Utilizing heterologously overexpressed endo-1,3-fucanase to investigate the structure of sulfated fucan from sea cucumber (Holothuria hilla)

Sea cucumber sulfated fucan (SC-FUC) attracted increasing interests in the recent decades. Endo-1,3-fucanase has been employed in the structural clarification and structure-function relationship investigations of SC-FUC. Nevertheless, the preparation of wild-type endo-1,3-fucanase is costly and time-consuming, which hinders its further utilization. In this study, a heterologously overexpressed endo-1,3-fucanase (FunA) was introduced into structural identification of SC-FUC. FunA was efficiently prepared within one day and utilized in the investigation of sulfated fucan from sea cucumber Holothuria hilla (Hh-FUC). By using enzymatic degradation, glycomics and NMR analysis, the major structure of Hh-FUC was identified to be composed of a tetrasaccharide repeating unit →3-α-L-Fucp-1 → 3-α-L-Fucp2,4(OSO₃⁻)-1 → 3-α-L-Fucp2(OSO₃⁻)-1 → 3-α-L-Fucp2(OSO₃⁻)-1→. Due to the efficient acquisition of enzyme and the superior oligosaccharide recovery, 0.6 mL of E. coli broth and 10 mg of Hh-FUC were sufficient for the structural identification. The results demonstrated the superiority of heterologously overexpressed fucanase over its wild-type enzyme in structural investigation of sulfated fucan.