Aromatic hydroxylation catalyzed by toluene 4-monooxygenase in organic solvent/aqueous buffer mixtures

Toluene 4-monooxygenase is a four-protein component diiron enzyme complex. The enzyme catalyzes the hydroxylation of toluene to give p-cresol with sim ilar to 96% regioselectivity. The performance of the enzyme in two-phase re action systems consisting of toluene, hexane, or perfluorohexane and an aqu eous buffer was rested. In each of the cosolvent systems, containing up to 93% (v/v) of solvent, the enzyme was active and exhibited regioselectivity indistinguishable from the aqueous reaction. Using the perfluorohexane/buff er system, a number of polycyclic aromatic hydrocarbons were oxidized that were not readily oxidized in aqueous buffer. An instability of the hydroxyl ase component and a substantial uncoupling of NADH utilization and product formation were observed in reactions that were continued for longer than si milar to3 min. More stable enzyme complexes will be needed for broad applic ability of this hydroxylating system in nonaqueous media.