Cloning, Heterologous Expression, and Biochemical Characterization of a Novel Glycoside Hydrolase 16 Family Enzyme for Biorefinery of Furcellaria lumbricalis.

Abstract

Carrageenan has strong structural heterogeneity, resulting in the production of several hybridized forms in nature. Furcellaran is a typical hybrid type of carrageenan that includes both κ-carrageenan and β-carrageenan motifs in its structure. The discovery and characterization of a novel furcellaranase is of great significance for investigating and determining the structures of carrageenan. Herein, a new GH 16 enzyme CeFurA, with furcellaran and porphyran degrading activities, was cloned, and it included 350 amino acid residues and has a predicted theoretical molecular weight of 40.45 kDa. The enzyme displayed the highest biological activity (824.64 U/mg) on furcellaran at 35 °C and pH 9.0. Notably, CeFurA has excellent temperature stability throughout the wide 25 to 40 °C temperature range. It is useful and promising to efficient prepare hybrid bk-carrageenan oligosaccharides and elucidate the fine structure of the hybrid polysaccharide and oligosaccharides. TLC and ESI-MS indicate that CeFurA, as an endo-type enzyme, can specifically act on DA-Gβ1 → 4DA-G and DA-G4Sβ1 → 4DA-G4S glycosidic linkages within the furcellaran, producing disaccharides, tetrasaccharides, and hexasaccharides as the primary products. The CeFurA exhibited a sandwich-like structure according to structural modeling, which contains an embedded catalytic chamber formed by the β folded sheets placed in a reversing manner by acting on the internal DA-G4Sβ1 → 4DA-G4S glycosidic link. These exceptional properties make CeFurA a powerful tool for studying the heterogeneity of carrageenan structures and producing COS in the industry.