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.