Characterization of a Novel Alkaliphilic ι-Carrageenase with a pH- Regulated Hydrolytic Mode of Action

The ι-carrageenase is crucial for producing ι-carrageenan oligosaccharides, which are promising for use in food and pharmaceuticals. Here, a novel ι-carrageenase, Cgi82_Fl, was identified and characterized. Cgi82_Fl exhibits optimal activity at 20 °C and pH 10.5, making it the only known ι-carrageenase efficient under strongly alkaline conditions until now. Structural analysis revealed an open active-site cleft lacking a lid, correlating with its random endotype cleavage at optimal pH. Remarkably, its mode of action is found to be pH-dependent, and it switches to a processive endotype mode at neutral pH. Molecular dynamics simulations showed stable binding of ι-carrageenan hexasaccharides mainly via electrostatic interactions. Site-directed mutagenesis identified the conserved E270 as a key catalytic residue, while the other catalytic residue is nonconserved, suggesting a noncanonical mechanism. This study provides an efficient enzyme for preparing ι-carrageenan oligosaccharides under alkaline conditions and highlights the potential to modulate the hydrolytic mode of glycoside hydrolases by pH.