New method for the immobilization of pullulanase onto hybrid magnetic (Fe3O4-κ-carrageenan) nanoparticles by electrostatic coupling with pullulanase/chitosan complex.

We present a simple method to immobilize pullulanase onto hybrid magnetic (Fe3O4-κ-carrageenan) nanoparticles, involving the in situ synthesis of magnetic carrageenan nanoparticles and the formation of pullulanase/chitosan complex. The complex behavior of pullulanase with chitosan as a function of pH and protein-polysaccharide ratio was studied by turbidimetric titration. Then, the as-prepared immobilized enzymes were characterized by vibrating-sample magnetometer, transmission electron microscopy, Fourier transform infrared spectroscopy, X-ray diffractometer, and thermogravimetric analysis. It was found that the activity retention of immobilized pullulanase and amount of enzyme loaded reached 95.5% and 96.3 mg/g, respectively, under optimal conditions. The immobilized enzyme exhibited great operational stability (retaining approximately 61% residual activity after ten consecutive reuses), demonstrating that enzyme leakage during the catalysis reaction was efficiently reduced. Furthermore, the activity of immobilized pullulanase was significantly (p < 0.01) higher than that of free pullulanase in a low pH range (pH < 3.0) and temperature over 60 °C, and the immobilized enzymes retained 45% of their initial activity after 5 h at 60 °C, compared to 21% for the free enzyme. These results indicated that immobilized pullulanase was efficient in terms of catalytic activity and can be applied to continuous starch processing applications in the food industry.