Directed evolution of toluene-o-xylene monooxygenase for green chemistry and bioremediation

Date of Completion

January 2005


Biology, Microbiology|Engineering, Chemical|Environmental Sciences




Toluene-o-xylene monooxygenase (ToMO) from Pseudomonas stutzeri OX1 is a multi-component enzyme that oxidizes toluene to 3- and 4-methylcatechol, benzene to phenol as well as degrades all the chlorinated ethenes individually and as mixtures. ToMO was found here to also form catechol and 1,2,3-trihydroxybenzene from phenol, m-nitrophenol (72%) and p-nitrophenol (28%) from nitrobenzene, 4-nitrocatechol from m-nitrophenol and p-nitrophenol, and 3-nitrocatechol (12%) and nitrohydroquinone (88%) from o-nitrophenol. ^ To synthesize novel dihydroxy and trihydroxy derivatives of benzene, methylbenzene, and nitrobenzene, to enhance the degradation of chlorinated aliphatics, as well as to study the role of the active site residues I100, Q141, T201, and F205 of the alpha hydroxylase fragment of ToMO (TouA), DNA shuffling and saturation mutagenesis were used to generate random mutants. Mutants with higher rates of activities and with different regiospecificities were identified from TouA positions I100, A101/M114, A107/E214, A110/E392, M180/E284, T201, F205, and E214/D312/M399. To study the importance of TouA positions M180 and E214, saturation mutagenesis was performed. It was discovered that position TouA M180 influences the regiospecificity of hydroxylation of substituted aromatics, and TouA E214 influences the catalysis rate. By substituting glycine, alanine, valine, glutamine, phenylalanine, and tryptophan at TouA position 214, it was found this residue acts as a gate. TouA positions A101 and A110 was also performed, and it was found saturation mutagenesis of these positions influence catalysis. ^ Eleven novel, industrially-important double- and triple-hydroxylated substituted aromatics, 3-nitrocatechol, 4-nitrocatechol, nitrohydroquinone, methylhydroquinone, 4-methylresorcinol, 3-methylcatechol, resorcinol, hydroquinone, catechol, 1,2,4-trihydroxybenzene, and 1,2,3-trihydroxybenzene, were synthesized. Nonspecific ToMO was converted into a para enzyme, which rivals toluene 4-monooxygenase of P. mendocina KR1 and toluene ortho-monooxygenase of Burkholderia cepacia G4 in its specificity. TouA variant I100Q was identified having 2.8 times better trichloroethylene degradation activity and variant E214G/D312N/M399V having 2.5 times better cis-1,2-dichloroethylene degradation activity. It was also discovered that P. stutzeri OX1 is chemotactic toward chlorinated ethenes and that expression of ToMO from touABCDEF appears to be required for positive chemotaxis. The attraction is stronger with the touR (ToMO regulatory gene). ^