Comparative Study of Enzymatic Lipolysis Using Nanofructosome-Coated CalB Lipase Encapsulated in Silica and Immobilized on Silica-Coated Magnetic Nanoparticles

This study evaluates the enzymatic lipolysis performance of nanofructosome-coated CalB lipase (CalB@NF) encapsulated in silica and immobilized on silica-coated magnetic nanoparticles (Si-MNP) for converting natural olive oil to oleic acid. The nanofructosome coating, composed of levan, a nanosized fructan polymer, was applied to enhance the heat and acid resistance of the CalB enzyme. To further improve functionality, CalB@NF was encapsulated in silica (CalB@NF@SiO₂) or immobilized on Si-MNP using a chloropropylsilane linker. The silica-encapsulated CalB@NF (CalB@NF@SiO₂) was synthesized via a sol-gel process, resulting in an average particle size of 304 nm, while the immobilized CalB@NF on Si-MNP exhibited a smaller average particle size of 58 nm. Quantitative determination of CalB in both formulations was conducted using the Bradford assay, yielding concentrations of 19.5 μg/mL for CalB@NF@SiO₂ and 44.9 μg/mL for CalB@NF@Si-MNP. Enzymatic lipolysis was evaluated by measuring the production of oleic acid from natural olive oil. CalB@NF@Si-MNP achieved complete lipolysis within 3 h, whereas CalB@NF@SiO₂ required 24 h to reach the same result. The lipolysis rates were 0.92 mmol/h for CalB@NF@Si-MNP and 0.21 mmol/h for CalB@NF@SiO₂, indicating that CalB@NF@Si-MNP was 4.5 times faster. Regarding reusability, CalB@NF@SiO₂ retained 20% more activity compared to CalB@NF@Si-MNP. While the reusability of CalB@NF@Si-MNP decreased to 76% after the first cycle, CalB@NF@SiO₂ maintained nearly 100% reusability across multiple cycles. These results highlight the complementary strengths of the two formulations: CalB@NF@SiO₂ offers controlled lipolysis rates, high stability, and excellent reusability, whereas CalB@NF@Si-MNP excels in rapid lipolysis. Both silica encapsulation and silica-coated magnetic nanoparticles demonstrate substantial potential for optimizing enzyme activity, stability, and reusability in diverse applications.