A novel method for pullulanase immobilized onto magnetic chitosan/Fe3O4 composite nanoparticles by in situ preparation and evaluation of the enzyme stability

A novel immobilization of pullulanase has been developed by using magnetic chitosan/Fe3O4 nanoparticles (CS-MNPs). The CS-MNPs were synthesized by in situ mineralization of Fe3O4 in chitosan hydrogel and characterized by transmission electron microscopy, vibrating sample magnetometer, Fourier transform infrared spectroscopy, thermogravimetric analyzer and X-ray diffractometer. The resultant material (CS-MNPs) was used as support for the covalent immobilization of pullulanase with glutaraldehyde as cross-linker. In the process of pullulanase immobilization, both the amount (g) of immobilized pullulanase and the activity retention(%) increased with enzyme concentration increasing (15-60 mu g/ml), and the optimum activity retention (93.5%) was obtained at pH 3.5. In enzymatic properties, the immobilized enzyme exhibited a broadened pH profiles and higher thermal stability than the free enzyme. The optimum pH of immobilized pullulanase (3.5) was lower than that of the free enzyme (4.4), and the immobilized enzymes could retain about 84% of their initial activity after incubated for 5 h at 60 degrees C in contrast with 58% of free enzyme. furthermore, the immobilized pullulanase retained more than 56% of its initial activity after eight consecutive reuses. These results prove that the CS-MNPs are an effective support for the immobilization of pullulanase. (C) 2014 Elsevier B.V. All rights reserved.