Enhanced enzymatic hydrolysis of chitosan via MOF-mediated activation of chitosanase

Chitosanases are attractive biocatalysts for producing bioactive chito-oligosaccharides (COS), but their applications are limited by insufficient catalytic efficiency, poor stability, and challenging recovery. Although many chitosanases can be activated by metal ions, the direct addition of free ions into reaction systems suffers from low efficiency and separation challenges. This study presented a MOF-based strategy to create a Cu-enriched coordination microenvironment, enabling simultaneous immobilization and activation of marine polysaccharide hydrolases. Taking chitosanase OUC-CsnCA as a model, the immobilization on Cu-trimesic acid (Cu-BTC) doubled its activity (413.5 ± 34.7 U/mg) and increased k_cat from 361.8 ± 8.0 to 704.4 ± 16.1 min^-1, along with improved thermostability and pH tolerance. MALDI-TOF-MS and HPLC confirmed deeper substrate depolymerization upon immobilization. Kinetic and spectroscopic analyses revealed that Cu²⁺-binding modulated enzyme conformation and promoted enzyme-substrate interactions. Molecular dynamics simulations further indicated that Cu²⁺ coordination was associated with adjustments in catalytic cleft geometry (D102-E84) and reinforcement of hydrogen-bond interactions within the substrate-binding pocket. Enhanced activities were further observed for immobilized ι-carrageenase and α-agarase, demonstrating the potential applicability of this strategy to other metal-responsive marine polysaccharide hydrolases.