# Exploring Metalloprotease from _Dunaliella_ sp.: Production, Regulation, and Structural Insight
## 3 Results and Discussion
### Protein Production Optimization
[92] and [PERSON] and [PERSON] [93] observed a higher protein content induced by increased light intensity in _Chlorella vulgaris_ (30 umol photons/m\({}^{2}\)/s compared to 70 and 150 umol photons/m\({}^{2}\)/s) and _Auxenochlorella pyrenoidosis_ (formerly _Chlorella pyrenoidosis_) (Chlorophyta) (250 umol photons/m\({}^{2}\)/s compared with 100 umol photons/m\({}^{2}\)/s). Several studies have highlighted the importance of photoperiod and investigated its effect on cell growth and metabolite production (lipids, proteins, pigments) [94]. Overnight reductions were noted in the biomass concentration and carbohydrate content of _Auxenochlorella pyrenoidosis_, alongside an increase in protein content. These changes indicate that in the absence of light, stored carbohydrates in the cells are metabolized for energy. This energy is partly used for cell maintenance and partly directed toward protein synthesis. These fluctuations peak during the exponential growth phase, diminish as the culture ages, and are influenced by both diurnal and nocturnal culture conditions [93]. The night biomass loss is greater in cultures grown under high light intensity. In the dark, when light intensity is insufficient to support cell growth, respiration occurs. Temperature affects both the growth of microalgae and the production of metabolites, especially proteins. Unfortunately, there is a dearth of literature addressing the influence of temperature on protein production. This study tested protein production at temperatures of 16 \({}^{\circ}\)C, 22 \({}^{\circ}\)C, and 28 \({}^{\circ}\)C. It was found that 28 \({}^{\circ}\)C was the optimal temperature for maximizing protein production. Another study indicated that _Chorella vulgaris_ exhibited better protein and polysaccharide productions at a temperature of 25 \({}^{\circ}\)C compared with 15 \({}^{\circ}\)C and 20 \({}^{\circ}\)C [95]. However, some studies have suggested that a temperature range of 22 to 25 \({}^{\circ}\)C is the most suitable for the growth of _D. salina_[96, 97, 98]. [PERSON] and [PERSON] [99] reported that the optimal growth temperature for _Dunaliella_ sp. is about 32 \({}^{\circ}\)C, with a broad range of effective growth temperatures spanning from 25 \({}^{\circ}\)C to 35 \({}^{\circ}\)C. The response of microalgae protein content to temperature varies among species, showing no consistent trend. Therefore, identifying the temperature response specific to each species is crucial for optimizing protein production rates. One important factor tested for its influence in this experimental design is bicarbonate. _Dunaliella salina_ strains are strictly photoautotrophic and capable of CO\({}_{2}\) uptake. They convert HCO\({}_{3}\)- to CO\({}_{2}\) using extracellular carbonic anhydrase. In this study, concentrations of 0.5, 1, and 1.5 g/L of NaHCO\({}_{3}\) were used. The results found in this study proved that protein synthesis by _D. salina_ was enhanced with increasing NaHCO\({}_{3}\) concentration, reaching maximum production at 1.5 g/L (equivalent to 17.8 mM). A study was carried  
Figure 2: Response surface (3D) plots depicting the combined effects of temperature and luminosity (**A**,**G**), temperature and NaHCO\({}_{3}\) concentration (**B**,**H**), temperature and NaCl concentration (**C**,**I**), luminosity and NaHCO\({}_{3}\) concentration (**D**,**J**), luminosity and NaCl concentration (**E**,**K**), NaHCO\({}_{3}\) concentration and NaCl concentration (**F**,**L**). The left panel shows protein accumulation (%), and the right panel shows specific proteolytic activity (U/mg of proteins) of _Dunaliella salina_. out on three strains of _Dunaliella salina_ to better understand the influence of several factors, including bicarbonate. The results indicated that a high concentration of NaHCO\({}_{3}\) (1.5 g/L) resulted in better growth compared with a low concentration (0.5 g/L) [100].