**1. Introduction**

Chitosan, a natural cationic polysaccharide, has key roles in many biological processes, such as artificial skin, absorbable surgical sutures, and wound healing accelerators [1]. Chitosanase (EC 3.2.1.132) catalyzes the hydrolysis of β-1,4-linked glycosidic bond in the chitosan chain, releasing chitooligosaccharides (CHOS) as products [2]. In recent years, CHOS have increasingly gained more attention because of their important biological activities, such as anti-tumor [3,4], immuno-enhancing [5], anti-fungi [6], anti-bacterial [7], and anti-diabetic effect [8]. These activities are dependent on chemical structures and molecular sizes of the oligosaccharides [9].

It has been identified that chitosanases can be isolated from various organisms, including fungi, plants, and bacteria [10,11]. Based on the classification in Carbohydrate-Active enZYmes (CAZy) databases [12] (http://www.cazy.org), chitosanases are included into seven different glycoside hydrolase (GH) families: 3, 5, 7, 8, 46, 75, and 80. Currently, most known chitosanases belong to GH family 46, which comprise only chitosanases. Thus far, several chitosanases from different organisms have been identified [13–16]. According to the sequence alignments, chitosanases are classified into seven GH families: 3, 5, 7, 8, 46, 75, and 80 [11]. Among which, chitosanases, which belong to GH family 46, have been fully characterized, and several crystal structures of chitosanases have been determined (PDB codes: 1CHK, 1QGI, and 2D05). The catalytic mechanism of chitosanases from GH family 46 has been elucidated; it was identified to follow an "inverting" catalytic mechanism [12–14]. The 3-D crystal structure of chitosanase-substrate complex (CsnOU01) shows that the −2, −1, and +1 subsites of chitosanase from GH family 46 play a predominant role for the formation of hydrogen bond intermediate during substrate binding and catalysis [12,13]. Because chitosanases with special characteristics have potential applications in industry and biotechnology, a rapid and efficient purification method that can be used to purify chitosanases, from which their biochemical characteristics are determined, from different microorganisms is essential. According to the literature, the purification protocols of native chitosanases generally involve ultrafiltration, ammonium sulfate precipitation, salting out, hydrophobic interaction chromatography, ion-exchange chromatography, or gel filtration chromatography [13–16]. These traditional methods not only require a large number of steps, but are also time-consuming and difficult to scale up. Biomimetic affinity chromatography is potentially the most selective method in protein purification [17,18]. This technique requires lower number of steps, while resulting in higher yields, so can be considered economical. Therefore, it can be beneficial to develop a rapid and efficient one-step affinity purification protocol for chitosanase.

Affinity purification of enzymes often design affinity ligands that function as substrate analogues or specific inhibitors [16,18,19]. In this study, CHOS-based resin was synthesized by coupling chitosan-disaccharide (CHDS) to epoxy-activated Sepharose 6B using cyanuric chloride as a spacer. The resin was then used in the development of one-step affinity purification of chitosanases, in which three typical enzymes, GH families 46, 75, and 80, were purified. The developed purification protocol was highly efficient and resulted in high purity enzymes (more than 95% purity). The method was also applied to directly purify chitosanase from bacterial culture medium.
