Immobilization of HRP onto PAN Membrane 1 Applied Biochemistry and Biotechnology Vol. 110, 2003 1 *Author to whom all correspondence and reprint requests should be addressed. Copyright © 2003 by Humana Press Inc. All rights of any nature whatsoever reserved. 0273-2289/03/110/0001/$20.00 Horseradish Peroxidase Immobilized Through Its Carboxylic Groups onto a Polyacrylonitrile Membrane Comparison of Enzyme Performances with Inorganic Beaded Supports P. R. S. LEIRIÃO, L. J. P. FONSECA, M. A. TAIPA, J. M. S. CABRAL, AND M. MATEUS* Centro de Engenharia Biológica e Química, Instituto Superior Técnico, 1049-001 Lisboa, Portugal, E-mail: marilia.mateus@ist.utl.pt Received April 2002; Revised June 2002; Accepted July 2002 Abstract A hydrophilic polyacrylonitrile (PAN) flat sheet membrane was aminated (8.5 μmol of NH 2 /mg of dry support) for covalent binding of horseradish peroxidase (HRP), mediated by the soluble carbodiimide l-ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDC). Silica microbeads derivatized by silanization, to yield an aminated support, and commercial aminated glass microbeads were also coupled to HRP with EDC or activated with glut- araldehyde. The immobilized enzyme activities were determined in a batch enzyme reactor with an external loop, the highest specific immobilized HRP activity being obtained on the glass support (55.8 U/mg of protein). Continu- ous operational stability studies showed that hydrophilic PAN membrane led to the highest retention of HRP activity after an overall period of 35 h, with a normalized productivity of 59.5 μmol of H 2 O 2 reduced/(h·U immob HRP ). Index Entries: Horseradish peroxidase activity; stability; immobilized enzyme productivity; polyacrylonitrile membrane. Introduction Peroxidases can be used for rapid (or online) detection methods of several analytes in mammalian body fluids, in pharmaceutical and biotech- nological processes, or in commercial food samples. A stable peroxidase is