# Exploring Metalloprotease from _Dunaliella_ sp.: Production, Regulation, and Structural Insight
## 3 Results and Discussion
### Protein Production Optimization
Protein synthesis in _Dunaliella salina_ typically begins with a brief latent phase of approximately three days, after which it accelerates during the exponential growth phase under both standard and optimized conditions. Notably, the optimized conditions led to a substantial increase in protein content, elevating it from 11.98% \(\pm\) 0.26 to 18.39% \(\pm\) 0.1 (Figure 3A). [PERSON] and [PERSON] [82] examined the biochemical composition of _Chlorella vulgaris_ and _Tetradesmus obliquus_ (formerly _Scenedesmus obliquus_) (Chlorophyta) and two Cyanobacteria, namely, _Anacystis nidulans_ and _Microystis aeruginosa_, across growth phases. They found that all exhibited peaks in protein content during the exponential phase, followed by a significant decline in the stationary segment. These findings align with the pattern observed in _Dunaliella_ sp. during the present study. Additionally, a similar pattern was also found in the microalgae _Isochrysis galbana_ (Haptophyta), grown under varying nitrogen sources and temperatures [83; 84]. It has been reported that the fluctuating protein content, increasing during the exponential phase and decreasing during the stationary period, is primarily influenced by nitrogen availability. Insufficient nitrogen during the stationary phase leads to a reduction in protein synthesis [82; 83; 85; 86]. At this stage, the metabolism shifts towards directing excess carbon obtained from the photosynthesis process into storage compounds instead of protein [87; 88]. This suggests that microalgal biomass grown under insufficient nitrogen concentrations exhibits high protein levels [84]. Similarly, protein accumulation in _Chlorella vulgaris_ (Chlorophyta) and _Microchloropsis gaditana_ (formerly _Nannochloropsis gaditana_) (Eustigmatophyceaeae) was highest during exponential growth but decreased in _Chlorella vulgaris_ four days after entering the stationary phase [89]. These findings reaffirm the significant impact of the growth phase on the final protein content of the biomass. Overall, these findings emphasize that the exponential phase offers peak protein productivity, making it an optimal harvest point for generating protein-rich biomass. When cultivated at 135 g/L NaCl salinity (equivalent to 2.31 M), _D. salina_ showed the highest intracellular protein production. These results are consistent with the work of [85], who showed that a culture of _D. salina_ at a salinity of 2 M yielded the best results, achieving the highest biomass production and productivity, as well as protein content. The ideal cultivation salinities for a Chinese _Dunaliella_ sp. are 0.5 M and 0.9 M. At a salinity of 0.9 M, the microalgae achieved its peak protein accumulation, approximately 50%, during the stationary phase [90]. _Dunaliella tertioleta_, in contrast, has demonstrated peak biomass production at a NaCl salinity of 0.9 M [91]. Nevertheless, given that optimal cultivation conditions for microalgae vary significantly by strain, each one must undergo testing to determine its ideal performance capabilities [85]. Regarding the protein biosynthesis in this study, _Dunaliella_ sp. cultivated at a salinity of 135 g/L exceeded the salinities of 27 g/L and 81 g/L for protein accumulation. Light intensity stands as the primary fundamental requirement influencing the photoautotrophic growth of microalgal cells. Their cellular composition, particularly their pigment profile, depends on the availability of light energy. In this study, three light intensities (100, 250, and 400 umol photons/m\({}^{2}\)/s) were investigated. [PERSON] et al. [78] tried a two-phase cultivation strategy to enhance the concurrent yield of high-quality proteins and carotenoids from _D. salina_ CCAP 19/18. Transitioning from the first phase, where microalgal cells were exposed to a light intensity of 70 umol photons/m\({}^{2}\)/s, to a second culture phase, with increased light intensity at 110 umol photons/m\({}^{2}\)/s, promoted cell growth, despite nitrogen or phosphorus being limiting factors. [PERSON] et al.