Abstract
Chitooligosaccharides (CHOS) have gained increasing attention because of their important biological activities. Enhancing the efficiency of CHOS production essentially requires screening of novel chitosanase with unique characteristics. Therefore, a rapid and efficient one-step affinity purification procedure plays important roles in screening native chitosanases. In this study, we report the design and synthesis of affinity resin for efficient purification of native chitosanases without any tags, using chitodisaccharides (CHDS) as an affinity ligand, to couple with Sepharose 6B via a spacer, cyanuric chloride. Based on the CHDS-modified affinity resin, a one-step affinity purification method was developed and optimized, and then applied to purify three typical glycoside hydrolase (GH) families: 46, 75, and 80 chitosanase. The three purified chitosanases were homogeneous with purities of greater than 95% and bioactivity recovery of more than 40%. Moreover, we also developed a rapid and efficient affinity purification procedure, in which tag-free chitosanase could be directly purified from supernatant of bacterial culture. The purified chitosanases samples using such a procedure had apparent homogeneity, with more than 90% purity and 10–50% yield. The novel purification methods established in this work can be applied to purify native chitosanases in various scales, such as laboratory and industrial scales.
Highlights
Chitosan, a natural cationic polysaccharide, has key roles in many biological processes, such as artificial skin, absorbable surgical sutures, and wound healing accelerators [1]
Chitooligosaccharides (CHOS) are natural cationic saccharides, while the catalytic domain of chitosanases is rich in acidic amino acids [12,13]; the acid-base interactions between the two molecules can provide affinity force during affinity purification
Contains an amine group at the C-2 position of the sugar ring, we focused our efforts on the design of CHOS-based affinity resin for purification of chitosanase
Summary
A natural cationic polysaccharide, has key roles in many biological processes, such as artificial skin, absorbable surgical sutures, and wound healing accelerators [1]. 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]. 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.
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