Optimized expression and purification of toluene 4-monooxygenase hydroxylase

Toluene 4-monooxygenase is a four-protein complex that catalyzes the O-2- a nd NADH-dependent oxidation of toluene to p-cresol, The influence of variou s expression systems on the host cell growth characteristics, purified prot ein yields, and specific activity of the hydroxylase (T4moH) component of t he complex was evaluated by considering the cell mass obtained per liter of fermentation culture medium, the purified protein obtained per gram of cel l mass, and the specific activity of purified T4moH, The specific activity of purified T4moH was determined to be 1200-1250 nmol of p-cresol formed pe r minute per milligram of T4moH in air-saturated 50 mM phosphate buffer, pH 7.5, at 25 degrees C in the presence of optimal concentrations of the othe r protein components of the complex, saturating toluene (5.8 mM at 25 degre es C), and saturating NADH (1 mM), This value was obtained for T4moH purifi ed from several different expression systems and apparently represents the maximal specific activity of the enzyme complex for toluene hydroxylation. By manipulation of vectors and gene inserts to eliminate adventitious catal ytic turnover of NADH, up to 60-fold increase in the volumetric yield of T4 moH activity was obtained from recombinant fermentations in Escherichia col i BL21(DE3). (C) 2000 Academic Press.