Preparation and characterization of a novel nanocomposite with double enzymes immobilized on magnetic Fe 3 O 4 -chitosan-sodium tripolyphosphate

In this study, a novel and efficient synchronously immobilization of double enzymes (DE, alcalase and trypsin) were developed by using the chitosan and sodium tripolyphosphate-coated magnetic nanoparticles (Fe3O4@CS-TPP) as support. The physicochemical properties of DE-Fe3O4@CS-TPP NPs were characterized by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), transmission electron microscopy (TEM) and magnetic measurements. The preparation conditions and stabilities were also optimized and assessed. Results showed that the optimum preparation conditions were as follows: ratio of alcalase and trypsin of 3:1, enzyme concentration of 6% (w/w), temperature of 50 °C and pH of 9.0, the enzyme concentration absorbed by Fe3O4@CS-TPP NPs of 6 mg mL−1, immoblization time of 4 h, and glutaraldehyde content of 3% (w/w). The Km and Vmax values were determined as 0.5035 mg mL−1, 6.900 mg mL−1 min−1 for the immobilized enzymes, respectively. DE-Fe3O4@CS-TPP NPs was more stable than the free enzyme above 40 °C. The activity of DE-Fe3O4@CS-TPP NPs was preserved 86% after 35 days storage and retained more than 60% of its initial activity after ten times of successive reuse. Application of DE-Fe3O4@CS-TPP NPs in various protein hydrolysis showed favorable degrees of hydrolysis, yields and antioxidant activity. The results demonstrated that the DE-Fe3O4@CS-TPP NPs were suitable for application in food protein hydrolysis.