Isolation and characterization of a micromycete, a producer of neutral catechol oxidases, from tropical soils with elevated dioxine content

—Samples of South Vietnamese soils intensely treated with Agent Orange defoliant were tested for the presence of fungi and actinomycetes with an elevated phenol oxidase activity. As a result, a fast-growing nonsporulating strain producing neutral phenol oxidases was isolated and identified asMycelia sterilia INBI2-26. The strain formed extracellular phenol oxidases during surface growth on a liquid medium in the presence of guayacol and copper sulfate, as well as during submerged cultivation in liquid medium containing wheat bran and sugar beet pulp. Isoelectric focusing of the culture liquid revealed two major catechol oxidases (PO1 and PO2) with pI 3.5 and 8, respectively. The enzymes were purified by Ultrafiltration, ion exchange chromatography, and exclusion HPLC. Both were stable between pH 3 and 8. At pH 8 and 40°C., they retained at least 50% of activity after incubation for 50 h. At 50°C., PO2 was more stable and retained 40% of activity after 50 h, whereas PO1 was inactivated in 3–6 h. The pH-optimutns for PO1 and PO2 toward catechol were 6 and 6.5; and theK _m values were 1.5±0.35 and 1.25±0.2 mM, respectively. PO1 and PO2 most optimally oxidized 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) at pH 3 withK _m values 1.6±0.18 and 0.045±0.01 mM, respectively, but displayed no activity toward tyrosine. The PO2 absorbance spectrum had a peak at 600 nm, thus indicating the enzyme to be a member of the laccase family.