*3.2. Synthesis of Affinity Resins*

CHDS-based affinity resins were synthesized according to our previous published method [20,21,27]. The synthesis scheme is shown in Figure 1. Originally, activated amino-sepharose resins were formed by modifying Sepharose 6B (100 g) using epichlorohydrin (Figure 1a). Briefly, Sepharose 6B (100 g) was

first thoroughly washed with distilled water at a 10:1 ratio. After being drained and aired, Sepharose 6B was suspended in 50 mL activating solution (0.8 M NaOH aqueous solution, containing 25% DMSO and 10 mL epichlorohydrin) for 2 h at 40 ◦C. To form aminated Sepharose 6B, activated Sepharose 6B was suspended in 350 mL of distilled water and 35% saturated ammonia was added (150 mL) for mixing. The mixture was incubated for 6 h at 30 ◦C on a rotary shaker (Figure 1b). After that, cyanuric chloride (2,4,6-trichloro-1,3,5-triazine) was linked as a scaffold for the amino groups. The mixture was shaken in ice-salt bath, then 8 g of cyanuric chloride (44 mmol from Sigma-Aldrich; St. Louis, MO, USA), dissolved in 350 mL acetone, was slowly added (Figure 1c). About 100 mL NaOH aqueous solution (1 M) was slowly added to maintain the neutral pH. To clear away free cyanuric chloride, 50% (*v/v*) acetone was utilized to wash the resins. The ninhydrin test was applied to examine the density of free amino groups and the linkage of cyanuric chloride to the amino groups, according to the previously described procedure [18,20,21]. Briefly, a small aliquot of gel was smeared on filter paper, sprayed with ninhydrin solution (0.2%, *w/v*, in acetone), and heated briefly with a hair dryer. Purple color indicated the presence of free amino groups and color disappearance indicated that cyanuric chloride had been linked to the amino groups. Subsequently, dichlorotriazinylated Sepharose 6B resins were added with two-fold molar excess of CHDS dissolved in 2 M sodium carbonate and stirred for 24 h at room temperature (Figure 1d). The coupling efficiencies and yields for the CHDS ligands were also determined by ninhydrin test. Control resins were synthesized from chitotrisaccharide (CHTS) or glucosamine, according to the method described above (Figure 2A,B). Control resins with 5-atom or 10-atom spacer arms were also synthesized from CHDS-modified Sepharose 6B resins, according to the previously published method [21]. The schemes are shown in Figure 2C,D.

#### *3.3. Expression and Purification of Three Typical Chitosanase*

Currently, GH families 46, 75, and 80 comprise only chitosanases in the CAZy database [10,11,28]. Genes encoding three typical chitosanases, including CsnOU01 from GH family 46 (Genbank number ABM91442), Csn from GH family 75 (Genbank number AFG33049), and ChoA from GH family 80 (Genbank number BAA32084) were cloned into the pET22b (containing 6×His tag) vector and expressed in *Escherichia coli* BL21(DE3). The genes were optimized for *E. coli* and synthesized by BGI (Qingdao, China). The DNA fragment was digested to introduce *Nco* I and *Xho* I sites, then ligated into the *Nco* I and *Xho* I sites of plasmid pET22b. The recombinant plasmid was transferred into *E. coli* BL21 (DE3). Cells were cultured in LB medium containing 30 μg/mL ampicillin at 37 ◦C, until the OD600 reached 0.6. Afterwards, the expression of the target gene was induced by 0.1 mM isopropyl-β-thiogalactoside (IPTG) at 20 ◦C and 100 rpm for 18 h. These chitosanases with 6 his-tags were purified using a Ni-NTA Sepharose 6B column (GE Healthcare, Madison, WI, USA) at an AKTA avant 150 platform. After centrifugation for 10 min at 12,000 rpm, the supernatant was loaded into 10 mL equilibrated affinity column and washed with washing buffer (0.1 M Tris–HCl buffer, pH 7.6) until the elute exhibited no detectable absorbance at 280 nm. Then, the elution buffer 1 (0.02 M Tris-HCl, pH 7.6, with 10 mM imidazole) was used to deplete the impure protein. The target protein was eluted by elution buffer 2 (0.02 M Tris-HCl, pH 7.6, with 150 mM imidazole). The flow rate of the mobile phase was 3.0 mL/min. The concentrations of each elution peak were assayed by the Bradford method, using BSA as a standard. Chitosanses without 6 his-tag were purified by the traditional method and CHDS-based method, as shown in Section 3.5. Molecular weight and purity of the enzymes were confirmed by SDS-PAGE or HPLC with size-exclusion chromatography.

#### *3.4. Calculation of Desorption Constant of Chitosanase*

The characterization of the interactions between chitosanases and affinity resins was carried out using equilibrium adsorption study. Scatchard analysis model was used for analysis of the desorption constant (*K*d) and the theoretical maximum adsorption capacity (*Q*max) of different affinity resins [22,29]. Various concentrations of enzymes (10 mL, 0.1–0.9 mg/mL in 20 mM Tris-HCl buffer, pH 8.0) were combined with 5 g of each type of resin to reach the adsorption equilibrium in a shaken

condition. Mixed culture was subsequently centrifuged at 1500× *g* for 5 min at 4 ◦C. Afterward, the residual activity of chitosanase and protein concentration in the supernatants was measured and analyzed according to the following Equation:

$$Q = \frac{Q\_{\text{max}}[\mathbb{C} \text{ \*}]}{K\_{\text{d}} + [\mathbb{C} \text{ \*}]} \tag{1}$$

where *Q* represents the amount of chitosanase adsorbed to the affinity resin (mg/g wet resin), *Q*max represents the theoretical maximum absorption of chitosanase to the affinity resin (mg/g wet resin), [C\*] represents the protein concentration of chitosanase in the mixed solution (mg/mL), and *K*<sup>d</sup> represents the desorption constant. Scatchard plot represents one of the linearized forms of Equation (1). Equation (1) could be transformed into the following Equation (2):

$$\frac{\mathcal{Q}}{\left[\mathcal{C} \ast\right]} = \frac{\mathcal{Q}}{\mathcal{K}\_{\text{d}}} + \frac{\mathcal{Q}\_{\text{max}}}{\mathcal{K}\_{\text{d}}} \tag{2}$$

According to the Scatchard model, a plot of *Q*/[C\*] against *Q* should yield a straight line. The batch adsorption of CsnOU01 towards the affinity medium showed that the respective correlation coefficient R2 ranged from 0.921 to 0.991. These results indicate that the data fit well with the model.
