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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 <i>Arthrobacter</i> sp. CS01 was cloned, expressed in <i>Pichia pastoris</i> 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 &#176;C and pH 6.5 and maintained stability at a broad range of pH (5&#8211;7.5) and temperature (below 35 &#176;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.