Functionalized superparamagnetic graphene oxide nanosheet in enzyme engineering: A highly dispersive, stable and robust biocatalyst

Abstract

Immobilized enzymes are used as biocatalysts for analytical purposes in diagnostics and for preparative purposes in large scale industrial processes. Immobilization can increase the half-life, improve the stability, and increase the catalytic activity of an enzyme. It facilitates the separation and recovery of the catalyst from the reaction products, and allows for multiple use of the biocatalysts. Graphene oxide nanosheets were decorated with superparamagnetic iron oxide nanoparticles (SPGO) and functionalized with cyanuric chloride (SPGO-CC) to serve as solid support for the covalent immobilization of enzymes. Xylanase was attached to the functionalized nanocomposite with a yield of 215mg protein pergram SPGO-CC. The kinetic constants of the immobilized (of the free soluble) xylanase are Km=4.9 (4.1)mg/mL, vmax=1.6 (1.7)μmolmin−1mL−1, and kcat=82 (91)min−1, indicating a minimal impairment of catalytic activity by covalent coupling. The immobilized xylanase has shallow pH and temperature optima of 6.5 and 60°C. It retains 70% of the original activity after 10 cycles of 15min incubation with substrate (polymeric xylan) at 60°C. It retains 80% of the original catalytic activity after 3.5months storage at 4°C, whereas the free, soluble enzyme retains only 50%.