Preparation of different immobilized/stabilized biocatalyst of Alcalase by modification with glutaraldehyde

In this work, different strategies for the stabilization of commercial Alcalase based on glutaraldehyde modification were investigated. First, the free Alcalase was modified with 0.01, 0.1, or 1% (v/v) glutaraldehyde, which led to a significant increase in thermal stability without loss of enzyme activity. Subsequently, two immobilization strategies on aspartic-functionalized agarose (Asp-agarose) were evaluated: (i) immobilization of the glutaraldehyde-modified enzyme, and (ii) glutaraldehyde modification of Alcalase after its cation exchange onto the support. In strategy (i), full immobilization was achieved, but was accompanied by a reduction in expressed activity (∼40%), attributed to intense intermolecular crosslinking on the support as confirmed by SDS-PAGE. In strategy (ii), only ∼60% of the enzyme activity could be immobilized on the support, yet glutaraldehyde modification post-immobilization produced no activity loss and yielded the most stable biocatalyst. In both cases, SDS-PAGE analysis of the supernatants after boiling in SDS showed a marked reduction in protein, indicating extensive intermolecular crosslinking of the immobilized enzyme molecules. These results demonstrate that glutaraldehyde-based chemical modification is an effective approach for Alcalase stabilization, both in free and immobilized forms. The post-immobilization modification strategy offers the best balance between stability and retained activity.