Alkaline protease (22.5 kDa) from Bacillus licheniformis was denatured using 3-6 M Guanidine hydrochloride followed by renaturation with cobalt metal ion in buffer solution (pH 7.5) in order to design cobalt dependent nitrile hydratase AP-xMGdn-Co, where x = 3, 4, 5 and 6. The native alkaline protease showed a 20% residual molar ellipticity at 212 nm while on treatment with Gdn-HCl, it got reduced to 12% due to nearly complete collapse of the globular structure of the protein. The observed circular dichroism spectra of the renatured proteases with Co metal ion are different from the native enzyme, because those molecules contain many metal chelating sites through its coordinating amino acid residues. The introduction of cobalt centered new active site was evidenced by the fluorescence spectroscopy (appearance of a new peak at 360 nm) and ICP analysis (0.0476-0.0553 mg/L). The specific activities of the modified enzymes were measured using the selective catalytic hydrolysis of 3-cyanopyridine to nicotinamide. The highest specific activity of renatured enzymes (AP-xMGdn-Co) were obtained at optimum pH of 7.5 and temperature of 50 degrees C, due to the proper co-ordination of amino acid residue with cobalt metal ion and the appropriate arrangement of alpha-helix, beta-strand, turn and unordered segment in the globular structure. (C) 2012 Elsevier B.V. All rights reserved.