Influence of cationic species on β-agarase-mediated depolymerization of agarans

This study explores the enzymatic degradation of red algal galactans-agarose, porphyran, and funoran-using 19 commercial hydrolases, including β-galactosidases, β-galactanases, β-agarases, and β-porphyranases. While agarose and porphyran hydrolysis is well studied, funoran degradation, particularly under ionic conditions, remains poorly understood. We evaluated the impact of mono- (Na⁺, K⁺, NH₄⁺) and divalent (Mg²⁺, Ca²⁺, Ba²⁺) cations on β-agarase 86A (GH86). Divalent ions, especially Ba²⁺, significantly enhanced funoran hydrolysis, with optimal activity at 20 mM BaCl₂. This enhancement is attributed to Ba²⁺ shielding -OSO₃^- groups in funoran, facilitating structural rearrangements that improve enzyme access. Notably, β-agarase 86A exhibited broad substrate specificity, cleaving β-(1→4) linkages in all three galactans. NMR analysis confirmed cleavage between G6S and LA residues in funoran, releasing LA-G6S disaccharides via an endo-acting mechanism. This is the first report demonstrating Ba²⁺-mediated enhancement of funoran degradation. These results provide mechanistic insights into ion-assisted enzymatic depolymerization of sulfated agarans. The findings broaden the substrate range of GH86A enzymes and highlight Ba²⁺ as a key modulator for improving enzymatic accessibility to complex sulfated galactans, with potential applications in biocatalysis and bioactive oligosaccharide production.