Engineering of aniline dioxygenase for bioremediation and industrial applications

Abstract

Enhancement of the bioremediation capabilities of aniline dioxygenase (AtdA), a multi-component enzyme, using biomolecular engineering techniques has been hindered by the lack of knowledge of the structural determinants of its substrate specificity. This project aims to probe for the substrate specificity determinants of AtdA, and improve its bioremediation capabilities by directed evolution. Saturation mutagenesis of the active site residues of subunit AtdA3, identified using a homology model, enhanced the promiscuity of AtdA to accept 2-isoppropylaniline, which was not accepted by the wild type enzyme. Further rounds of saturation and random mutagenesis on AtdA3 improved the activity of AtdA for aniline, 2,4-dimethylaniline, and 2-isoppropylaniline. This study improved the understanding of the structural determinants of the substrate specificity of AtdA, and enhanced the substrate range and activity of AtdA, making it a better enzyme for bioremediation. The AtdA mutant created has potential to be applied for industrial applications as well.