## 4 Discussion  
L-Fucl-encoding genes are distributed throughout a wide range of L-fucose-metabolizing bacteria, many of which are found in the phyla _Firmicutes_, _Bacteroides_, _Proteobacteria_, and _Actinobacteria_ (Figure 5). L-Fucl enzymes originate not only from typical mesophilic bacteria, such as _E. coli_[1,23], but also from unique extremophiles [3,5]. Among extremophiles, _H. orenii_ represents one of the rarest cases owing to its dual ability to tolerate both high temperature and salinity [18,19]. Organisms that grow optimally at temperatures higher than 50 \({}^{\circ}\)C and 1.5 M NaCl are defined as thermophiles and halophiles, respectively. In this regard, _H. orenii_ that shows optimal growth at 60 \({}^{\circ}\)C and 1.7 M NaCl is classified as a thermohalophile [18,19]. As enzymes derived from such organisms often have distinct features (i.e., high thermostability and halostability) that are beneficial for biotechnological applications [3,5], it is of key interest to investigate their biochemical properties. Halophiles have evolved to adapt to hypersaline environments by equilibrating intracellular osmotic pressure with their external conditions based on two strategies [24]. One is the \"salt-in strategy\" that increases the intracellular ion strength up to 3-6 M in response to the external environment; hence, high concentrations of ions like Na+, K+, and Cl-, are accumulated in the cytoplasm [27]. Generally, proteins experience disruption of electrostatic interactions at intracellular molar concentrations of salt, losing their functional conformation [24,28]. In contrast, proteins from organisms utilizing the salt-in strategy are able to maintain their active conformation in the hypersaline cytoplasm. This may be due to the presence of excessive acidic amino acids and negatively charged acidic surfaces, which allow proteins to be sufficiently solvated in limited conditions of water availability by forming electrostatic interactions between the residues and salts [24,26,29]. Consequently, the presence of excessive acidic amino acids (e.g., glutamate and aspartate) over basic amino acids (e.g., lysine and arginine) has been regarded as a unique feature of such halophilic proteins [25,30]. In contrast to the salt-in strategy that increases the intracellular inorganic ions, the \"salt-out strategy\" exports salts back while accumulating organic solutes in the cytoplasm by synthesizing or acquiring them from the environment to counterbalance the intracellular osmotic pressure [31]. These organic solutes are represented by glycine betaine and ectoine, which are compatible with protein functionality and cellular metabolism [32]. Proteins from halophilic organisms that rely on salt-out strategies do not display acidic signatures. From the NaCl and temperature profiles in this study, _Ho_Fucl was revealed as a poly-extremozyme, evidenced by its thermophilic and halophilic properties in synthesizing L-fucose, with optimal activity at 50-60 \({}^{\circ}\)C and 0.5-1 M NaCl (Figures 3 and 4a). This correlates well with the growth behavior of the original organism, _H. orenii_, which has adapted to multi-extreme environments (i.e., hot and salty conditions) [18,19]. It is noteworthy that despite its maximized activity in the presence of a molar salt concentration, _Ho_Fucl retained considerable activity even with no or low salt concentrations. Keeping in mind that the halophilic proteins from organisms employing the salt-in strategy tend to be deactivated by low salt concentrations caused by the repulsive electrostatic forces among the excessive acidic amino