Enzymatic synthesis of selenium (Se)-enriched peptides is vital for their application in supplementing organic Se. However, the poor stability and reusability of the free enzyme impedes the reaction. In this work, a highly stable immobilized Alcalase was synthesized by immobilizing Alcalase on tannic acid (TA) and polyethyleneimine (PEI) modified Fe3O4 nanoparticles (NPs). The optimal immobilization conditions for immobilized Alcalase were found at a TA/PEI (v/v) ratio of 1 : 1, pH of 10, and temperature of 40 °C, and the results from scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier Transform Infrared (FTIR) and X-ray photoelectron spectroscopy (XPS) characterization confirmed the successful immobilization of Alcalase. The results of an enzyme property test showed that immobilized Alcalase had higher thermal and pH stability than free Alcalase, and retained 61.0% of the initial enzyme activity after 10 repetitions. Furthermore, the organic Se content of Se-enriched peptide prepared through enzymatic hydrolysis of Cardamine violifolia (CV) protein with immobilized Alcalase was 2914 mg kg-1, and the molecular weight was mainly concentrated in 924.4 Da with complete amino acid components. Therefore, this study proposes the feasibility of immobilized enzymes for the production of Se-enriched peptides.
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