## 3 Results
### Optimization of Reaction Parameters for Maximum Pectinase Activity  
The results of the pectinase property analysis are shown in Figures 4 and 5. The highest activity (100%) of the enzyme was recorded at 70 g L\({}^{-1}\) NaCl (98 U/mg). With increasing NaCl concentration, the activity of the enzyme remained similar but at 150 g L\({}^{-1}\) NaCl, it slightly decreased (78 U/mg). These results demonstrate its salt tolerance (Figure 4a). It was found that as the substrate concentration increased, the enzyme activity also increased, and with 10 g L\({}^{-1}\) citrus pectin, a maximum value of 100 U/mg was reached, followed by a decrease to 80 U/mg: 80% of the relative activity at higher substrate concentrations (Figure 4b). This result may be due to substrate inhibition when the active site of the enzyme was completely saturated with a substrate. Reaction time can significantly affect enzyme activity through its effects on enzyme activation associated with conformational changes, substrate availability, stability, and inactivation. In our study, the enzyme activity increased with increasing reaction time up to 30 min and then remained unchanged in the subsequent incubation period (Figure 4c). The pH is one of the main factors that play a crucial role in enzyme activity and stability by influencing enzyme conformation, substrate interaction, structural integrity, and susceptibility to denaturation. Our results showed that 106 U/mg pectinase activity at pH 9.0 was the highest recorded value. Pectinase activity increased when the pH of the reaction systems increased from 7.5 to 9.5, and at pH 10.0, the activity decreased to about 75% of the maximum (80 U/mg). However, the shift of pH towards the acidic region drastically reduced the enzymatic activity of the pectinase and caused a loss of its initial activity at pH 6.0 to 80% (Figure 5a). The stability of the enzyme at different pH values is important for its commercialization, so it was also tested. The enzyme was found to be stable over a wide range of pH values and retained 100% activity at pH 7.0 to 9.5 for up to 60 min. The alkaliiphic nature of the enzyme was confirmed by the 50% (54 U/mg) residual activity found at alkaline pH 10 after half an hour of pretreatment (Figure 5b). \begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Purification Step** & **Total Activity** & **Total Protein** & **Specific Activity** & **Purification** & **Purification** \\  & **(U)** & **(mg)** & **(U/mg)** & **Fold** & **Yield** \\ \hline Crude enzyme & 1825 \(\pm\) 0.04 & 200 \(\pm\) 0.03 & 9.1 \(\pm\) 0.02 & 1 & 100 \\ \hline Ultrafiltration & 1015 \(\pm\) 0.02 & 65 \(\pm\) 0.01 & 15.6 \(\pm\) 0.02 & 1.7 & 56 \\ \hline Gel filtration & 521.5 \(\pm\) 0.01 & 5 \(\pm\) 0.01 & 104.3 \(\pm\) 0.01 & 11.5 & 29 \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Purification of _V. salarius_ strain 434 pectinase. Figure 4: Effect of different enzyme reaction factors on the activity of the purified pectinase. (**a**)—NaCl concentration; (**b**)—substrate concentration; (**c**)—reaction time. Each experiment was performed in triplicate. The data are presented as their mean values. Error bars indicate the standard deviation, \(p\) < 0.05. These results showed that the investigated enzyme may be alkaline in nature. Alkaline pectinases are suitable for application in the degumming of ramie fibers and in processes of pretreatment of wastewater for food processing. Therefore, this enzyme can be used in various biotechnological processes that require an alkaline pH. Our study demonstrated that a temperature of 40 \({}^{\circ}\)C appeared to be the optimal for reaching the maximum activity of the purified pectinase--100 U/mg. When the enzyme reaction took place at higher temperatures, the pectinase activity decreased, and at 70 \({}^{\circ}\)C was about 40% of the maximum activity reported at 40 \({}^{\circ}\)C (Figure 5c). The enzyme was stable at 40 \({}^{\circ}\)C, retaining 100% of its initial activity after 60 min.