Engineering of aniline dioxygenase for bioremediation and industrial applications.

摘要

The aniline dioxygenase (AtdA) is a multi-component enzyme that has potential uses in bioremediation of aromatic amines and biorefining processes. However, the lack of characterization of the enzyme has limited its development as a practical biocatalyst. The overall objective of this project was to first determine the substrate specificity of AtdA, and probe for the molecular determinants of its substrate specificity as well as its activity. Biomolecular engineering techniques were then used to improve the activity of AtdA as well as to expand its substrate range for application in bioremediation and biorefining.; The first part of the dissertation presents the development of the tools for the engineering of AtdA. An expression system was established and a screening method, which was sensitive and efficient enough to allow for screening of the large genetic libraries generated, was then developed.; Saturation mutagenesis of the active site residues of subunit AtdA3, identified using a homology model, enhanced the promiscuity of AtdA to accept the substrate 2-isopropylaniline (2IPA), which was not accepted by the wild type enzyme. A single V205A mutation was found to be responsible for creating the enhanced substrate range of the mutant 1-K31. This is the first study on the molecular determinants for substrate specificity of a five subunit Rieske-dioxygenase, AtdA, and it was shown that the alpha-subunit of the enzyme (AtdA3) indeed plays a part in controlling the substrate specificity and activity of the enzyme. Using knowledge gained from these findings, saturation and random mutagenesis was then employed to enhance the activity of 1-K31.; Another round of saturation mutagenesis on active site residues with 1-K31 as parent followed by random mutagenesis yielded the mutant 3-R21. Whole cell activity of 3-R21 for AN, 24DMA and 2IPA were 27.7, 9.8, and 12 nmol/min/mg protein respectively. The activities of 3-R21 for AN, 24DMA and 2IPA were improved by 8.9, 98.0, and 2.0-fold respectively over its parent 1-K31. In particular, the activity of the final mutant 3-R21 was improved by 3.5-fold over the WT AtdA enzyme. Overall, mutant 3-R21 had three mutations---V205A, 1248L, and S404C.