3.3.3. Cross-Stabilities of Extremophiles

Many examples are reported in the recent literature that highlight the enhanced and cross-stabilities of extremophilic enzymes. Thermostability can be accompanied with pH, salts, metals, or solvent resistance [108,109]. Key enzymatic reaction may benefit from these cross-stabilities. Transformation of cellulose into biofuels is a promising eco-friendly process. However, it requires the enzymes involved in the process to be thermostable, halostable and organic solvent-stable. A cellulase presenting all these characteristics was identified through metagenomic from a deep sea foil reservoir which could be a valuable candidate [110]. Hydrogen is considered as an energy vector for a low carbon economy. Its production and conversion by enzymes should be promoted through operation on a wide range of temperatures. The hyperthermophilic hydrogenase (HASE) from *Aquifex aeolicus* was demonstrated not only to be able to operate at high temperatures for H<sup>2</sup> oxidation but was also much more stable at room temperature than the HASE from *Ralstonia eutropha*, a mesophilic homologue [111] (Figure 7). The structure of this hyperthermostable enzyme reveals more salt-bridges compared to mesophilic HASE, that contribute to its thermostability [112].

**Figure 7.** *A. aeolicus* and *R. eutropha* are two homologous NiFe HASEs with different thermostability. (**Left**) Structural alignment of mesophilic *R. eutropha* (blue) and hyperthermophilic *A. aeolicus* (green) HASEs, (**right**) remaining activity of the two HASEs after 360 s incubation at increasing temperatures. Adapted with permission from [111].

## **4. Strategies for Enzyme Stabilization in the Immobilized State**

The discussion above has emphasized that the less flexible enzymes are, the most stable they are. In line with this assessment, it is quite straightforward that the immobilization of an enzyme on a support will decrease the movement of the protein, tightening the structure by single or multipoint binding and hence will increase its stability. The extent of the stabilization will depend on the enzyme, the nature of the support and the mode of enzyme attachment to the support (simple adsorption, covalent attachment, entrapment in pores, etc.) This means that the support must be amenable to surface modification for further enzyme attachment. The material acting as a support must also be biocompatible, stable, and able to host high enzyme loadings. A delicate balance between stability and activity of the enzyme will be engaged, with an additional advantage of immobilization being that the support itself can help in the stabilization by consumption of inhibitors or by providing buffer properties as examples [47]. In the following, we will especially discuss immobilization strategies that can enhance enzyme stability. We will not report an exhaustive list of the coatings that can simply prevent enzyme leaching, but we will emphasize the role of the support structuration on the enzyme conformation, hence its stability, although both concepts are closely linked. We will extend the discussion to modes of immobilization that do not require any carrier, like cross-linking of enzyme aggregates (Figure 8).

**Figure 8.** Strategies for enzyme stabilization in the immobilized state.

*4.1. Some Fundamentals on Enzyme Immobilization on a Solid Support*

4.1.1. Various Types of Interactions

The key point to develop immobilization strategies able to enhance protein stability is to understand how the interactions between the support and the immobilized enzyme may modulate the enzyme internal motion and conformation. The specific structural features of enzymes make their immobilization process very different from rigid particles that simply attach to or detach from a support with certain adsorption and desorption probabilities. The interactions that drive protein adsorption range from high energy covalent bonds (disulfide bridges 320 kJ/mol) to electrostatic interactions (35–90 kJ/mol), hydrogen bonds (8–40 kJ/mol), van der Waals interactions and hydrophobic interactions (4–12 kJ/mol) [113]. Using the concept of "hard" and "soft" proteins based on structural rigidity scale, it can be categorized that adsorption of "hard" proteins will be driven by electrostatic interactions, while "soft" proteins will be able to adsorb either on hydrophobic or hydrophilic surfaces [114].
