Identification and Characterization of Marine Bacteria-Derived Chondroitinase CHa3 in the PL35 Family

Chondroitinase (CSase) derived from microorganisms is valuable for structural studies and preparation of chondroitin sulfate (CS) oligosaccharides. In this study, a new chondroitinase C, CHa3, which belongs to the PL35 family, was identified. The optimal activity of CHa3 was observed in NaH₂PO₄-Na₂HPO₄ buffer (pH 8.0) at 20 °C. Its specific activity toward chondroitin sulfate A, chondroitin sulfate C, chondroitin sulfate D, and chondroitin sulfate was 1.11, 4.78, 2.63, and 1.91 U/mg protein, respectively. CHa3 digests substrate chains, acts as an endolytic lyase, and shows a significant preference for C-unit-enriched substrates such as CSC; thus, it is classified as chondroitinase C. The catalytic activity of CHa3 was resisted by O/A/D/E units when the CS substrates were degraded and was resisted by 2-O sulfated regions when the heparin substrate was digested. Tetrasaccharides in the final products of CS, which were degraded by CHa3, included ΔC-A, ΔC-C, ΔC-D, and ΔC-E. The residues Tyr²⁶⁷ and His⁴¹⁷ played crucial roles in the catalytic process, and Arg¹⁰⁹, Arg¹¹⁶, Asn²¹², Asn²¹³, Trp²¹⁴, and Ser⁴¹⁶ may participate in the binding process for CHa3. This study of CHa3 contributes to our understanding of the PL35 family and provides a foundation for investigating the structure of CS.