3.1.2. Salt Effect

Salt addition affects protein stability according to a combination of binding effect, screening of protein surface electrostatic potential, and effect on protein/water interface. Salts may contribute to hydrophobic interaction strengthening in proteins, hence to stabilization, but salt binding to amino acids on the protein surface decreases the repulsion between proteins and induces aggregation. The salting out process, or salt-induced precipitation, depends on the structure of the protein and in particular on the population of hydrophilic amino acid residues. It also largely depends on the salt concentration. The effect of salts on protein stabilization has been tentatively explained on the basis of the Frank Hofmeister series [4,57–59] (Figure 3A). This series early ordered anions and cations composing salts according at first to their ability to precipitate lysozyme, and later to their ability to stabilize protein secondary and tertiary structure. They were respectively classified at that time as water-structure formers (kosmotropes) or water-structure breakers (chaotropes). Kosmotropes and chaotropes would be nowadays more understood as a characterization of the degree of hydration, and different models are developed to consider the various effects observed experimentally [60]. Ionic liquids (IL) would follow the same tendency as salts where kosmotropic anions and chaotropic cations stabilize the enzyme, while chaotropic anions and kosmotropic cations destabilize it [61]. While they can be considered as eco-friendly solvents, many proteins are inherently inactive in ILs, requiring the addition of water for activity recovery. This suggests ILs could affect the internal water shell [61,62].

Recent examples in the literature illustrate the effect of salts on enzyme stability, and most of them agree with the Hofmeister series. Changes in the secondary structure of two proteins with helical and beta structural arrangement, respectively, were followed by Fourier Transform Infra-Red spectroscopy (FTIR) in the presence of various salts. It was shown that the stabilization effect of the salt follows the Hofmeister series of ions, although some exceptions were observed with formation of intermolecular β-sheets typical of amorphous aggregates [62]. Glucose oxidase (GOx) stability was studied using microcalorimetry

in the presence of various salts [63]. At high salt concentrations (over 1 M), it was shown that the Hofmeister effect on the temperature of inactivation was determined by the ionspecific effect on the protein/water interface. Correlation between stability and activity of lysozyme in the presence of various salts from the Hofmeister series suggested a role of local stability/flexibility in enzyme activity [64]. The thermal stability of *Aspergillus terreus* glucose dehydrogenase (GDH) was substantially improved by kosmotropic anions, retaining more than 90% activity after 60 min of heat treatment at 60 ◦C. The stabilizing effect followed the Hofmeister series and was anion concentration-dependent and strongly related to the structural stabilization of the enzyme, which involved enzyme compaction [65] (Figure 3B). It was further shown that salts can stabilize proteins not only in vitro but also in vivo or intracellularly, the stabilization level correlating with the Hofmeister series of ions [66].

β

**Figure 3.** Salt effect on enzyme stability. (**A**) Hofmeister series and effect on protein properties. (**B**) Stabilization effect of kosmotropic anions and cations on *Aspergillus terreus* GDH. Residual activity after 1 h incubation at 50 ◦C as a function of added anions and cations. Reproduced with permission from [65].
