*3.1. Addition of Stabilizers in the Medium*

#### 3.1.1. Chemical Stabilizer Addition

It has been known for long time that addition of polyols and polysaccharides such as trehalose, glycerol, dextran, etc. helps stabilizing enzymes [29–32]. Protective effect of polyols was reported in many recent studies in different experimental situations and exposition to stresses (high pressure or high temperature, presence of non-aqueous solvents) [33–37].

Generally speaking, polyols stabilize proteins either in the dried state by serving as water substitutes and preventing dehydration through hydrogen bonding, or in solution by altering protein-solvent interactions [38]. Several mechanisms are discussed to account for the effect of such chemical additives on the protein stabilization [23]. Hydrophilic polyols tend to strengthen hydrophobic interactions among nonpolar amino acid residues resulting in a more compact and spherical enzyme form with smaller surface area, preventing protein dynamics and enhancing stability. Compressibility of proteins, a crucial thermodynamic parameter that determines flexibility, is affected by polyol addition [39]. One accepted mechanism is based on the different sizes between water and stabilizing molecules that preferentially exclude the latter from the protein surface. The preferential hydration of proteins causes an unfavorable free energy change that the proteins tend to minimize by favoring the more compact state over the structurally expanded state. Protective effect is also explained in terms of influence of additives on water activity that results in an increase of structural organization of water molecules contributing to the conservation of low energy interactions favoring native protein conformation [25].

Surfactants, maltodextrine, sodium azide, or special buffers have also been used as additives to maintain the native structure of proteins through purification steps [25]. Dimethyl sulfoxide (DMSO) acts as a stabilizer because it is preferentially excluded from the protein surface [22,40,41]. Addition of DMSO but also of glycerol or ethylene glycol serve as cryoprotectants preventing protein solutions from freezing at −20 ◦C and allowing multiple use of a unique sample without freeze-thaw cycles [38,42]. Adding polymers in solution such as poly(ethylene glycol) (PEG), alginate, or chitosan maintains a hydration shell around the protein according to the exclusion mechanism [38,43–45]. Addition of polymers also prevents protein aggregation by modifying protein-protein interactions and increasing medium viscosity, thus decreasing enzyme motion. Macromolecular crowding, which is the natural environment of enzymes in vivo, can explain the stabilizing effect of some additives [46–49]. It can be mimicked in vitro through the addition of high concentrations of macromolecules such as dextran or PEG [50] and would favor the folded state of proteins and compact conformation through the excluded volume effect [51–55]. However, the stabilizing effect of in vitro crowding would not be universal as the properties of all macromolecules in vivo are finely regulated under different physiological conditions [56]. Aggregation under crowded conditions can be enhanced because the activity of water is decreased, and the refolding rate of proteins as a consequence of increased viscosity is also decreased.
