A Chitosan-Binding Protein Mediated the Affinity Immobilization of Enzymes on Various Polysaccharide Microspheres

In this study, we developed an innovative method for one-step enzyme purification and immobilization utilizing polysaccharide-based microspheres through a chitosan-binding module that mediated affinity adsorption. The chitosan-binding domain derived from Paenibacillus sp. IK-5 was genetically fused with multiple target enzymes (lysine decarboxylase, glutamate oxidase, and formate dehydrogenase), all of which were successfully expressed in soluble forms. Three distinct polysaccharide microspheres with optimized surface characteristics were engineered to facilitate the concurrent purification and immobilization of these fusion enzymes. Comprehensive characterization using organic elemental analysis, fluorescence microscopy, and thermogravimetric analysis confirmed the efficient immobilization of fusion enzymes. Remarkably, the immobilized enzymes demonstrated exceptional operational stability, maintaining over 80% of their initial catalytic activity after ten consecutive reuse cycles. This study establishes a robust and versatile platform for enzyme immobilization, providing significant advantages in biocatalyst engineering applications.