# Exploring Metalloprotease from _Dunaliella_ sp.: Production, Regulation, and Structural Insight
## 3 Results and Discussion
### Specific Proteolytic Activity Optimization
Cysteine proteases produced by _Clostridium histolytica_ and _Streptococcus_ spp., as well as protozoans like _Tetrahymena thermophila_, have demonstrated relatively low activities, around 5 U/mL [106; 107]. _Candidia buniensis_ has been reported to produce a notably high neutral protease activity of 573 U/mL [108]. _Mucor subtilisismus_ and _Bacillus licheniformis_ exhibited fibrinolytic activity of 22.53 U/mL and subtilisism activity of 2450 U/mL [109; 110], respectively. Protease \begin{table}
\begin{tabular}{l c c c} \hline \hline
**Factor** & **Regression Coefficient** & \(p\)**-Value** \\ \hline Constant & & 5.4636 & 0.000 \\ X\({}_{1}\) & & 0.8304 & 0.000 \\ X\({}_{2}\) & & \(-\)0.6898 & 0.000 \\ X\({}_{3}\) & & 1.2445 & 0.000 \\ X\({}_{4}\) & & \(-\)0.1360 & 0.283 \\ X\({}_{1}^{2}\) & & 0.4226 & 0.038 \\ X\({}_{2}^{2}\) & & 0.8218 & 0.001 \\ X\({}_{3}^{2}\) & & 0.3243 & 0.099 \\ X\({}_{4}^{2}\) & & \(-\)0.8443 & 0.001 \\ X\({}_{1}\)\(\times\) X\({}_{2}\) & & \(-\)0.6606 & 0.008 \\ X\({}_{1}\)\(\times\) X\({}_{3}\) & & 1.5993 & 0.000 \\ X\({}_{1}\)\(\times\) X\({}_{4}\) & & \(-\)2.2958 & 0.000 \\ X\({}_{2}\)\(\times\) X\({}_{3}\) & & \(-\)0.2791 & 0.207 \\ X\({}_{2}\)\(\times\) X\({}_{4}\) & & \(-\)0.9399 & 0.001 \\ X\({}_{3}\)\(\times\) X\({}_{4}\) & & 0.3687 & 0.104 \\ \hline Source of variation & Degree of freedom & Sum of square & Mean square & F-value & \(p\)-value \\ \hline Regression & 14 & 82.9160 & 5.9226 & 33.74 & 0.000 \\ Residual & 12 & 2.1062 & 0.1755 & \\ Lack of fit & 10 & 2.0775 & 0.2078 & 14.51 & 0.066 \\ Pure Error & 2 & 0.0286 & 0.0143 & \\ Total & 26 & 85.0222 & & \\ \hline R\({}^{2}\) = 97.52\% & & & \\ \hline \hline \end{tabular}
\end{table} 
Table 4: Specific proteolytic activity (U/mg of proteins): Regression coefficients and ANOVA assessment. production by _Arthrospina platensis_ was reported by [PERSON] et al. [111], who isolated, purified, and partially characterized a novel serine protease involved in the selective proteolysis of phycobiliproteins, and by [PERSON] et al. [112], who purified a novel arginine-specific protease that hydrolyzed gelatin and fibrin but not phycocyanin. High protease activity (21.7 U/mL) produced by _Chlorella vulgaris_ UTEX 1803 was obtained under optimized conditions of 1.1% glycerol and 0.9% corn steep liquor, demonstrating the potential viability of using corn steep liquor to produce fibrinolytic enzymes from this microalga [113]. [PERSON] et al. [114] reported that cultivating _D. tertioleta_ UTEX LB999 under mixotrophic conditions with 0.5% corn steep liquor resulted in the highest fibrinolytic activity (102 \(\pm\) 1 U/mL) of the extract obtained through homogenization. The enzyme extracted from the microalgal biomass was subsequently purified five-fold using salting out and ion-exchange chromatography, resulting in a 20% yield and a specific activity of 670 U/mg. The literature reveals a significant gap in our understanding of how various factors affect protease production in microalgae, underscoring the early stage of research in this area. While current studies offer limited insights into the influence of environmental conditions on protease activity, particularly in bacteria and fungi, there is scant information specific to microalgae. This preliminary work offers a glimpse into the effects of temperature, light intensity, NaCl concentration, and NaHCO\({}_{3}\) concentration on protease synthesis in microalgae.