Enhanced catalytic performance of penicillin G acylase by covalent immobilization onto functionally-modified magnetic Ni_0.4Cu_0.5Zn_0.1Fe₂O₄ nanoparticles

by Zhixiang Lv, Zhou Wang, Shaobo Wu, Xiang Yu

With the emergence of penicillin resistance, the development of novel antibiotics has become an urgent necessity. Semi-synthetic penicillin has emerged as a promising alternative to traditional penicillin. The demand for the crucial intermediate, 6-aminopicillanic acid (6-APA), is on the rise. Enzyme catalysis is the primary method employed for its production. However, due to certain limitations, the strategy of enzyme immobilization has also gained prominence. The magnetic Ni_0.4Cu_0.5Zn_0.1Fe₂O₄ nanoparticles were successfully prepared by a rapid-combustion method. Sodium silicate was used to modify the surface of the Ni_0.4Cu_0.5Zn_0.1Fe₂O₄ nanoparticles to obtain silica-coated nanoparticles (Ni_0.4Cu_0.5Zn_0.1Fe₂O₄-SiO₂). Subsequently, in order to better crosslink PGA, the nanoparticles were modified again with glutaraldehyde to obtain glutaraldehyde crosslinked Ni_0.4Cu_0.5Zn_0.1Fe₂O₄-SiO₂-GA nanoparticles which could immobilize the PGA. The structure of the PGA protein was analyzed by the PyMol program and the immobilization strategy was determined. The conditions of PGA immobilization were investigated, including immobilization time and PGA concentration. Finally, the enzymological properties of the immobilized and free PGA were compared. The optimum catalytic pH of immobilized and free PGA was 8.0, and the optimum catalytic temperature of immobilized PGA was 50°C, 5°C higher than that of free PGA. Immobilized PGA in a certain pH and temperature range showed better catalytic stability. V_max and K_m of immobilized PGA were 0.3727 μmol·min^-1 and 0.0436 mol·L^-1, and the corresponding free PGA were 0.7325 μmol·min^-1 and 0.0227 mol·L^-1. After five cycles, the immobilized enzyme activity was still higher than 25%.