Biotransformation of Phenol to Catechol by Recombinant Phenol Hydroxylase

Abstract

The phenol hydroxylase gene from Pseudomonas putida NCIB10015 was cloned and expressed in a high-copy number plasmid in E. coli DH5α. Expression of the gene was found to be positively regulated. Induction of the phenol hydroxylase activity was shown to be maximal after 3 h of culture. Phenol and cresol isomers were shown to be good inducers of the recombinant phenol hydroxylase and, to a lesser extent, other aromatic compounds such as catechol, 2-amino-phenol and 2-chlorophenol were also inducers of gene expression. In optimal culture conditions, catechol was produced at concentrations up to 4.1mM in cultures of E. coli DH5α carrying the recombinant phenol hydroxylase gene. Phenol hydroxylase activity was strongly inhibited in vivo by high levels of both phenol and catechol. Catechol stability in the culture medium was strictly dependent on the pH of the medium, being optimal at pH 6.0. Several membrane-permeabilizing compounds were tested for their effect on catechol production, although none of them showed a positive effect on the process.