Gordonia sp. strain P8219, a strain able to decompose di-2-ethylhexyl phthalate, was isolated from machine oil-contaminated soil. Mono-2-ethylhexyl phthalate hydrolase was purified from cell extracts of this strain. This enzyme was a 32,164-Da homodimeric protein, and it effectively hydrolyzed monophthalate esters, such as monoethyl, monobutyl, monohexyl, and mono-2-ethylhexyl phthalate. The K m and V max values for mono-2-ethylhexyl phthalate were 26.9 ؎ 4.3 M and 18.1 ؎ 0.9 mol/min · mg protein, respectively. The deduced amino acid sequence of the enzyme exhibited less than 30% homology with those of meta-cleavage hydrolases which are serine hydrolases but exhibited no significant homology with the sequences of serine esterases. The pentapeptide motif GXSXG, which is conserved in serine hydrolases, was present in the sequence. The enzymatic properties and features of the primary structure suggested that this enzyme is a novel enzyme belonging to an independent group of serine hydrolases.Phthalate diesters (PAEs), such as di-2-ethylhexyl phthalate (DEHP) and diisononyl phthalate, are used as plasticizers on a massive scale in the industrial production of plastics. The annual amount of PAEs produced was estimated to be several hundred kilotons for Japan alone in 1995 and is estimated to have been increasing. These compounds arise from domestic and industrial wastewater because PAEs easily leak from plastic goods, such as tableware, and might be discharged from plastic-producing plants. PAEs are recalcitrant compounds and have accumulated as environmental contaminants, and they have been detected in the water and sediments of rivers, seas, and lakes at many points around the world (10,28,35). PAEs are suspected to be endocrine-disrupting chemicals (5,13,16,32,37). Reports of developmental toxicity and teratogenic effects of these compounds on animals have been increasing (4,17,36).Because PAEs are considered to be difficult to decompose, removal of these compounds from wastewater has become an issue. It was shown first that biodegradation of PAEs occurs in river water and in acclimated activated sludge (31). After this, microorganisms that could grow using PAEs as a sole carbon source were isolated from soils and enriched wastewater samples. The microorganisms were identified and were reported to be Rhodococcus erythropolis, Pseudomonas species, and members of the genus Corynebacterium (3, 21).It was reported previously that phthalate esters are hydrolyzed by specific and/or nonspecific esterases in many tissues of mammals and plants and in microorganisms (6,18,24,29,33). Phthalate diesters are metabolized to monoesters in many tissues of mice (18) and in human blood plasma (33). The purified specific esterases from mouse hepatic microsomes and human saliva have activity that hydrolyzes phthalate diesters to the corresponding monoesters (18,29). Purified wheat plant esterase exhibits activity that hydrolyzes DEHP to mono-2-ethylhexyl phthalate (MEHP) (20). Higher organisms, such as mammals and plants, metabolize ...