Size-Controlled Fabrication of Alginate Hydrogel Microbeads Optimized for Lipase Entrapment

Enzyme entrapment in alginate hydrogel microbeads is an effective method of immobilization for industrial applications, but many fabrication methods for alginate microbeads involve oil, organic solvents, or high temperatures that reduce enzymatic activity. In this study, we employed an oil- and solvent-free gas-shearing technique to prepare alginate microbeads for the entrapment of Candida rugosa lipase (CRL), thereby minimizing thermal- and solvent-induced inactivation. To enhance immobilization efficiency and reusability, the effects of gas flow rate, alginate concentration, and cross-linking metal ions were systematically investigated. CRL entrapped in Ba- and Fe-alginate microbeads showed superior immobilization yield, activity retention, and activity recovery compared with CRL entrapped in conventional Ca-alginate microbeads. Notably, both Ba- and Fe-alginate microbeads exhibited significantly enhanced stability, with half-lives up to 127-fold greater than that of free CRL at 60 °C, and maintained substantially higher pH stability across the tested range. Ba-alginate microbeads provided greater pH stability and substrate affinity, whereas Fe-alginate microbeads demonstrated enhanced thermal stability and catalytic turnover. These findings highlight gas-shearing as a scalable and gentle fabrication method for producing high-performance alginate microbeads with tunable properties, making them suitable for enzyme entrapment in diverse biocatalytic applications.