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Abstract
Chondroitin AC lyase (ChSaseAC) is one of the essential polysaccharides lyases in low molecular chondroitin sulfate production. In this work, a novel PrChSaseAC from Pedobacter rhizosphaerae was successfully cloned, expressed in Escherichia coli. After optimizing the induction, the recombinant PrChSaseAC could be expressed efficiently at 0.1 mM IPTG, 25°C, and 12 h induction. Then, it was purified with Ni-NTA affinity chromatography. The characterization of the purified PrChSaseAC showed that it had high specific activity and good storage stability, which would favor the production of low molecular weight chondroitin sulfate. It also displayed activity toward chondroitin sulfate C and hyaluronic acid. PrChSaseAC had the highest activity at pH 7.5, 37°C, 10 mM Ca2+, and 5 mg/ml of chondroitin sulfate A. Molecular docking of substrate and enzyme showed the interactions between the enzyme and substrate; it revealed that the enzyme showed high activity to CS-A and hyaluronic acid, but lower activity to CS-C attributed to the structure of the binding pocket. The high stability and specific activity of the enzyme will benefit the industrial production or clinical treatment.
Highlights
Chondroitin sulfate (CS) belongs to the glycosaminoglycan family formed with linear polysaccharides with repeated disaccharide units consisting of hexuronic acid and hexosamine (Mizumoto et al, 2013; Bhotmange and Singhal, 2015)
PrChSaseAC shows more than 60% identity to FhChSase, which means that they have high homology
According to the previous report (Féthière et al, 1999), the residues His225, Arg288, Arg292, and Lys298 might be involved in the binding to the substrate and contribute to the catalysis residues highly conserved compared to the template 1cb8
Summary
Chondroitin sulfate (CS) belongs to the glycosaminoglycan family formed with linear polysaccharides with repeated disaccharide units consisting of hexuronic acid and hexosamine (Mizumoto et al, 2013; Bhotmange and Singhal, 2015). The demand for CS worldwide keeps increasing dramatically recently due to the increasing aging population
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