Immobilization of cyclodextrin glucanotransferase on aminopropyl-functionalized silica-coated superparamagnetic nanoparticles

Abstract

Background: Cyclodextrin glycosyltransferase (CGTase) from Amphibacillus sp. NPST-10 was successfully covalently immobilized on aminopropyl-functionalized silica coated superparamagnetic nanoparticles; and the properties of immobilized enzyme were investigated. The synthesis process included preparing of core magnetic magnetite (Fe₃O₄) nanoparticles using solvothermal synthesis; followed by coating of Fe₃O₄ nanoparticles with dense amino-functionalized silica (NH₂-SiO₂) layer using in situ functionalization method. The structure of synthesized Fe₃O₄@NH₂-SiO₂ nanoparticles was characterized using TEM, XRD, and FT-IR analysis. Fe₃O₄@NH₂-SiO₂ nanoparticles were further activated by gluteraaldehyde as bifunctional cross linker, and the activated nanoparticles were used for CGTase immobilization by covalent attachment.

Results: Magnetite nanoparticles was successfully synthesized and coated with and amino functionalized silica layer (Fe₃O₄/NH₂-SiO₂), with particle size of 50-70 nm. The silica coated magnetite nanoparticles showed saturation magnetization of 65 emug^-1, and can be quickly recovered from the bulk solution using an external magnet within 10s. The activated support was effective for CGTase immobilization, which was confirmed by comparison of FT-IR spectra of free and immobilized enzyme. The applied approach for support preparation, activation, and optimization of immobilization conditions, led to high yields of CGTase immobilization (92.3%), activity recovery (73%), and loading efficiency (95.2%); which is one of the highest so far reported for CGTase. Immobilized enzyme showed shift in the optimal temperature from 50 to 55 ºC, and significant enhancement in the thermal stability compared with free enzyme. The optimum pH for enzyme activity was pH 8 and pH 7.5 for free and immobilized CGTase, respectively, with slight improvement of pH stability of immobilized enzyme. Furthermore, kinetic studies revealed that immobilized CGTase had higher affinity toward substrate; with k_m values of 1.18 ± 0.05 mg/ml and 1.75 ± 0.07 mg/ml for immobilized and free CGTase, respectively. Immobilized CGTase retained 87% and 67 of its initial activity after 5 and 10 repeated batches reaction, indicating that immobilized CGTase on Fe₃O₄/NH₂-SiO₂ had good durability and magnetic recovery.

Conclusion: The improvement in kinetic and stability parameters of immobilized CGTase makes the proposed method a suitable candidate for industrial applications of CGTase. To best of our knowledge, this is the first report about CGTase immobilization on silica coated magnetite nanoparticles.