Design and Synthesis of a Chitodisaccharide-Based Affinity Resin for Chitosanases Purification

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.

Enhancing the Thermo-Stability and Anti-Biofilm Activity of Alginate Lyase by Immobilization on Low Molecular Weight Chitosan Nanoparticles

“…The quantification of amino group in the surface of CS-NPs was determined spectrophotometrically by a method of modified Ninhydrin assay in order to identify the conjugation [42]. Briefly, a small aliquot of beads was smeared on filter paper, sprayed with ninhydrin solution (0.2% ( w/v ) in acetone), and heated briefly with a hair dryer.…”

Characterization of a New Chitosanase from a Marine Bacillus sp. and the Anti-Oxidant Activity of Its Hydrolysate

“…However, the reaction products of most reported chitosanases are a mixture of DP2-DP6, which are difficult to separate. In our previous study, we have designed and synthesized an affinity resin to screen and isolate native chitosanases directly from bacterial culture supernatants [19]. Using this protocol, a chitosanase, CsnQ, was isolated and purified from the marine bacterium Bacillus sp.…”

Cloning and Characterization of a New Chitosanase From a Deep-Sea Bacterium Serratia sp. QD07

“…Of particular importance are its admirable biological activities including anti-microbial ( Fernandes et al, 2008 ), anti-fungal ( Mei et al, 2015 ), anti-oxidant ( Ma et al, 2020 ), anti-inflammatory ( Kunanusornchai et al, 2016 ), anti-obesity ( Huang et al, 2015 ), anti-tumor ( Suzuki et al, 1986 ; Zhao et al, 2019 ), anti-hypertensive ( Huang et al, 2005 ), anti-HIV-1 ( Artan et al, 2010 ), anti-Alzheimer’s ( Eom et al, 2013 ), and immune-enhancing effects ( Zhang et al, 2014 ). It also shows promise as a drug/DNA delivery agent ( Kumari et al, 2018 ; Li et al, 2019a ; Wang et al, 2020 ). Therefore, COS is a promising candidate with potential application in several fields, including the medical and pharmaceutical industries.…”