*3.3. ScCDA2 Activity Assay and Biochemistry Properties*

The purified *Sc*CDA2 was studied to determine its enzymatic properties and deacetylation patterns. The colloidal chitin (water-soluble chitin), colloidal chitin and chitin oligomers dimers to hexamers (A2 to A6) were used as substrates [14]. The reaction mixture for *Sc*CDA2 enzyme activity assay containing 20 mM Tris-HCl buffer (pH8.0), including 1 mM CoCl2, 0.5 mg/mL substrate and 0.75 μM purified soluble protein (*Sc*CDA2) or distilled water as a control was incubated at 37 ◦C for 30 min. The reaction was terminated by the addition of 10 μL 5% formic acid [14]. Determination of CDA activity by measuring the amount of acetate released by a coupled enzyme assay using an acetate assay kit [14]. The total reaction volume of the coupled enzyme reaction was 266 μL, which was measured spectrophotometrically at 340 nm [14].

In order to determine the optimum pH of *Sc*CDA2, protein in different buffers (final concentration 20 mM) was incubated at 37 ◦C for 30 min at the pH range of 4.0 to 10.0, in either sodium citrate disodium hydrogen phosphate buffer (pH 3.0–5.0), sodium phosphate dibasic sodium dihydrogen phosphate buffer (pH 6.0–7.0), Tris-HCl buffer (pH 8.0) or sodium carbonate sodium bicarbonate buffer (pH 9.0–10.0). The optimum temperature was determined in the 20 mM Tris-HCl buffer, at the optimum pH of 8.0, and each protein solution was incubated at 37 ◦C, 50 ◦C and 65 ◦C for 60 min. Subsequently, the remaining enzyme activity was measured using standard activity assays. The effects of different metal ions on enzyme activity were verified by adding 1 mM of different metal ion solutions (NaCl, NH4Cl, BaCl2, CoCl2, MnCl2, ZnCl2, CuCl2, MgCl2 and FeCl3) to the reaction mixtures [17].
