## 2 Materials and Methods
### Enzyme Activity and Protein Quantitation  
Lyase mode of action of the enzyme extract was assayed as described by [PERSON] et al. (1988) [21]. Briefly, 1.5 mL enzyme solution was added to 1.5 mL of 10 g L\({}^{-1}\) citrus pectin solution in 50 mM Tris/HCl buffer, containing 70 g L\({}^{-1}\) NaCl with pH 8.5. After one-hour incubation at 40 \({}^{\circ}\)C, the changes in absorbance were measured using a Pye Unicam Spectrophotometer (Thermo Fisher Scientific, Mundelein, IL, USA) at 235 nm. The amount of enzyme that is need for obtaining of unsaturated oligogalacturonides--equivalent to 1 \(\upmu\)mol/min--was defined as one unit of enzyme activity under standard assay conditions. Pectin methyl esterase activity was determined as the enzyme amount that released 1 \(\upmu\)mol of methanol from pectin per min at pH 8.5 and 40 \({}^{\circ}\)C. First, 1 mL of supernatant was added to the reaction mixture consisting of 0.5 mL 50 mM Tris/HCl buffer, containing70 g L\({}^{-1}\) NaCl, pH 8.5, and then supplemented with 2 mL of 10 g L\({}^{-1}\) citrus pectin and 0.15 mL 0.01% (\(w/v\)) of bromothymol blue. The enzyme activity was determined by measuring the absorbance at 620 nm [22]. Polygalacturonase activity was investigated by DNSA assay [23]. The citrus pectin (10 g L\({}^{-1}\)) was dissolved in 50 mM Tris/HCl buffer, containing 70 g L\({}^{-1}\) NaCl and pH 8.5. The reaction mixture of 0.05 mL substrate and 0.05 mL enzyme solution was incubated at 40 \({}^{\circ}\)C and pH 8.5 for 30 min. After addition of DNSA reagent, the mixture was incubated at 100 \({}^{\circ}\)C for 5 min. Finally, the mixture was cooled to room temperature, diluted with distilled water (1 mL), and then vortexed. One unit of enzyme activity (U) was defined as the amount of enzyme required to release 1 \(\upmu\)mol of reducing sugar per minute under the assay conditions (1 U = 1 \(\upmu\)mol/min/mL). Galacturonic acid in a concentration of 0-1 mg/mL was used as a standard. All assays were conducted at least in duplicate. Data were presented as mean \(\pm\) standard deviation. The specific activity of pectinase was presented as units per milligram of protein. The total protein content was estimated by the Lowry assay using bovine serum albumin as a standard in a concentration range of 0-1000 \(\upmu\)g/mL [24]. Partial Purification and Molecular Weight Determination of the Pectinase from V. salarius Strain 434  
Extracellular pectinase was purified from the culture liquid of the strain by centrifugation at 10,000\(\times\)\(g\) for 20 min for cells removal. One liter of culture supernatant was concentrated to 50 mL through ultrafiltration using Amicon(tm) stirred cell with 30 kDa cellulose membrane (Millipore, Darmstadt, Germany). The concentrated sample (1.0 mL) was loaded on to a glass column packed with Sephadex G-100 (40 \(\times\) 1.6 cm, bed volume 60 mL). Tris-HCl buffer (50 mM, pH 8.5), containing 70 g L\({}^{-1}\) NaCl, was used as an elution buffer. Fractions of 4.0 mL volume were collected and their protein content and pectinase activity were determined according to the methods described above. The pectinase-containing fractions were pooled and used for electrophoresis. Purity degree and molecular weight were examined by SDS-PAGE in 10% resolving and 5% stacking gel, respectively, each containing 0.1% SDS by the method described by [PERSON] (1970) [25], using a Bio-Rad electrophoresis apparatus (Merck KGaA, Darmstadt, Germany). The gel was run on a constant voltage of 80 V. Molecular markers 10-170 kDa (Fisher BioReagents, Waltham, MA, USA) were used as a standard. Gels were stained with 0.1% Coomassieie Brilliant Blue R-250 in methanol/acetic acid/dH\({}_{2}\)O (in 5:4:1 ratio) for 1.5 h, and destained by washing with methanol/acetic acid/dH\({}_{2}\)O (in a ratio of 5:4:1) several times. The molecular weight of the pectinase from _V. salarius_ strain 434 was also confirmed through gel filtration. A column (40 \(\times\) 1.6 cm) of Sephadex G-100 was prepared.