*2.3. Direct Purification of Chitosanases from Bacterial Culture Medium*

In order to establish a rapid purification protocol for native chitosanase, nine bacterial strains with high chitosanase activity (more than 50 U/mL) were chosen as target strains in the affinity purification of chitosanase using the established CHDS-based materials. After bacterial culture was centrifuged at 10,000× *g* for 15 min, the supernatant was loaded onto 10 mL pre-equilibrated column and washed with washing buffer (0.1 M Tris-HCl buffer with 100 mM NaCl, pH 8.0) until the eluate showed no detectable absorbance at 280 nm. After that, the target protein was eluted with elution buffer (0.1 M acetic acid buffer, pH 5.6, 0.8 M NaCl). As shown in Figure 5, CHDS-based resin was able to purify chitosanases (with certain homogeneity) from culture medium.

**Figure 5.** SDS-PAGE analysis of purified chitosanases direct from bacterial supernatant.

The activity recovery and purity of the purified enzymes were shown in Table 3. The purified chitosanase samples using such procedure had apparent homogeneity with more than 90% purity and 10–50% yield. Different strains showed different activity recoveries. This result may be caused by multi-factors. In this study, our affinity purification condition was only used for rapidly screening chitosanases. The optimal purification protocol toward the special enzyme needs further optimization. After characterizing these purified enzymes, the chitosanase from *Serratia* sp. QD07 showed high thermo-tolerant property and suitability for industrial usage (data not shown). The characterization data of this thermo-tolerant enzyme will be reported in the next paper.


**Table 3.** Affinity purification of chitosanases direct from bacterial supernatant.

Chitosanases that have special characteristics can potentially be applied in biotechnology industry and other fields. As has been described in the literature, the purification of native chitosanases (without His-tag) usually involves ultrafiltration, ammonium acetate precipitation, salting out, ion-exchange chromatography, and hydrophobic interaction chromatography [13–16]. These conventional methods usually involve a large number of steps, which are time-consuming and difficult to scale up. As far as we know, biomimetic affinity chromatography specially designed for native chitosanase has not been established. In this study, we developed the rapid and efficient affinity purification procedure, in which native chitosanase could be directly purified from supernatant of bacterial culture. The novel purification methods established in this work can be applied to screen and purify chitosanases, both in laboratory and industrial scales.

#### **3. Materials and Methods**

#### *3.1. Materials*

Chitosan, with degree of deacetylation (DDA) ≥ 95%, was purchased from Aladdin, China. To obtain chitodisaccharide (CHDS) and chitotrisaccharide (CHTS), chitosan (0.1%) was degraded by GH family 46 chitosanase CsnOU01 at a final enzyme concentration at 20 mg/mL for 6 h. CHDS and CHTS were purified from the degradation products of chitosan using a Biogel P-2 column (GE Healthcare, Madison, WI, USA). Briefly, 100 mg degradation product of CHDS and CHTS was loaded into the Biogel P-2 column (1.6 × 130 mm), using 0.2 M ammonium acid carbonate as mobile phase. The flow rate was 1 mL/min. Then, the effluent was collected every 1 min and the sugar content was determined by phenol sulfate method. Finally, oligosaccharides were collected and identified by TLC. Sigma-Aldrich (St. Louis, MO, USA) provided cyanuric chloride (2,4,6-trichloro-1,3,5-triazine) and glucosamine. Beijing Weishibohui Chromatography Technology Co., China, provided activated Sepharose 6B with two different spacer arm lengths (5-atom and 10-atom). Sinopharm Chemical Reagent (Shanghai, China) provided other analytical grade reagents.
