A polymeric support based on the natural silk fibers was prepared and characterized for covalent immobilization of -galactosidase from Escherichia coli. The silk fibers were grafted using polyacrylonitrile in presence of benzophenone as a photo-initiator. The grafted fibers were then activated by treatment with hydrazine hydrate followed by glyoxal cross-linker. FTIR spectra, scanning electron microscope (SEM) in addition to the staining test derived from the Coomassie protein assay were utilized for investigation of the modification and immobilization steps. Also, the activity of both free and immobilized -galactosidase was evaluated as a function of the various important parameters like grafting percentage, pH, and temperature. In addition, the kinetic parameters K-m and v(max) for both free and immobilized enzyme were anticipated using Michaelis-Menten equation. The results in this study indicated that the prepared modified woven silk fibers could be used effectively as a polymeric support for immobilization of -galactosidase. (c) 2013 Wiley Periodicals, Inc.