Heterogeneous dual-enzyme cascade platform based on COF and MOF for targeted valorization of rice protein

Rice protein is a high-quality plant-based resource, but its poor solubility limits applications. Deep enzymatic hydrolysis is required to improve functionality and produce bioactive peptides. However, industrial implementation is hindered by the instability and non-reusability of free proteases. Herein, a substrate-matched heterogeneous dual-enzyme cascade platform was constructed by placing two proteases in distinct framework microenvironments according to their catalytic roles and substrate-accessibility requirements. α-Chymotrypsin was covalently immobilized onto magnetic Fe₃O₄@DQTp nanocarriers to maintain an exposed catalytic interface for primary hydrolysis and enable magnetic recovery, achieving a high loading of 218.4 mg/g and 5.1-fold enhanced thermal stability (92.2% activity retained after four cycles). Flavorzyme was encapsulated within ZIF-8 via in-situ biomineralization, which provided rigid framework confinement against thermal and pH stresses, broadening the pH tolerance and remarkably extending stability (86.5% activity retained after five cycles, 84.4% activity retained after 28 days). Through optimized sequential hydrolysis, a final degree of hydrolysis of 58.2% was achieved. This work provides a robust and reusable platform for deep protein hydrolysis, facilitating the targeted valorization of rice protein.