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 with saturation magnetization of 65
emug-1, and can be quickly recovered from the bulk solution using an
external magnet within 10 sec. 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\ubaC, 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 km
values of 1.18 \ub1 0.05 mg/ml and 1.75 \ub1 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