CO2 bubble-regulated porous architecture of CS@ZIF-8 microspheres for enhanced alcalase immobilization with improved walnut protein hydrolysis.

Alcalase shows potential for hydrolyzing hydrophobic proteins, but its use is limited by poor stability and reusability. This study fabricated porous chitosan/ZIF-8 (CS@ZIF-8) microspheres through gas-shearing microfluidics coupled with CO2 bubble regulation. Alcalase was immobilized on microspheres to form a microreactor (Alcalase@CS@ZIF-8) for walnut protein (WP) hydrolysis. SEM showed that the ratio of NaOH to Na2CO3 controlled the pore structure of CS@Zn2+ microspheres. The optimized CS@ZIF-8 microspheres, prepared with a NaOH to Na2CO3 volume ratio of 7:3, displayed a specific surface area of 32.46 m2/g and an average pore diameter of 4.80 nm, with an enhanced enzyme loading rate and activity recovery of 12.43% and 5.76%, respectively. FTIR and XPS analysis confirmed that alcalase was immobilized on CS@ZIF-8 microspheres through hydrogen bonding, covalent interactions and metal coordination. Secondary structure analysis showed that immobilization on CS@ZIF-8 microspheres increased alcalase β-sheet content from 38.41% to 59.54%, leading to a higher pH stability and thermal stability compared with free alcalase and Alcalase@CS, and retaining 12.24% of its initial activity after eight consecutive reuse cycles. The hydrolysis degree and ABTS radical scavenging activity of WP hydrolysate were enhanced by Alcalase@CS@ZIF-8. These results proved that porous CS@ZIF-8 microspheres are effective supports for enzyme immobilization and protein hydrolysis.