Enzyme immobilization on anodic aluminum oxide/polyethyleneimine or polyaniline composites

Anodic aluminum oxide (AAO) membranes were coated with either polyethyleneimine (PEI) or polyaniline (PANI) and used as supports for enzyme immobilization. The AAO-PEI composite was prepared by cross-flow filtration of the polymer solution through the membrane, whereas AAO-PANI was synthesized using surface chemical polymerization, where MnO2 was used as oxidizing agent for aniline polymerization. After glutaraldehyde activation, horseradish peroxidase and trypsin were immobilized on the composites. The immobilized enzyme activities were determined either in a batch mode or flow-through system. The AAO-PEI-trypsin derivative yielded filtering membrane catalytically active with initial velocities of 2.95 nmol min−1 and 3.8 nmol min−1 measured in batch mode and flow-through system (0.46 mL min−1), respectively. Furthermore, AAO-PEI-trypsin prepared with membranes of different porosities showed that the enzymatic activity increased as the inside diameter of the pore enlarged. Additionally, it was demonstrated that covalent coupling presents higher activity as compared to attachment by adsorption on a membrane without PEI functionalities, which had 2.9 times less activity. Horseradish peroxidase was successfully immobilized onto the AAO-PANI and retained 74% of its initial activity after five batch cycles and 98% following 24 min under continuous flow. It was also observed by electron microscopy that membrane pores remained unclog after polyaniline coating. Therefore, these results showed the viability of these composites as attractive nano-structured matrices for biotechnological applications.