Alumina−Pepsin Hybrid Nanoparticles with Orientation-Specific Enzyme Coupling

Abstract

Hybrid alumina nanoparticles with pepsin were prepared in a controlled and efficient manner. Phosphorylated pepsin can be coupled to alumina through the interaction between phosphoserine on pepsin and the alumina surface in an orientation-specific manner. A comparison of data obtained with nanoparticles and microsized alumina particles reveals that the conjugated pepsin retained much higher enzymatic activity when it was immobilized on nanoparticles mainly because of the lack of diffusion limitations of the substrate. Additionally, upon attachment to the alumina nanoparticles, the thermal stability of pepsin is enhanced. The coupled enzyme can be quantitatively released by simply incubating the hybrid nanoparticles with phosphate buffer.Introduction. Nanotechnology has emerged as a powerful tool in the fabrication of materials having superior and often unique properties. 1-4 The incorporation of biological molecules into these materials should expand the range of potential applications to include nanoscale biosensors and biocatalysts. 5 Previous studies on the conjugation of nanoparticles with biomolecules such as proteins and DNA used mainly gold-, silver-, silica-, and nickel-based nanoparticles as well as quantum dots. 6 Several methods have been employed for the attachment of biomolecules on nanoparticles including binding through a thiol group to gold 7,8 and maleimido-modified fullerenes, 9 through amino groups to carboxyl-functionalized paricles, 10 through a polyhistidine tag to nickel, 11 or through electrostatic interactions to charged nanoparticles. 12 Herein, we report a new class of hybrid nanoparticles composed of nanosized alumina functionalized with an enzyme. We demonstrate, using pepsin as a model enzyme, an orientation-specific, efficient, and reversible means to couple phosphorylated proteins to alumina nanoparticles through the interaction between the phosphoryl group and the alumina surface. When alumina nanoparticles are used in this manner, the decrease in enzymatic activity is minimal. A unique property of these hybrid nanoparticles is the ability to release the coupled pepsin quantitatively (98%) in a controlled manner.Pepsin is an enzyme essential to the digestion process in animals. The optimal pH for its activity is 2.0, which is compatible with that of alumina. The enzyme has a total of

Preparation of carboxylated magnetic particles for the efficient immobilization of C-terminally lysine-tagged Bacillus stearothermophilus aminopeptidase II

“…It is believed that surface immobilization of the enzyme on magnetic particles can somehow increase the thermal stability, probably by increasing its molecular rigidity [25], thus preventing any undesirable change in the tertiary molecular structure due to heating. Stability enhancement of immobilized enzymes as a function of temperature has also been observed for other enzymes immobilized on diVerent supports [26][27][28].…”

Immobilization and biocatalytic activity of fungal protease on gold nanoparticle‐loaded zeolite microspheres

“…The covalent linkage between the gold nanoparticles bound to the zeolite surface and F-prot would therefore lead to reduced conformational freedom with respect change in environmental parameters such as temperature and pH. Such enhancement in stability of immobilized enzymes as a function of temperature and pH has been observed for pepsin bound to alumina nanoparticles (Li et al, 2003), and for other enzymes within different supports (Akgol et al, 2002;Arica et al, 1995;Takahashi et al, 2000). Figure 6 shows the effect of temperature on the biocatalytic activity of free F-prot in solution (triangles) and F-prot immobilized on the nanogold-zeolite template (circles) at increasing temperatures in the range of 35j to 65jC, pH 3, for 30 min by using Hb as substrate.…”

Integrated Nanoparticle–Biomolecule Hybrid Systems: Synthesis, Properties, and Applications

“…[35] Reversible binding with specific alignment was observed with pepsin as a model enzyme on alumina NPs through the interaction of the phosphoryl group and the alumina surface. [66] One phosphoryl group is attached to the Ser68 residue in the native porcine pepsin. The crystal structure of the enzyme indicates that this modified residue is positioned at the protein surface, far away from the active site that comprises Asp32 and Asp215.…”