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