncovalent interactions, are closed in a support. Encapsulated _Candida rugosa_ lipase showed great activities in esterification and hydrolysis reactions. IL was added to the reaction mixture during the immobilization procedure [42]. CLEAs technique is based on the creation of aggregates of enzymes, with the use of glutaraldehyde, which reacts with amino groups in the protein structure. Enzymes are cross-linked in the aqueous solutions with some additives, e.g., ILs. _Burkholderia cepacia_ lipase, as CLEA, presented higher catalytic activity than in the native form [43]. The last technique--binding to the solid matrix, via physical adsorption, in the presence of ILs--is known as a supported ionic liquid phases (SILPs) method, and will be described in the next section. Besides the enzyme immobilization technique, there are also methods such as the modification of enzymes with polyethylene glycol (PEG) technique, and propanol-rinsed enzyme preparation (EPRP), but in these cases, ionic liquid is usually used as an additive or solvent [31]. The second category of methods increasing the activity and stability of the enzymes, is environment modification. One of these techniques is the water-in-IL microemulsion (w/IL), which can be described as water droplets, with enzymes dispersed in non-polar ILs. In addition, microemulsion can be stabilized by surfactants, in order to increase the efficiency of emulsifying and protect the protein from denaturation. For example, microemulsion w/IL was tested for _Burkholderia cepacia_ lipase in the ester hydrolysis reaction. The catalytic activity of BCL was higher in the w/IL microemulsion than in the w/isooctane microemulsion [44]. Coating proteins with ILs is another method of increasing enzyme stability. Novozyme 435 was coated with ionic liquid and showed higher activity in the synthesis of citronelly esters than without IL [45]. The modification of the enzyme's environment can also be assigned the designing of ILs structure, for increasing the enzyme's activity and stability [30; 46; 47]. As mentioned before, the structure of ionic liquid can be designed by selecting a proper cation and anion. Different combinations of ions result in the tailoring of the properties of IL, and interactions between IL and enzymes. Main properties of ionic liquid that are important for enzyme stabilization are polarity, anion nucleophilicity, alkyl chain length in the cation, hydrogen bonds, and viscosity [30; 31; 46; 47]. The folded three-dimensional structure of the enzymes can be easily destroyed in non-aqueous media.