Graphene based magnetic nanocomposites as versatile carriers for high yield immobilization and stabilization of β-galactosidase

Abstract

The present study demonstrates an efficient method for high yield immobilization of Aspergillus oryzae β-galactosidase onto graphene-iron oxide nanocomposites (Gr@Fe₃O₄ NCs) by a simple adsorption mechanism. The synthesized nanocomposites were characterized by X-ray diffraction and FT-Raman spectroscopy, and by vibrating sample magnetometry. The binding of the enzyme onto the nanocomposites was confirmed by transmission electron microscopy, scanning electron microscopy, and Fourier transform-infrared spectroscopy. The bound enzyme showed 90% immobilization yield. The adsorbed and free enzyme both exhibited the same pH-optima at pH 4.5. However, the immobilized enzyme showed enhanced pH stability toward acidic and basic extremes, as well as increased temperature resistance as compared to native β-galactosidase. The Michaelis constant, K_m decreased, while V_max increased, which indicates a higher affinity and activity retention by immobilized β-galactosidase. The bound enzyme retained 83% activity even after its 8th successive reuse. The adsorbed enzyme lost only 21% of its initial activity during storage at 4 °C, while the free β-galactosidase retained only 49% activity under similar storage conditions. The genotoxicity assessment revealed that the nanocomposites showed negligible toxicity to pBR³²² DNA plasmid and human lymphocytes. In view of its easy production, non-toxic nature, improved stability against various denaturants and excellent reusability, the versatile Gr@Fe₃O₄ NCs can serve as an ideal support for the immobilization of other enzymes as well, and it may find applications in constructing biosensors and producing lactose-free dairy products to feed lactose intolerant patients.