A Novel α2,3-Sialyltransferase Enabling the Synthesis of para α2,3-Sialylated Human Milk Oligosaccharides via Immobilized Cascade Enzymes

Abstract Sialylated oligosaccharides are critical components of human milk oligosaccharides (HMOs). Rapid and efficient synthesis of these oligosaccharides is essential for investigating their functions and applications. In this study, a novel α2,3-sialyltransferase MsST was successfully identified through molecular docking and virtual screening. MsST was cascaded with CMP-sialic acid synthase to form a one-pot multienzyme system, which achieved a conversion rate exceeding 90% for various neutral core HMOs. Subsequently, the cascade enzymes were purified and immobilized in one step using nickel ferrite magnetic nanoparticles. The immobilized cascade enzymes retained 86% of their initial activity after 12 cycles and 70% at 37 °C after storage for 4 days, demonstrating high recyclability and storage stability. Therefore, seven sialylated HMOs, including three para α2,3-sialylated hexasaccharides, were synthesized from the neutral core HMOs in 91–96% recovery yields. This system provides an efficient platform for producing structurally defined α2,3-sialylated HMOs, facilitating the investigation of future functional food applications.