Construction of porous chitosan macrospheres via dual pore-forming strategy as host for alkaline protease immobilization with high activity and stability

Fabrication of highly-efficient enzymatic supports having excellent affinity to enzymes and superior mass transfer properties is highly desirable for enzymatic bio-catalysis. Herein, newly engineered chitosan macro -spheres having interconnected and interlaced network pores are prepared via dual pore-forming strategy and applied as novel host for the effective immobilization of alkaline protease. The synergetic effect of SiO2 templates and gas-induced pore-forming agents play an important role in inhibiting the over-crosslinking of chitosan chains and promoting the elevation of interior porosity. Benefited from the highly exposed surface and abundant available binding sites, the as-developed porous support P2CSM achieves a maximum loading capacity of 43.8 +/- 0.8 mg/g and ultra-high activity recovery of 92.4 % for alkaline protease. P2CSM is competent to effectively stabilize the structural conformation of alkaline protease from inactivation through the flexible covalent inter-action. Considering these attributes, Protease@P2CSM demonstrates remarkably better structural stability, reusability and SDS-resistance than free alkaline protease, as well as excellent proteolytic ability, and the residual activity of Protease@P2CSM is evaluated as high as 70.3 % after 7 consecutive reuses. This work provides a promising avenue to construct highly-active enzyme-composites for widespread utilization in various practical applications.

Automated summary

Fabrication of chitosan macro-spheres with interconnected and interlaced network pores through a dual pore-forming strategy enables effective immobilization of alkaline protease. The resulting porous support P2CSM exhibits high loading capacity and activity recovery for alkaline protease. Protease@P2CSM demonstrates superior structural stability, reusability, SDS-resistance, and proteolytic ability, making it a promising enzyme-composite for practical applications.