Simultaneous One-Step\nPreparation of β/κ-Carrapentaose\nand 3,6-Anhydro-D-galactose by Cascading κ‑Carrageenase\nand an Exo-α-3,6-Anhydro-D-galactosidase

Carrageenan oligosaccharides have shown promising bioavailability\nand possess a variety of physiological activities, making them highly\nsuitable for use in the food, pharmaceutical, and agricultural industries.\nThe preferred method for producing carrageenan oligosaccharides is\nusing various carrageenolytic enzymes, as it offers mild reaction\nconditions, high efficiency, and product specificity. However, there\nis still a lack of specific applications for using these enzymes to\nprepare odd-numbered carrageenan-oligosaccharides (OCOSs). Our previous\nresearch identified a more convenient route for simultaneously preparing\nOCOSs and 3,6-anhydro-D-galactose (D-AHG) using only two types of\ncarrageenolytic enzymes: κ-carrageenase and exo-α-3,6-anhydro-D-galactosidase\n(D-ADAGase). In this study, we utilized a CipA-based self-assembly\nsystem to cascade κ-carrageenase CaKC16A and D-ADAGase ZuGH129A\nfor one-step preparation of β/κ-carrapentaose, G-(DA-G4S)<sub>2</sub>, and D-AHG from degrading β/κ-carrageenan. This\nself-assembled enzyme, namely CipA-CaKC16A-ZuGH129A, can be easily\nobtained through a simple centrifugation process. The final optimized\nenzymatic process produced 0.74 g/L G-(DA-G4S)<sub>2</sub> and 0.13\ng/L D-AHG. This cascade system of different types of carrageenolytic\nenzymes has the potential to achieve the preparation of various types\nof carrageenan oligosaccharides.