Circular dichroism (CD) was used to characterize the secondary structure of penicillin G acylase upon covalent immobilization on silica nanoparticles. Covalent immobilization was achieved by functionalizing the silica nanoparticles with glutardialdehyde and coupling to the free NH2 groups of the enzyme (lysine and arginine side chains). The loading of the covalently bound enzyme was increased up to saturation, which was reached at 54.6 mg immobilized enzyme per g silica nanobeads. For structural characterization of the commercially available enzyme its exact molecular mass was determined by mass spectrometry in order to enable precise evaluation of the CD data. The fraction of secondary structure elements of the free and immobilized enzyme were estimated from the respective CD spectra using standard algorithms (CONTINLL, CDSSTR, SELCON3). The fractions obtained by the different algorithms for the free enzyme agreed well with one another and also with data from X-ray diffraction described in the literature. Interestingly, the secondary structure fractions found for the immobilized enzyme were very similar to the free enzyme and nearly constant over all experiments. These results indicate that even a loading of up to 55.8 mg/g (enzyme per silica nanoparticles) causes only slight structural changes. However, the specific activity determined by a kinetic assay decreased by around 60%, when increasing the loading from 14.9 to 55.8 mg/g. Because of the fact that we found no major changes in the secondary structure, diffusion limitation seems to be the main reason for the decline of the specific activity. (C) 2007 Elsevier Inc. All rights reserved.