# Exploring Metalloprotease from _Dunaliella_ sp.: Production, Regulation, and Structural Insight
## 2 Materials and Methods  
### Sampling Site, Microalgae Isolation Process, and Maintenance of Monolgal Culture  
Sewaater samples were collected from Chott El Jerid, a salt flat between the Kebili and Tozeur governorates in the southwest of Tunisia (33\({}^{\circ}\)58\({}^{\prime}\)38.6\({}^{\prime\prime}\) N 8\({}^{\circ}\)20\({}^{\prime}\)50.3\({}^{\prime\prime}\) E). Samples were collected and subsequently stored at 4 \({}^{\circ}\)C in sterile glass test tubes. Later, they were subjected to filtration steps as described by [PERSON] et al. [37]. The filtered seawater was then immersed in 50 mL of F/2 medium composed of (per liter) NaCl (27 g), MgSO4\({}^{\prime}\)7H2O (6.6 g), MgCl2\({}_{\cdot}\)6H2O (5.6 g), CaCl2\({}_{\cdot}\)2H2O (1.55 g), KH2 PO4 (70 mg), NaHCO3 (0.04 g), NaHCO2 PO4 (5 mg), KNO3 (1 g), NaNO3 (75 mg), 0.5 mL of a vitamin solution (1 mg/L biotin, 1 mg/L vitamin B12, and 0.2 g/L vitamin B1), and 1 mL of a trace metal solution (6.3 mg/L Na2 MoO4\({}_{\cdot}\)2H2O, 3.15 g/L FeCl3\({}_{\cdot}\)6H2O, 4.36 g/L Na2 EDTA\({}_{\cdot}\)2H2O, 9.8 mg/L Cu5O4\({}_{\cdot}\)5H2O, 180 mg/L MnCl2\({}_{\cdot}\)4H2O, 0.18 g/L MnCl2\({}_{\cdot}\)4H2O, 0.022 g/L ZnSO4\({}_{\cdot}\)7H2O, and 0.01 g/L CoCl2\({}_{\cdot}\)6H2O) [38]. All chemicals and reagents were obtained from Sigma-Aldrich (St. Quentin Fallavier, France). To achieve a pH of 7.2 in the F/2 medium, 20 mL of Bis-TRIS buffer was incorporated. The culture medium was then sterilized via autoclaving (Raypa(tm) AE-Dry Autolave, Barcelona, Spain) for 20 min at 121 \({}^{\circ}\)C prior to the addition of vitamins. Samples were maintained at room temperature (25 \({}^{\circ}\)C) under continuous light conditions (100 \(\mu\)mol photon/m\({}^{2}\)/s) with stirring at 130 rpm. These samples were subject to successive decimal dilutions (10\({}^{-1}\)-10\({}^{-10}\)) to reduce cell concentration until reaching the lowest one, followed by streaking into Petri plates measuring 10 \(\times\) 1.5 cm and containing F/2 medium, solidified with 1.5% (\(w/v\)) agar. Each dilution (0.1 mL) was inoculated into the plates. Petri plates were then kept at room temperature (23 \({}^{\circ}\)C) at standard conditions: irradiance of 100 \(\mu\)mol photons/m\({}^{2}\)/s and photoperiod of 16 h light and 8 h darkness. Approximately 10 days post-inoculation, small colonies of microalgae were observed and subcultured every 15 days until unialgal cultures were obtained. In fact, only the green colonies were subjected to a series of subsequent dilutions, and eight medium renewals finally resulted in the growth of single colonies of microalgae using antibiotic and antifungal treatment (comprising final concentrations of 100 \(\mu\)g/mL of ampicillin, 50 \(\mu\)g/mL of kanamycin, 50 \(\mu\)g/mL of streptomycin, 50 \(\mu\)g/mL of chloramphenicol, and 100 \(\mu\)g/mL cycloheximide) [39]. The purity of the microalgal colony was confirmed through periodic monitoring using an optical microscope and streaking on a solid nutrient agar medium [40]. The isolated green microalga was grown in 500 mL glass Erlenmeyer flasks containing F/2 medium. The flasks were incubated under the following initial culture conditions: 23 \(\pm\) 2 \({}^{\circ}\)C, agitated manually twice a day under the light intensity of 100 \(\mu\)mol photons/m\({}^{2}\)/s with a photoperiod of 16 h light/8 h dark, to obtain a final cell concentration of around 7 \(\times\) 10\({}^{5}\) cell/mL. This culture was used as a standard inoculum for all cultivations in various culture media.