In this work, a papain inhibitor extracted from seeds of Adenanthera pavonina was covalently immobilized onto glutaraldehyde modified polyaniline (PANIG). The immobilization was very efficient, presenting 54.24% inhibitor retention in the following conditions: immobilization time of 30 min, at 4 degrees C, pH 7.0, inhibitor concentration of 20 mg and 5 mg of PANIG. The resultant material (PANIG-I) was tested as stationary phase for the cysteine proteases papain, ficin and bromelain purification through bioaffinity chromatography. A commercial preparation of papain was efficiently purified, resulting in a single band with 25 kDa. SDS-PAGE of purified bromelain showed a characteristic band around 28 kDa and this procedure resulted in 0.89-fold purification and 32.4% yield. The purification process was more effective for ficin reaching 2.6-fold purification and a single band of 25 kDa in the SOS-PAGE. These results showed that the stationary phase containing A. pavonina inhibitors immobilized onto PANIG is a very promising material for a single step purification of cysteine proteases through bioaffinity chromatography. (C) 2013 Elsevier B.V. All rights reserved.