*3.5. Affinity Purification of Chitosanases Using CHDS-Based Resin*

The synthesized CHDS-based affinity resins were pre-equilibrated with sample loading buffer (0.1 M Tris-HCl buffer, pH 8.0). Before sample was loaded, the supernatant of expression strains were centrifugated at 10,000× *g* for 10 min to remove the impurity. Approximately 100 mL samples were loaded onto 10 mL column with synthesized resins. Next, the column was washed with 30 mL washing buffer (0.1 M Tris-HCl buffer with 100 mM NaCl, pH 8.0). Elution buffers (0.1 M acetic acid buffer, pH 5.6, 0.8 M NaCl) were used for eluting the target proteins. The flow rate was 5.0 mL/min. As a control, the traditional purification protocols of three different chitosanses were also developed. The traditional purification protocol of CsnOU01 (GH46) was composed of five steps, including ultrafiltration with a Millipore Amicon® Ultra-10 kDa in 15 mL filter, 60% saturation ammonium sulfate precipitation in an ice-bath and still stirring for more than 2 h, desalting with a 5 mL desalting column (GE Healthcare, Madison, WI, USA), DEAE anion-exchange chromatography, and superdex 75 gel-filtration chromatography. The traditional purification protocol of Csn (GH75) was composed of three steps, including 60% saturation ammonium sulfate precipitation in an ice-bath and still stirring for more than 2 h, desalting with a 5 mL desalting column (GE Healthcare, Madison, WI, USA), and DEAE anion-exchange chromatography. The traditional purification protocol of ChoA (GH80) was composed of six steps, including 40% saturation ammonium sulfate precipitation in an ice-bath and still stirring for more than 2 h, phenyl hydrophobic chromatography (GE Healthcare, Madison, WI, USA), desalting with a 5 mL desalting column, DEAE anion-exchange chromatography, Superdex 75 gel-filtration chromatography, and Superdex 200 gel-filtration chromatography. Protein concentrations of elution peaks were determined by Bradford assay. The purified enzymes were analyzed by 10% SDS-PAGE and HPLC with size-exclusion chromatography.

#### *3.6. Direct Affinity Purification of Chitosanases from Marine Bacteria*

In our previous study, sixty-two strains of marine bacteria with chitosan degradation ability were isolated. Among these, twenty-three strains produced chitosanase, nine of which showed high chitosanase activity (>50 U/mL). In this study, the nine bacterial strains with high chitosanase activity were chosen as target strains in affinity purification of chitosanase using the CHDS-based resins. The bacteria were cultured in a medium (containing 30 g/L NaCl, 3 g/L MgSO4·7H2O, 0.2 g/L CaCl2, 0.1 g/L KCl, 0.02 g/L FeSO4, 1.5 g/L Na2HPO4, 1 g/L NaH2PO4, and 2 g/L chitosan) at 25 ◦C for 48 h on a rotary shaker (speed, 150 rpm). After that, cultures were centrifuged at 10,000× *g* for 15 min

to remove strains. Approximately 500 mL of supernatants was loaded onto a 10 mL pre-equilibrated column. Washing buffers (0.1 M Tris-HCl buffer with 100 mM NaCl, pH 8.0) were used to remove the uncombined proteins. After that, elution buffers (0.1 M acetic acid buffer, pH 5.6, 0.8 M NaCl) were used for eluting the target protein. The flow rate was 5.0 mL/min.
