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

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.