# Exploring Metalloprotease from _Dunaliella_ sp.: Production, Regulation, and Structural Insight
## 3 Results and Discussion
### Protein Production Optimization  
Focusing on optimizing protein production is crucial because the global demand for protein ingredients is steadily increasing driven by factors such as population growth, rising incomes, and urbanization. Traditionally, animal-derived products (such as dairy, eggs, and meat) have met the majority of human dietary protein needs. However, with the global population projected to reach 9.6 billion by 2050, identifying sustainable alternative protein sources to supplement animal proteins is essential. Moreover, studies on the genus _Dunaliella_ have highlighted its remarkable ability to accumulate valuable \(\beta\)-carotene (up to 14% of a cell's dry weight) [78]. Consequently, it is important to focus on the study of proteins. The cultivation of microalgae and their biochemical composition are closely linked to environmental factors (temperature, light intensity, amount and type of nutrients, CO\({}_{2}\) levels, and pH) [79; 80]. Among these factors, salinity appears to have a powerful influence on the growth and protein production of _Dunaliella salina_, known for its remarkable ability to thrive in highly saline environments, like salt lakes and coastal lagoons [81]. Response surface methodology (RSM) and a Box-Behnken design were successfully applied to optimize the four chosen variables by analyzing their impact on soluble protein production by _Dunaliella_ sp. For this purpose, twenty-seven experiments were conducted. The concentration of soluble proteins ranged from 4.10% to 14.45% (_w/w_) (Supplementary Data). The experimental data were fitted to a second-degree polynomial equation (Equation (3)) that included only the significant factors. \[ \begin{array}{l}\text{Proteins content }(\%,\ w/w)=9.77392+2.46564\ \times\ \text{X}_{1}-0.66642\ \times\ \text{X}_{3}-2.23708\ \times\ \text{X}_{1}^{2}+1.23719\ \times\ \text{X}_{2}^{2}\\ +1.36795\ \times\ \text{X}_{1}\ \times\ \text{X}_{2}-1.34743\ \times\ \text{X}_{1}\ \times\ \text{X}_{3}+2.71626\ \times\ \text{X}_{2}\ \times\ \text{X}_{4}\end{array}  \tag{3}\] \begin{table}
\begin{tabular}{l l c c c c c c c c c} \hline \multirow{2}{*}{
\begin{tabular}{c} **Microalgae** \\ **Species** \\ \end{tabular} } & \multirow{2}{*}{**Medium**} & \multicolumn{8}{c}{**Composition (\% Dry Weight)**} & \multirow{2}{*}{**Reference**} \\ \cline{3-3} \cline{5-10}  & & **Chlorophyll a** & & **Chlorophyll b** & & **Carotenoid** & **Ash** & **Moisture** & **Protein** & **Carohydrate** & **Lipid** \\ \hline _Dunaliella salina_ & F/2 & & & 0.493 \(\pm\) & 32.937 & 0.824 & 11.823 \(\pm\) 0.004 & 10.585 \(\pm\) & 32.930 \(\pm\) \\  & medium & 1.199 \(\pm\) 0.056 & 0.399 \(\pm\) 0.022 & 0.026 & \(\pm\) 1.56 & \(\pm\) 0.04 & (only soluble) & 0.832 & 3.048 & This study \\ \hline _Dunaliella salina_ & Wahae & & & & & & & & & & \\  & medium & - & - & - & - & - & 24.1 & - & 19.5 & [67] \\ \hline _Dunaliella salina_ & Commercial & & & & & & & & & & \\  & powder & - & - & 3.46 & 48.74 & 6.63 & 10.03 & 25.31 & 3.49 & [68; 69] \\ \hline _Dunaliella salina_ & - & - & - & 1.7 & 43.3 & 2.1 & 19.5 & 24.5 & 7.2 & [70] \\ \hline _Dunaliella salina_ & - & - & - & 4.5 & - & - & 36.4 & 33.0 & 7.8 & [71] \\ \hline _Dunaliella salina_ & - & - & - & - & - & 57 & 32 & 6 & [72] \\ \hline _Dunaliella salina_ & - & - & - & - & - & 15.89 & 25.67 & 40.21 & 18.02 & [73] \\ \hline _Dunaliella salina_ & Commercial & & & & & & & & & \\  & powder & - & - & - & 0.03 & 1.26 & 8.46 & 85.58 & - & [74] \\ \hline _Dunaliella salina_ & - & - & - & 1.70 & 20.30 & 5.1 & 19.50 & 24.20 & 7.20 & [70] \\ \hline _Dunaliella salina_ & Commercial & & & & & & & & & & \\  & powder & - & - & 3.46 & 48.74 & 6.63 & 10.03 & 25.31 & 3.49 & [75] \\ \hline _Dunaliella salina_ & - & - & - & - & - & - & 57 & 32 & 6 & [76; 77] \\ \hline \end{tabular}
\end{table} 
Table 2: Biochemical composition of _D. salina_ microalgal biomass. where the variables X\({}_{1}\), X\({}_{2}\), X\({}_{3}\), and X\({}_{4}\) correspond to the temperature, luminosity, NaHCO\({}_{3}\) concentration, and NaCl concentration, respectively.