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 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 55oC, 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 ± 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.
Highlights
Cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) is one of most industrially important enzyme that able to convert starch and related sugars, via a cyclization reaction, into non-reducing cyclic oligosaccharides called cyclodextrins (Biwer et al 2001)
The activated support was effective for Cyclodextrin glycosyltransferase (CGTase) immobilization, which was confirmed by comparison of Fourier transform infrared spectroscopy (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
Summary
Cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) is one of most industrially important enzyme that able to convert starch and related sugars, via a cyclization reaction, into non-reducing cyclic oligosaccharides called cyclodextrins (Biwer et al 2001). Recent breakthroughs in nanotechnology have made various nanostructured materials more affordable for a broader range of applications including enzyme immobilization (Wang, 2006; Ansari and Husain, 2011). In this regard, nanoscale materials provide the upper limits in balancing the key factors that determine the efficiency of biocatalysts, including surface area/volume ratio, mass transfer resistance, and effective enzyme loading (Kim et al 2006a; Mateo et al 2007; Lei et al 2008; Lee et al 2009). To best of our knowledge, this is the first report about CGTase immobilization on silica coated magnetite nanoparticles
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