*3.7. Assay of Enzyme Activity*

The 3,5-dinitrosalicylic acid (DNS) method was used for assay of the enzyme activity of chitosanase. Briefly, 100 μL of enzyme was mixed with 900 μL of chitosan substrate (0.3% *w/v*, prepared in 50 mM sodium acetate buffer, pH 6.5). Reaction solution was incubated at 40 ◦C in a water bath for 10 min. Immediately, 750 μL of DNS solution was added into the reaction solution. After that, the mixtures were heating at 100 ◦C for 10 min. The reaction mixture was cooled down and then centrifuged at 10,000 × *g* for 2 min to remove the remaining insoluble chitosan. The reducing sugars in the supernatant were analyzed at 520 nm. Each reaction was carried out in triplicate; standard deviation were calculated and used for analysis. D-glucosamine was used as standard. One unit of enzyme activity was defined as the amount of enzyme that releases 1 μmol D-glucosamine-equivalent reducing sugars per minute under the assay conditions.

#### *3.8. Analysis of Protein Purity*

Protein purity was determined by SDS-PAGE analysis. The purity of the purified chitosanase was estimated based on the intensity of the protein band using Gelpro Analyzer 3.2, a commonly used gel imaging analysis system. HPLC analysis was conducted using Agilent 1260, equipped with a TSK3000SW column (Tosoh Co., Tokyo, Japan), wherein protein was monitored by absorbance at 280 nm [27]. The mobile phase was 0.1 M PBS, pH 6.7, 0.1 M Na2SO4, 0.05% NaN3. The flow rate was 0.6 mL/min.

#### **4. Conclusions**

In this study, a highly efficient affinity resin designed for chitosanase purification was synthesized and characterized. Among other purification protocols, the synthesized resins using CHDS to couple with Sepharose 6B resin via cyanuric chloride spacer were used in the direct purification of native chitosanase without any tags from bacterial culture. This protocol has several significant advantages, for instance, higher purity, fewer steps, and better activity recovery. Coupled with accessible reagents, efficacy, and time-saving procedures, this efficient affinity purification protocol can be a potentially important tool for screening native chitosanases that possible have unique characteristics.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1660-3397/17/1/68/s1, Table S1: Different loading and elution condition on the recovery yield and specific activity of CsnOU01. Table S2: The sum of traditional protocol for three different chitosanase.

**Author Contributions:** S.L. and Y.H. conceived the study. S.L. and L.W. performed the experiments. S.L., M.S., X.C., and Y.H. analyzed the data. S.L. wrote the paper. All the authors reviewed the manuscript.

**Funding:** This project was funded by Open Research Fund Program of Shandong Provincial Key Laboratory of Glycoscience and Glycotechnology (Ocean University of China) (KLGGOUC201703); National Natural Science Foundation of China (41376175).

**Conflicts of Interest:** The authors have declared that no competing financial interests exist.

#### **References**


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