Overexpression and biochemical characterization of a truncated endo-α (1 → 3)-fucoidanase from alteromonas sp. SN-1009

Abstract

Endo-fucoidanases are important in structural analysis of fucoidans and preparation of fuco-oligosaccharides. However their enzymological properties and analysis of degradation products are scarcely investigated. Truncated endo-α (1 → 3)-fucoidanase Fda1 (tFda1B from Alteromonas sp. was overexpressed and characterized, showing highest activity at pH 7.0, 35 °C, and 1.0 M NaCl. Its K_m and k_cat were 3.88 ± 0.81 mg/mL and 0.82 ± 0.17 min^-1. Fe³⁺ and Mn²⁺ enhanced activity by 100% and 19.5% respectively. Co²⁺ and Cu²⁺ completely inactivated tFda1B, whereas Ni²⁺, Mg²⁺, Zn²⁺, Pb²⁺, Ca²⁺, Ba²⁺ and Li⁺ decreased activity by 58.8%, 56.0%, 50.6%, 47.7%, 28.9%, 15.6% and 37.5%, respectively. Catalytic residues were identified through structure and sequence alignment, and confirmed by mutagenesis. Degradation products of Kjellmaniella crassifolia fucoidan by tFda1B were characterized by LC-ESI-MS/MS, confirming tFda1B belongs to endo-(1 → 3)-fucoidanases, and backbone of K. crassifolia fucoidan is 1 → 3 fucoside linkage. This endo-α (1 → 3)-fucoidanase would be useful for elucidating fucoidan structures, and be used as a food enzyme.

Keywords: Endo-α (1→3)-fucoidanase; Fuco-oligosaccharides; Kjellmaniella crassifolia fucoidan.