Nano-engineered Alginate Lyase–Chitosan conjugates as a novel therapeutic strategy against biofilm-associated infections

Background: Alginate lyase plays a critical role in the degradation of alginate, a major component of bacterial biofilms, particularly in Pseudomonas aeruginosa. This study aimed to isolate, purify, and characterize alginate lyase from a local P. aeruginosa isolate, followed by its immobilization onto chitosan nanoparticles to enhance biological activity and biocompatibility. Methods: Forty local isolates were initially screened on alginate agar, from which the most efficient producer was selected based on hydrolysis zone ratios. Enzyme production was optimized through systematic evaluation of carbon sources, pH, temperature, and incubation duration, yielding maximum activity at 0.5% glucose, pH 7.0, 35°C, and 24 hours, respectively. Purification steps—including ammonium sulfate precipitation, DEAE-cellulose ion-exchange chromatography, and Sephadex G-100 gel filtration resulted in a 5.26-fold purification and a specific activity of 1.378 U/mg. Chitosan nanoparticles were synthesized using ionic gelation with Sodium Tripolyphosphate (TPP), followed by successful enzyme loading, as confirmed by UV–visible spectroscopy, FTIR analysis, and XRD patterns demonstrating semi-crystalline structural features. Results: The nanoenzyme formulation (A.L. CsNPs) exhibited superior antibacterial and antibiofilm properties compared with the free enzyme, showing the highest inhibition against Klebsiella pneumoniae and Staphylococcus haemolyticus. Hemolysis assays indicated excellent biocompatibility, with minimal cytotoxicity toward human erythrocytes. Conclusion: These findings demonstrate that chitosan-based nano-immobilization substantially enhances the stability, efficacy, and biomedical applicability of alginate lyase, providing a promising platform for treating biofilm-associated infections.