An optimized and efficient method for immobilization and stabilization of penicillin G acylase onto PEI-coated magnetic Fe3O4 nanoparticles.

Objective: The penicillin G acylase (PGA) enzyme, found in bacteria, yeast, and fungi, is used to produce 6-aminopenicillanic acid (6-APA) and beta-lactam antibiotics. To improve the catalytic activity and reusability of PGA, an efficient immobilization protocol was recruited.

Methods: In this research, Fe₃O₄ magnetic nanoparticles (Fe₃O₄ MNPs) were functionalized through polyethylene imine, and the PGA was immobilized on nanoparticles using a glutaraldehyde linker. The nanoparticles were monodispersed with spherical shape and size around 35nm and analyzed by SEM and DLS methods. The optimization process was performed by Design Expert 10.0 software based on the RSM method and CCD design. The immobilization of the enzyme was confirmed by FT-IR.

Results: At optimal stabilization conditions, the maximum amount of 6-APA intermediate substance was obtained at a temperature of 10 °C and a time of 336 minutes. The V_max and K_m were obtained around 0.024 mM and 1.04 mM for free PGA, and 0.47 mM and 1.53 mM for immobilized PGA. The stabilization increased the maximum speed of penicillin hydrolysis by a 2-fold. The antibiotic ampicillin was synthesized using 6-APA and phenylglycine methyl ester (PGME), and the immobilized enzyme maintained 45.87% of the initial activity after 10 reuse cycles, indicating that the immobilized enzyme had good stability and reusability.

Conclusions: Overall, our results showed that this nanoparticle could be considered a promising matrix for PGA immobilization, with the advantages of high catalytic efficiency and enhanced stability and reusability.