4.6.3. Effects of Metal Ions and Chemicals on Cadex Activity

The effects of different solutions containing chloride metal ion salts (final concentrations of 1 mM and 5 mM) and chemicals on purified Cadex activities were determined. The relative enzyme activity in the presence of metal ions and chemicals was calculated based on activity in the absence of reagent.

#### 4.6.4. Substrate Specificity

To determine the substrate specificity of Cadex, dextranase activity in the enzymatic hydrolysis of carbohydrates with various glycosidic linkages was determined using the method described by Wu. et al. [22]. Dextranase was incubated in 50 mM Tris-HCl (pH 8.0) with various carbohydrates at 40 ◦C for 20 min. Relative activity was expressed in percentage values of the highest activity, which was set as 100%.

#### 4.6.5. Products of Cadex Hydrolysis

First, Cadex hydrolysis took place in dextran T70 solution, and the products were analyzed by TLC using a silica gel GF254 plate developed in a chloroform: acetic: acid: water ratio of = 5:7:1 (*v*:*v*:*v*). An oligosaccharide kit was used as the standard.

Second, using the optimum dextranase temperature and pH, 3% dextran T70 samples were digested for different periods (15 min, 30 min, 1 h, 3 h, and 5 h). The products were identified and analyzed with the Waters 600 and Waters Sugar-Pak1 (300 mm × 7.8 mm; Waters, Milford, MA, USA) HPLC with a differential refraction detector. The mobile phase was water at 0.4 mL/min, the column temperature was 85 ◦C and the injection volume was 20 μL. The standard sugars were glucose, maltose, maltotriose, isomaltotriose, isomaltotetraose, isomaltopentose, and isomaltohexose. For quantification, the peak areas were determined. Data acquisition and processing were conducted using Empower GPC software (Waters, Milford, MA, USA).
