Biochemical Characterization of a GH16_13 Carrageenase and Identification of Key Amino Acids Governing Product Length

Among the various carrageenases identified to date, only a few GH16_13 family members have been functionally characterized, and these are generally recognized as being specific for hybrid κ/β-carrageenan. Here, we report the expression and biochemical characterization of a GH16_13 carrageenase, AfCar, derived from Aureibaculum flavum . Substrate specificity analysis revealed that AfCar exhibits multifunctional hydrolytic activity toward κ/β-carrageenan, κ-carrageenan, and λ-carrageenan. Notably, AfCar displayed the highest activity on KC among the three substrates. Mechanistically, Tyr123 is a key residue that locks the nonreducing terminal sugar unit to maintain high catalytic activity. Molecular dynamics simulations reveal that the Y123A mutation expands the substrate-binding channel and enhances loop flexibility adjacent to the binding pocket, shifting production toward oligosaccharides with higher degrees of polymerization. With its broad substrate specificity, unique activity profile, thermal adaptability, and tailorable product outcomes, AfCar serves as a promising biotechnological tool for the production of various carrageenan oligosaccharides.