Improvement of the Stabilization and Activity of Protocatechuate 3,4-Dioxygenase Isolated from Rhizobium sp. LMB-1 and Immobilized on Fe3O4 Nanoparticles

Protocatechuate 3,4-dioxygenase (P34O), which is isolated from Rhizobium sp. LMB-1, catalyzes the ring cleavage step in the metabolism of aromatic compounds, and has great potential for environmental bioremediation. However, its structure is very sensitive to different environmental factors, which weaken its activity. Immobilization of the enzyme can improve its stability, allow reusability, and reduce operation costs. In this work, the relative molecular mass of the native P34O enzyme was determined to be 500 kDa by gel filtration chromatography on Sephadex G-200, and the enzyme was immobilized onto (3-aminopropyl) triethoxysilane-modified Fe 3 O 4 nanoparticles (NPs) by the glutaraldehyde method. The optimum pH of immobilized and free P34O was unaffected, but the optimum temperature of immobilized P34O increased from 60 to 70 °C, and the thermal stability of immobilized P34O was better than that of the free enzyme and showed higher enzymatic activity at 60 and 70 °C. In addition, with the exception of Fe 3+ , most metal ions and organic chemicals could not improve the activity of free and immobilized P34O. The kinetic parameters of the immobilized P34O were higher than those of the free enzyme, and immobilized P34O on Fe 3 O 4 NPs could be reused ten times without a remarkable decrease in enzymatic activity.