Enhancing the Catalytic Properties of a Novel Bifunctional κ-Carrageenanase through Spontaneous Self-Cyclization Based on the SpyTag/SpyCatcher System

Furcellaran is a distinct carrageenan polysaccharide. The unique oligosaccharides produced by its degradation exhibit lower sulfate group content and play a crucial role in elucidating the structure-activity relationship of carrageenan oligosaccharides. In this study, a novel κ-carrageenanase OUC-CgkA-Sn from <i>Stieleria neptunia</i> was identified to display dual substrate hydrolysis activity against κ-carrageenan and furcellaran. Significantly, the furcellaran-degrading activity of OUC-CgkA-Sn was 1.77-fold higher than that toward κ-carrageenan, and the primary degradation product was desulfurized κ-neocarrahexaose. To enhance its catalytic properties, the SpyTag/SpyCatcher system was employed to construct the fusion enzyme CKT, which is capable of spontaneous self-cyclization <i>in vitro</i>. After cyclization, CKT exhibited a dual enhancement in stability and enzyme activity simultaneously: 193.61% higher half-life and 195.21% greater enzyme activity. Spatial structure analysis revealed that this enhancement stemmed from increased structural stability and strengthened substrate interactions. As a result, CKT holds great promise for the efficient preparation of structurally diverse carrageenan oligosaccharides.