Genetic diversity of catechol 1,2-dioxygenase in the fecal microbial metagenome

Catechol 1,2-dioxygenase is the key enzyme that catalyzes the cleavage of the aromatic ring of catechol. We explored the genetic diversity of catechol 1,2-dioxygenase in the fecal microbial metagenome by PCR with degenerate primers. A total of 35 gene fragments of C12O were retrieved from microbial DNA in the feces of pygmy loris. Based on phylogenetic analysis, most sequences were closely related to C12O sequences from Acinetobacter. A full-length C12O gene was directly cloned, heterologously expressed in Escherichia coli, and biochemically characterized. Purified catPL12 had optimum pH and temperature pH 8.0 and 25 degrees C and retained 31 and 50% of its maximum activity when assayed at 0 and 35 degrees C, respectively. The enzyme was stable at 25 and 37 degrees C, retaining 100% activity after pre-incubation for 1h. The kinetic parameters of catPL12 were determined. The enzyme had apparent K-m of 67 mu M, V-max of 7.3U/mg, and k(cat) of 4.2s(-1) for catechol, and the cleavage activities for 3-methylcatechol, 4-methylcatechol, and 4-chlorocatechol were much less than for catechol, and no activity with hydroquinone or protocatechuate was detected. This study is the first to report the molecular and biochemical characterizations of a cold-adapted catechol 1,2-dioxygenase from a fecal microbial metagenome.