Immobilization and SPR-based binding characterization of L-asparaginase on metal-chelated polymeric nanoparticles.

In this study, poly(2-hydroxyethyl methacrylate-N-methacryloyl-(L)-histidine methyl ester-Cu2+) [PHMCu2+] nanoparticles were synthesized by emulsion polymerization and used as carriers for L-asparaginase (L-ASNase) immobilization. The nanoparticles were characterized using SEM-EDX, TEM, FTIR, Zeta potential analyses. The binding affinity of L-ASNase on metal-chelated polymeric nanoparticles was investigated via surface plasmon resonance (SPR) analysis. The immobilized enzyme (PHM-Cu2+-L-ASNase) achieved an immobilization yield of 86.4 %, activity yield of 73.6 %, and immobilization efficiency of 85.1 %. The optimum pH shifted from 8.5 (free) to 7.0 (immobilized), and the optimum temperature from 45 °C to 50 °C. The PHM-Cu2+-L-ASNase presented enhanced resistance to proteolysis. Kinetic studies revealed that Km and Vmax values decreased after immobilization, indicating increased substrate affinity. Immobilization also resulted in a slightly lower turnover number (kcat) compared to the free enzyme. The PHM-Cu2+-L-ASNase preserved 60 % activity after ten reuse cycles and maintained higher activity in the presence of various metal ions and organic solvents. Thermal, storage, and in vitro artificial human serum stability studies revealed that the PHM-Cu2+-L-ASNase exhibited slower inactivation and extended half-life compared to the free L-ASNase. SPR analysis demonstrated high binding affinity (KD = 0.016 IU/mL), fitting the Langmuir model. Consequently, the PHMCu2+ nanoparticles were effective carriers for L-ASNase enzyme, enhancing its catalytic activity, stability, and reusability for pharmaceutical and industrial use.