Pseudomonas aeruginosa TKU002, capable of growing significantly on acidic side pH 5.5 and benzoic acid as a sole carbon source, was isolated from enrichment culture accumulation of catechol. An intracellular catechol 1,2-dioxygenase (CD) produced by P. aeruginosa TKU002 was purified and characterized. The TKU002 CD was found to have unique characteristics different to other microbial CDs. The unique characteristics include the smallest molecular mass of 22 kDa, the most acidic pI value of lower than 4, and the highest cleavage activity to the substrate, pyrogallol. Different to other CD producing strains, P. aeruginosa TKU002 produced CD at a lower pH of 5.5 when benzoate was used as the sole carbon source. The TKU002 CD was a monomer with a K-m of 5.9 mu M. The TKU002 CD showed 36% and 14% sequence coverage rate with protocatechuate 3,4-dioxygenase beta-subunit of P. aeruginosa UCBPP-COG3485 and catechol 1,2-dioxygenase of P. aeruginosa PA01, respectively, and possessed one matched peptide (YLWDDFAYATR). In conclusion, this is the first report of a microbial CD with the smallest molecular mass, the most acidic pI value, the highest specific activity to the substrate pyrogallol, and the most acidic medium for CD production. (c) 2006 Elsevier Ltd. All rights reserved.