Background: Cyclodextrin glucanotransferase (CGTase) from Amphibacillus sp. NPST-10 was covalently immobilized onto amino-functionalized magnetic double mesoporous core-shell silica nanospheres (mag@d-SiO₂@m-SiO₂-NH₂), and the properties of the immobilized enzyme were investigated. The synthesis process of the nanospheres included preparing core magnetic magnetite (Fe₃O₄) nanoparticles, coating the Fe₃O₄ with a dense silica layer, followed by further coating with functionalized or non-functionalized mesoporous silica shell. The structure of the synthesized nanospheres was characterized using TEM, XRD, and FT-IR analyses. CGTase was immobilized onto the functionalized and non-functionalized nanospheres by covalent attachment and physical adsorption.

Results: The results indicated that the enzyme immobilization by covalent attachment onto the activated mag@d-SiO₂@m-SiO₂-NH₂, prepared using anionic surfactant, showed highest immobilization yield (98.1%), loading efficiency (96.2%), and loading capacity (58 µg protein [CGTase]/mg) nanoparticles which were among the highest yields reported so far for CGTase. Compared with the free enzyme, the immobilized CGTase demonstrated a shift in the optimal temperature from 50ºC to 50-55ºC, and showed a significant enhancement in the enzyme thermal stability. The optimum pH values for the activity of the free and immobilized CGTase were pH 8 and pH 8.5, respectively, and there was a significant improvement in pH stability of the immobilized enzyme. Moreover, the immobilized CGTase exhibited good operational stability, retaining 56% of the initial activity after reutilizations of ten successive cycles.

Conclusion: The enhancement of CGTase properties upon immobilization suggested that the applied nano-structured carriers and immobilization protocol are promising approach for industrial bioprocess for production of cyclodextrins using immobilized CGTase.