## 3 Results
### Optimization of Pectinase Production by V. salarius Strain 434  
Culture conditions were adjusted in view of obtaining a maximum yield of bacterial pectinase (Figure 2). The highest cell growth (determined by OD\({}_{660}\)) and enzyme production were found after 48 h (7.5 U/mg) of cultivation at 35 \({}^{\circ}\)C (9.0 U/mg--100% of relative activity) with 1% inoculum (10.0 U/mg), using 10 g L\({}^{-1}\) citrus pectin as a substrate, yeast extract (9.8 U/mg) as a nitrogen source, and glucose (9.5 U/mg) as a carbon source. Similarly, the highest pectinase activity (230 U/mL) was detected at 48 h of cultivation of the thermophilic bacterium _Anoxyhacillus gonensis_ strain 357 [28] and at 40 h of cultivation of the _Bacillus tequilensis_ CAS-MEI-2-33 (1370 U/mL) [29]. Maximal pectinase activity of 0.70 U/mL was observed at a fermentation period of 120 h for the haloalkalophilic strain _Halomonas pantellerinis_ SSL8 [30]. Growth curve observations and the results of measuring pectinase production from the extremely halophilic archaea _Halorubrum chaoviator_ (CEJTA37) and _Natrinema altunese_ (CE)JGTEA101) showed that the highest activity was recorded at 122 h (0.067 U/mL) and at 144 h (0.067 U/mL) of cultivation, respectively [31]. Figure 2: Effect of the different fermentation medium factors on pectinase activity and cell growth: (**a**)—inoculum; (**b**)—substrate concentration; (**c**)—carbon source; (**d**)—nitrogen source; (**e**)—NaCl concentration; (**f**)—pH; (**g**)—temperature. The orange bars represent the pectinase activity and the green bars represent the cell growth measured by OD\({}_{660}\). Each experiment was performed in triplicate. The data are expressed as their mean values. Error bars show the standard deviation, \(p<0.05\). The short incubation time makes production cost-effective--it streamlines the production process, reduces energy-related costs, minimizes pollution risks, increases productivity, and optimizes the use of resources. Glucose also appeared to be the best carbon source according to other authors [32,33]. On the other hand, [PERSON] et al. (2010) [34] reported citrus pectin and xylose as the best carbon sources for pectinase production by _Bacillus spharcicus_. In the same way, yeast extract was the most appropriate organic nitrogen source for the pectinase synthesis by _Bacillus subtilis_ SAV-21 [33], while this peptone and NH4 Cl were found to stimulate the pectinase production by _Bacillus_ sp. MFW7 [35]. The optimum pH of the medium was 7.5 (10.5 U/mg--100% of relative activity) and the optimal NaCl concentration was 70 g L\({}^{-1}\) (9.7 U/mg). Partial Purification and Molecular Weight Determination of the Pectinase Synthesized by V. salarius Strain 434  
The specific activity of the crude enzyme was 9.1 U/mg. It was then purified by gel filtration chromatography. The purified pectinase showed a specific activity of 104.3 U/mg with 11.5-fold higher purity and a final yield of 29%. The summarized results obtained are shown in Table 2. Similar are the results for the pectinase from _Bacillus subtilis_ 15 AB92, which was 11.6-fold purified, and the measured specific activity of the enzyme had an increase from 8.6 U/mg to 99.6 U/mg with a yield of 18.8% [1]. The purified pectinase from _V. salarius_ strain 434 migrated as a single band with a molecular weight (Mw) of about 68 kDa (Figure 3a) as determined by a standard curve of Rf vs. logMw of protein standards with known Mw (Figure 3b). The molecular weight of the pectinase was confirmed, applying gel filtration chromatography by a Sephadex G-100 column. After comparing the elution volume of the pectinase with that of known protein standards, its molecular mass was calculated to be 68 kDa (Figure 3c). The pectinase of _V. salarius_ strain 434 falls within the Mw range (25-115 kDa) of _Bacillus_ sp. pectinases defined by [PERSON] and [PERSON] (2014) [36]. Its molecular weight was comparable with other purified pectinases--89 kDa for _Bacillus cereus_ NRC20 [37] and 66 kDa for _Bacillus_ sp. MBRL576 [38].