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

The TOL plasmid from Pseudomonas putida encodes a pathway for the degr adation of toluene and xylenes. The first step of this degradative pat hway involves the oxidation of the methyl side chain of the substrates , which is catalyzed by xylene monooxygenase. Xylene monooxygenase con sists of two components, an oxygenase component or the XylM protein, a nd an electron transfer component or the XylA protein. Xylene monooxyg enase activity was found to be membrane-bound, and Fe2+-dependent. The activity could be solubilized by two detergents, octyl-beta-glucopyra noside and olamidopropyl)dimethylammonio]-1-propanesulfonate, After se paration of the solubilized enzyme by anion exchange chromatography, t he stability of xylene monooxygenase was drastically reduced. As a con sequence, the enzyme was characterized using the membrane vesicle frac tion. The monooxygenase had a pH optimum of 7 and catalyzed the oxidat ion of toluene, m-xylene, p-xylene and o-xylene, but no activity was o bserved towards benzyl alcohol.