The use of papain inhibitor immobilized onto polyaniline for bioaffinity chromatography of cysteine proteases

• Published in: Separation and purification technology Vol. 120; pp. 467 - 472
• Main Authors: Gamboa, Adriane G., Batista, Karla A., Lopes, Flavio M., Fernandes, Kátia F.
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 2013
• Subjects: Adenanthera; affinity chromatography; Bromelain; bromelains; ficain; Ficin; glutaraldehyde; Papain; polyacrylamide gel electrophoresis; Purification; purification methods; seeds; Stationary phase; Bromelain; Papain; Purification; Ficin; Stationary phase
• ISSN: 1383-5866; 1873-3794
• DOI: 10.1016/j.seppur.2013.10.027

•An efficient, fast, easy and inexpensive methodology of immobilization.•New stationary phase for bioaffinity chromatography of cysteine proteases.•A new single step method for purification of ficin, bromelain and papain. 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 30min, at 4°C, pH 7.0, inhibitor concentration of 20mg and 5mg 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 25kDa. SDS–PAGE of purified bromelain showed a characteristic band around 28kDa 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 25kDa in the SDS–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.