CHARACTERIZATION IN-VITRO OF THE HYDROXYLASE COMPONENT OF XYLENE MONOOXYGENASE, THE FIRST ENZYME OF THE TOL-PLASMID-ENCODED PATHWAY FOR THE MINERALIZATION OF TOLUENE AND XYLENES

The TOL plasmid from Pseudomonas putida encodes a pathway for the degradation of toluene and xylenes. The first step of this degradative pathway involves the oxidation of the methyl side chain of the substrates, which is catalyzed by xylene monooxygenase. Xylene monooxygenase consists of two components, an oxygenase component or the XylM protein, and an electron transfer component or the XylA protein. Xylene monooxygenase activity was found to be membrane-bound, and Fe2+-dependent. The activity could be solubilized by two detergents, octyl-beta-glucopyranoside and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, After separation of the solubilized enzyme by anion exchange chromatography, the stability of xylene monooxygenase was drastically reduced. As a consequence, the enzyme was characterized using the membrane vesicle fraction. The monooxygenase had a pH optimum of 7 and catalyzed the oxidation of toluene, m-xylene, p-xylene and o-xylene, but no activity was observed towards benzyl alcohol.