**2. Intrinsic (in)Stability of Enzymes**

The metabolisms essential for life are catalyzed by enzymes. Over time, these proteins may have undergone different changes resulting from mutations, which cause enzymes to evolve to function in a particular cellular environment and will be very sensitive to exogenous conditions. In addition, in the cell, enzymes can be subjected to various stresses, which can lead to their denaturation or the formation of aggregates. Thanks to a perfectly designed proteolysis machine, new fresh proteins are produced to replace the deactivated proteins. These two fundamental concepts mean that using enzymes outside a cell is expected to induce some loss of activity and eventually progressive denaturation, which can be irreversible. The will to exploit enzymes for other works than predicted by evolution, although highly attractive, imposes finding out strategies to preserve activity within durations compatible with fundamental laboratory studies and with applications [3]. Before reaching that objective, the prerequisite is to know which factors affect enzyme activity and stability. A subtle balance between stability and flexibility is required to maintain activity, so that stability can be the price to pay against activity. Each enzyme is composed of a linear chain of amino acids that fold to produce a unique three-dimensional structure which confers specific and unique enzymatic properties. The overall structure of an enzyme is described according to fours levels which contribute to its specific function and stability (Figure 1). The secondary structure refers to the arrangement of the polypeptide chain (the primary structure) as random coil, α-helix, β-sheet and turn stabilized by hydrogen bonds between amino acid residues. While the primary structure is stable thanks to the strength of the peptide bonds which are unlikely to be broken upon changes in the environmental conditions, the secondary structure can be altered even during protein storage. The tertiary structure is the three-dimensional arrangement of the amino acid residues, giving the overall conformation of the polypeptidic chain. Hydrophobic interactions between nonpolar chains as well as disulfide bonds and ionic interactions between charged residues are involved in the stabilization of the tertiary structure. To fulfill the requirement of enzyme localization in the cell, hydrophobic and hydrophilic groups are arranged within the protein. For soluble proteins, hydrophobic side groups tend to be hidden in the protein core while hydrophilic groups are exposed to the surrounding environment. These amino

acid residues on the surface of the enzyme are more likely to be sensitive to the external environment than the core amino acids.

**Figure 1.** The four levels of protein structures determine the enzyme function.

β α Denaturation occurs upon unfolding of the tertiary structure to a disordered polypeptide where residues are no longer arranged for functional or structural stabilizing interactions [22]. This process may be reversible. Irreversible loss of activity may also occur upon external stresses. This leads to two different concepts to evaluate in vitro stability of a protein. The thermodynamic stability refers to the ability for a protein to unfold/refold after being subjected to stresses (elevated temperature, extreme pH, or high organic solvent concentration). Methods to evaluate this stability range from scanning calorimetry to circular dichroism (CD) and fluorimetry. Kinetic stability represents the duration of enzyme activity before irreversible denaturation and is measured by activity assays. Actually, the native active state of an enzyme is in equilibrium with the partially denatured, enzymatically inactive state, and the folded/unfolded transition involves mainly intramolecular hydrogen bonds and hydrophobic interactions. This scheme is most certainly more complicated since it was shown that a protein can oscillate between many different folded/unfolded configurations. Each oscillation is governed by the second thermodynamic law, the entropy decreasing as the protein folds, and hydrogen bond formation contributing to the enthalpy. Each strategy that tends to increase rigidity in the enzyme architecture decreases entropy and enhances stability [23]. Thermodynamic instability of enzymes can be partly attributed to the lack of rigidity within the tertiary structure, caused by flexible random coils in contrast to stable β-sheets [24]. In addition to higher rigidity and compact packing, the presence of α-helices with antiparallel arrangement contributes to stability. Water is the natural environment in which protein molecules do exist and operate, and water molecules play a key role in maintaining the entropic stability of the enzyme structure.

Any engineering towards enzyme structural rigidification must maintain the hydration shell and flexibility required for activity. Based on enzyme structural features, many different agents are potential enzyme "killers". When temperature increases, the enzyme tends to unfold in a cooperative process. Disulfide bridges can be broken by reducing agents, extreme pHs are going to affect hydrogen bonding and salt bridges, high salt concentration may aggregate the protein, while charged components may form bonds with charged amino acids modifying the tertiary structure. Organic solvents or detergents will alter hydrophobic interactions, and stability of enzymes in organic solvents will be

dependent on the ability of the solvent to strip the essential hydration layer from the protein surface [25,26]. Last but not least, mechanical stress or radiation may disrupt the delicate balance of forces that maintain protein structure [27]. Stabilizing the enzyme means preventing these changes.
