*3.3. Screening of the Biodiversity and Outstanding Properties of Extremophiles*

Screening in the biodiversity for "exotic" enzymes such as extremophiles presenting unusual and/or outstanding properties is an attractive strategy to be considered. Although this research topic is increasingly growing, there is no doubt that Nature still retains many secrets that would allow new opportunities for stable biocatalysis. However, the harsh conditions required for the growth of extremophiles very often limit their laboratory studies. Extremophiles have evolved to survive in ecological niches presenting extreme temperatures (thermophiles and psychrophiles living at high (>80 ◦C) and low (<20 ◦C) temperatures, respectively), extreme pH (acidophiles and alkalophiles), high pressures (barophiles), high salt concentrations (halophiles), or in the presence of heavy metals (metallophiles)). These extreme environments are found in deep-sea hydrothermal vents, hot springs, volcanic areas, or mine drainage [88–90]. Ancestral microorganisms are another source of stable enzymes because distant ancestors of current organisms were thermophiles and would be composed of proteins that are more thermostable than their current homologues [91–93].

Extremophilic enzymes present specific structural characteristics that afford them to resist in extreme conditions. Interestingly, the same structural features often induce enhanced stability of such enzymes at normal conditions or in the presence of non-aqueous solvents. Moreover, "extremostable" enzymes, thermostable, halostable, acidostable, etc. isolated from extreme environments or obtained by protein engineering can support large number of mutations due to their robustness, leading to eventually even more stable enzymes.

#### 3.3.1. Thermophiles

Thermostability refers to two different concepts: thermotolerance, which is the transient ability to maintain activity at high T◦ , and thermostability, which is the ability to resist irreversible inactivation at high T◦ [94]. In addition to the ability to resist to high temperatures, thermostable enzymes usually also present higher resistance to chemical denaturants and extreme pHs. Such cross-adaptations are highly interesting to get stable enzymes in many different extreme conditions. Furthermore, due to their stability at elevated temperatures, enzymatic reactions are faster and less susceptible to microbial contaminations. Many different structural characteristics have been demonstrated to be

involved in thermostability. Compared to mesophiles, protein packing, a high number of hydrophobic residues, increased helical fold content, a high number of disulfide bonds, density of internal hydrogen bonds and salt bridges, and distribution of charged residues on the surface are some of the features often shared by thermostable enzymes [95–97]. Proportion of certain amino acids is also significantly different between extremophiles and mesophiles. For instance, in thermostable enzymes, lysines are replaced by arginines; asparagine/glutamine content is lower while proline content is higher [22,98–100]. The most widespread explanation is that these structural features contribute to reducing the flexibility of the enzyme and to allowing optimal conformation at higher temperatures than mesophiles, with no denaturation [101]. MD simulations confirmed that hydrophobic packing and electrostatic interaction network provided by salt bridges explain enzyme thermostability [102]. Dimerization can also be a key factor for enhanced thermostability [103].
