Abstract
Chondroitinase (ChSase), a type of glycosaminoglycan (GAG) lyase, can degrade chondroitin sulfate (CS) to unsaturate oligosaccharides, with various functional activities. In this study, ChSase AC II from a newly isolated marine bacterium Arthrobacter sp. CS01 was cloned, expressed in Pichia pastoris X33, purified, and characterized. ChSase AC II, with a molecular weight of approximately 100 kDa and a specific activity of 18.7 U/mg, showed the highest activity at 37 °C and pH 6.5 and maintained stability at a broad range of pH (5–7.5) and temperature (below 35 °C). The enzyme activity was increased in the presence of Mn2+ and was strongly inhibited by Hg2+. Moreover, the kinetic parameters of ChSase AC II against CS-A, CS-C, and HA were determined. TLC and ESI-MS analysis of the degradation products indicated that ChSase AC II displayed an exolytic action mode and completely hydrolyzed three substrates into oligosaccharides with low degrees of polymerization (DPs). All these features make ChSase AC II a promising candidate for the full use of GAG to produce oligosaccharides.
Highlights
Chondroitin sulfate (CS), a glycosaminoglycan (GAG), is a linear polysaccharide with repeated disaccharide units of β-D-glucuronic acid (GlcA) and N-acetyl-β-D-galactosamine (GalNAc), which are linked through a β-1,4-glycosidic bond [1,2]
The results showed that the enzyme activity of the recombinant ChSase AC II was highest in the P. pastoris X33 that the enzyme activity of the recombinant ChSase AC II was highest in the P. pastoris X33 compared compared to the other strains (Figure 2)
The enzyme showed maximal activity at 37 ◦ C and pH 6.5. It was stable at a broad pH range (5–7.5) and retained above 80% activity after incubation at 37 ◦ C for
Summary
Chondroitin sulfate (CS), a glycosaminoglycan (GAG), is a linear polysaccharide with repeated disaccharide units of β-D-glucuronic acid (GlcA) and N-acetyl-β-D-galactosamine (GalNAc), which are linked through a β-1,4-glycosidic bond [1,2]. Both GlcA and GalNAc residues can carry different numbers of sulfate groups in different positions. Some GlcA residues are epimerized into Iduronic acid (IdoA), and those containing repeating disaccharide units [IdoAα1-3GalNAc(4S)] are designated as dermatan sulfate (DS, CS-B) [4]. CS is mainly used for the treatment of osteoarthritis and inflammation.
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