Error prone pcr mutagenesis of toluene dioxygenase of Pseudomonas putida T57 to enhancedegradation of 4-chloroaniline

Abstract

Chloroanilines are important intermediates for the production of polyurethanes, rubber additives, pesticides and dye. 4-Chloroaniline (4CA) is one of chloroaniline group, which is an aromatic amine ubiquitously detected to accumulate during the growing of agricultural and industrial activities. 4CA is toxic toward human and living organism, so it requires appropriate technique for treatment. Bioremediation is an effective technique using whole-cell microorganisms or enzymes. The recent report demonstrated that Escherichia coli recombinant strain harboring todC1C2BA, the gene encoding toluene dioxygenase from Pseudomonas putida T57, was able to oxidize 4CA, but with limited efficiency. Consequently, this study aimed to enhance the enzyme efficiency toward 4CA using error prone PCR technique to random mutagenesis of todC1C2 gene. The degradation rate of the obtained E. coli recombinant strain (pET21atodC1C2’BA) was determined by the modified Gibbs method. The degradation rate of the mutants was then compared with that of the wild-type. The changing in amino acid residues of the mutated enzyme was identified by nucleotide sequence analysis. After one round of random mutagenesis, the mutants were selected with the higher degradation rate of 4CA or aniline using the modified Gibbs method. The selected mutants were confirmed the degradation rate by HPLC analysis. The kinetics parameter of mutants showed the high catalytic efficiency compared to the wild-type enzyme. The mutated position (E339 and V345) of todC1 revealed to be involved in substrate binding site.