Immobilization of β-Agarase onto Ni-NTA Magnetic Nanoparticles for Efficient Production of Functional Neoagarooligosaccharides

AgaDcat is a promising β-agarase for the production of functional neoagarooligosaccharides (NAOSs), but its limited thermal stability and poor reusability as a free enzyme increase enzyme consumption and production cost during repeated agarose hydrolysis. In this study, β-agarase AgaDcat was immobilized on nickel-nitrilotriacetic acid magnetic nanoparticles (Ni-NTA-MNPs) through His-tag/Ni-NTA affinity interactions. The successful preparation of Ni-NTA-MNPs and the immobilization of AgaDcat were verified by SEM, EDS, VSM, FT-IR, XRD, and TGA. Following optimization, the immobilized enzyme exhibited an activity retention of 80.47%. Thermal stability assays showed that the immobilized enzyme was more thermostable than the free enzyme. After incubation at 50 °C for 60 min, the immobilized enzyme retained 59.4% of its initial activity, whereas the free enzyme retained only 29.1%. Moreover, the immobilized enzyme displayed good reusability, retaining 90.59% and 58.71% of its activity after 3 and 7 cycles, respectively. HPAEC-PAD analysis showed that the immobilized enzyme reached reaction equilibrium within 4–8 h, with neoagarotetraose (NA4) and neoagarohexaose (NA6) identified as the major degradation products, whereas the free enzyme required 20 h, indicating significantly improved catalytic efficiency. These results indicate that IMAC-based immobilization improves the stability, reusability, and catalytic efficiency of β-agarase, providing a promising reusable biocatalytic strategy for the efficient production of functional neoagarooligosaccharides.