Characteristics of a New Catechol 1,2-Oxygenase fromTrichosporon cutaneumWY 2-2

Abstract

Successive feeding of phenol at concentrations of less than 5.5 mM into a thick suspension of Trichosporon cutaneum WY 2-2 precultured in MPY-medium resulted in a high yield (approximately 28.7 g wet cells/liter) of intact cells capable of decomposing phenol actively (3.7 μmol/min/g of wet cells). The effects of pH and additions of ethanol and 2-mercaptoethanol were tested on the stability of crude extracts from the strain grown on phenol. The crude extracts were stable at a pH range of 7.6 and 8.3, and were stable for 35 days when 10% ethanol and 5 mM 2-mercaptoethanol were added. A highly purified preparation of catechol 1,2-oxygenase was obtained from strain WY 2-2 grown on phenol. The purified enzyme was homogeneous on polyacrylamide disc-gel electrophoresis. The enzyme had a molecular weight of about 105,000 and gave rise to subunits of molecular weight of 35,000 by SDS gel electrophoresis. Therefore, the enzyme appears to be a trimer of subunits with identical molecular weight. The Michaelis constants were 9.0 μM for catechol and 6.8 μM for 4-methylcatechol. The enzyme exhibited higher activities towards 4-methylcatechol and hydroxyquinol than towards catechol, and had essentially the same substrate specificity as the crude extracts. 4-Methylcatechol completely inhibited the enzyme activity towards catechol.