Simultaneous One-Step Preparation of β/κ-Carrapentaose and 3,6-Anhydro-D-galactose by Cascading κ-Carrageenase and an Exo-α-3,6-Anhydro-D-galactosidase.

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