Enzymatic synthesis of antioxidant peptides with controllable and adjustable molecular weights using magnetically recyclable immobilized Alcalase.

Enzymatic hydrolysis of proteins to obtain bioactive peptides is increasingly attractive, but the poor stability and low reusability of enzymes remain unsolved. Here, the magnetically recyclable immobilized Alcalase (Alcalase@SGO-PEGA) was constructed by immobilizing the free protease of Alcalase to the superparamagnetic graphene oxide (SGO) whose surface was modified with polyethylene glycol diamine (PEGA). The results indicate that Alcalase@SGO-PEGA significantly improved the thermostability and pH tolerance of Alcalase, withstanding temperatures up to 70 °C and pH levels up to 12. Additionally, Alcalase@SGO-PEGA with a saturation magnetizations (Ms) of 20.64 emu/g allowed for efficient recovery using external magnetic fields, and its catalytic stability was demonstrated by retaining 50 % of its initial activity after seven cycles of reuse. Using Alcalase@SGO-PEGA for the enzymatic hydrolysis of soy protein isolate, casein, bovine, serum protein, β-lactoglobulin, sesame protein and flaxseed, bioactive peptides with different molecular weights were obtained by adjusting the hydrolysis temperature and time. Additionally, the antioxidative capacity of the bioactive peptides was confirmed by their ABTS+ free radicals scavenging rate and Fe2+ chelating activity. This paper presents a novel, sustainable strategy for obtaining antioxidant peptides with adjustable molecular weights using magnetically recyclable immobilized Alcalase, advancing its application and promoting cleaner protein processing.