Enhanced Stability and Reusability of Subtilisin Carlsberg Through Immobilization on Magnetic Nanoparticles.

Abstract

Introduction: Immobilizing enzymes on solid supports such as magnetic nanoparticles offers multi-dimensional advantages, including enhanced conformational, structural, and thermal stability for long-term storage and reusability.

Methodology: The gene encoding subtilisin Carlsberg was isolated from proteolytic Bacillus haynesii, a bacterium derived from salt mines. The nucleotide sequence encoding pro-peptide and mature protein were cloned into pET22(a)+ vector and expressed in E. coli. The extracted enzyme was subsequently immobilized on glutaraldehyde-linked-chitosan-coated magnetic nanoparticles.

Results: Fourier-transform infrared analysis revealed higher intensity peaks for the enzyme-immobilized nanoparticles indicating an increase in bonding numbers. X-ray diffraction analysis revealed a mild amorphous state for immobilized nanoparticles in contrast to a more crystalline state for free nanoparticles. An increased mass content and atomic percentage for carbon and nitrogen were recorded in EDX analysis for enzyme immobilized magnetic nanoparticles. Dynamic light scattering analysis showed an increase in average particle size from ~85 nm to ~250 nm. Upon enzyme immobilization, the Michaelis-Menten value increased from 11.5 mm to 15.02 mM, while the maximum velocity increased from 13 mm/min to 22.7 mm/min. Immobilization significantly improved the thermostability with 75% activity retained by immobilized enzyme at 70 °C compared to 50% activity by free enzyme at the same temperature. Immobilization yield, efficiency and activity recovery were 61%, 84% and 51%, respectively. The immobilized enzyme retained 70% of its activity after 10 cycles of reuse, and it maintained 55% of its activity compared to 50% activity by free enzyme after 30 days of storage.

Conclusion: The present study highlights the efficacy of magnetic nanoparticle-based immobilization in enhancing enzyme functioning and facilitates its incorporation into commercial applications necessitating high stability and reusability, including detergents, medicines, and bioremediation.