Improvement of biochemical properties of asparaginase by immobilization on cysteine functionalized magnetic Fe۳O۴@Au NPs

L-Asparaginase converts L-asparagine to L-aspartic acid and causes cancer cells to starve. The main idea of the current study was to improve the biochemical properties of this enzyme using immobilization onto modified magnetic nano-particles (NPs). To this end, Fe۳O۴ NPs were synthesized, coated with an Au shell, and conjugated with cysteine. The formation of NPs and core-shell structures and their morphology were confirmed using Fourier Transform Infrared spectroscopy (FTIR), Energy Dispersive X-Ray (EDX), VU-Vis, Scanning Electron Microscopy (SEM), and Transmission Electron Microscopy (TEM). Also, Circular Dichroism (CD) and fluorescence spectroscopy were employed for the analysis of the secondary and tertiary structures of the immobilized L-ASNase. The alterations in the kinetic parameters of the immobilized enzyme were analyzed using a Lineweaver-Burk plot. The results of instrumental chemistry analysis confirmed the formation of NPs and core-shell structure, and cysteine binding with the core-shell. Based on CD and fluorescence results, no significant changes were observed in the secondary and tertiary structures of the immobilized enzyme compared to the free one. Kinetic parameters of the immobilized enzyme improved compared to the free enzyme so that Km decreased from ۴.۴۳±۰.۰۵ to ۳.۷۵±۰.۱۲ mM and Vmax increased from ۱۸۷.۲۳±۱۱ to ۲۲۴.۷۸±۱۶ μM min-۱mg-۱. Also, the stability of the immobilized enzyme improved with acidic and alkaline pH values compared to the free one at temperatures higher than ۵۰ ۰C. In addition, the reusability of the immobilized enzyme was superior to the free enzyme, with the immobilized enzyme maintaining ۷۲% of its activity after ۱۵ cycles of catalytic reaction. The immobilized enzyme showed an ۸۶% residual activity after ۱۲۰ min incubation with trypsin, which was higher than the free enzyme (۳۷%). According to the results of this study, immobilization of L-ASNase onto magnetic NPs can be an efficient strategy to enhance the biochemical properties of this enzyme.

Keywords:

asparaginase , Immobilization , Core-shell , iron-gold nano-particles