## 4 Discussion
\begin{table}
\begin{tabular}{c c c} \hline \hline  & **Specific Activity (U/mg)** & **Condition** \\ \hline _Ho_Fucl [this study] & 106.2 \(\pm\) 8.7 & 60 \({}^{\circ}\)C, pH 7, w/o NaCl \\  & 151.1 \(\pm\) 11.9 & 60 \({}^{\circ}\)C, pH 7, w/o5 M NaCl \\  _R_dFucl [2] & 64.0 \(\pm\) 3.4 & 40 \({}^{\circ}\)C, pH 7, w/o NaCl \\  _T_Fucl [3] & 199.8 \(\pm\) 0.3 & 70 \({}^{\circ}\)C, pH 7, w/o NaCl \\ \hline \hline \end{tabular} \({}^{\dagger}\)Various concentrations of L-fuculose (10 mM) were incubated with 7 ug _Ho_Fucl in 20 mM sodium phosphate (pH 7) buffer at 60 \({}^{\circ}\)C for 5 min in the presence of Mn2+. 2 Experimental data represent the means \(\pm\) standard deviations in at least three replicates.  
\end{table} 
Table 3: Comparison of the specific activities of L-Fucls against L-fuculose \({}^{1,2}\). acids [26], such a phenomenon with _Ho_Fucl provides evidence that \(H\). _orenii_ does not adopt the salt-in strategy. This is also consistent with the behavior of AmyA (an amylase), one of the few characterized enzymes from the same bacterium, which still exerts 40% activity without salt, while optimal activity is observed at 2 M NaCl [33]. Furthermore, _Ho_Fucl is active in a broad range of salinities from 0 to 5 M, which is also consistent with the results of AmyA [33]. Such behaviors of these enzymes could be associated with the extreme seasonal fluctuations of salt in the habitat of \(H\). _orenii_ (i.e., Tunisian salt lake of the Sahara desert) [18; 19]. Since organisms dependent on the salt-out strategy are generally known to rapidly adjust to fluctuating salinity, the broad activity of _Ho_Fucl in saline conditions suggests that \(H\). _orenii_ may adopt a salt-out strategy [19]. Our bioinformatics analyses, carried out to better understand the halophilic properties of _Ho_Fucl, showed that _Ho_Fucl does not strictly follow the general trend of halophilic proteins that have been adapted to high ionic strength (i.e., salt-in strategy). First, there were no pronounced acidic residues, which is an exclusive feature of general halophilic proteins, in _Ho_Fucl when the amino acid composition was analyzed (Table 2). Next, because the negative surface electrostatic potentials are characteristic of proteins from organisms that rely on salt-in adaptation [23; 34], surface electrostatic potentials were investigated by comparing the model structure of _Ho_Fucl with those of its non-halophilic counterparts with known structures, _Ec_Fucl and _R_dFucl (Figure S2 in Supplementary Materials). No distinctive differences in surface electrostatic potentials of the three \(\mathsf{L}\)-Fucl (model) structures were observed, which further proves that the behavior of _Ho_Fucl is distinct from those of general halophilic enzymes. AmyA from \(H\). _orenii_ also did not show an acidic signature in either the amino acid composition or on its structural surface [33]. Nevertheless, it is too early to conclude that \(H\). _orenii_ relies on a salt-out strategy instead of a salt-in strategy because of the lack of related evidence, such as the identification of organic solute-synthetic enzymes [18; 19]. Therefore, further studies are required to elucidate the halophilic mechanisms of this bacterium and its enzymes, such as _Ho_Fucl. It is noteworthy that proteins from several halophilic organisms belonging to the same order as \(H\). _orenii_ (Halanaerobiales) are also very halophilic in enzymatic functioning, but do not follow the general rule regarding the primary and tertiary structures of halozymes, similar to _Ho_Fucl in this study [35]. Interestingly, a thermophilic signature was clearly observed in the amino acid composition of _Ho_Fucl. Briefly, there was a reduced frequency of thermolabile amino acids, such as histidine, glutamine, and threonine, whose behaviors were quite similar to those of thermophilic _Tf_Fucl and proteins from general thermophiles. Further analysis based on the phylogenetic tree also showed that _Ho_Fucl is highly evolutionarily related to thermophilic enzymes (Figure 5).