The discovery of a random endo-acting ι-carrageenase with bifunctional activities against ι-carrageenan and κ-carrageenan.

Carrageenans have attracted increasing research interests in recent decades for their various physicochemical and physiological properties. Random endo-acting carrageenases are promising tools for tailoring the molecular weight of carrageenan and preparing a series of carrageenan oligosaccharides. Although the processive ι-carrageenases in the GH82 family have been widely investigated, the random ι-carrageenase has not been reported. Herein, a novel GH82 family protein Cg82Mf, which was identified by AlphaFold2 as lacking a lid structure on the catalytic groove, was cloned and expressed in Escherichia coli. The analysis of hydrolysis pattern proved that Cg82Mf was the first random endo-acting enzyme against ι-carrageenan, and was capable of preparing the hydrolysis products with various degrees of polymerization. Cg82Mf exhibited higher substrate affinity among all characterized ι-carrageenases, reflected by its low K value (0.18 μM). Remarkably, Cg82Mf could also hydrolyze κ-carrageenan, that is, the subsites of the enzyme could tolerate κ-carrageenan disaccharide units, which demonstrated a novel cleavage specificity. The novel hydrolysis pattern and cleavage specificity shed light on the presence of diversity within the GH82 family, and indicated that Cg82Mf could be facilitated as a potential biocatalyst for the molecule tailoring of carrageenans.