Purification and characterization of the recombinant form of acyl CoA oxidase 3 from the yeast Yarrowia lipolytica

The Acyl CoA dependent oxidase 3 (Aox3p) from the yeast Yarrowia lipolytica , expressed in Escherichia coli, as an active protein with a 6 His tag at i ts N-terminal region has been purified to electrophoretic homogeneity. The purified enzyme exhibits a specific activity of 1.95 muM/min/mg using hexan oyl-CoA as substrate, and it remains active for at least 1 month upon stora ge at -30 degreesC in the presence of 35% (V/V) glycerol. The pH and temper ature optima of the enzyme are 7.4 and 28-38 degreesC, respectively. Aox3p catalyzes the oxidation of both aliphatic acyl-CoA substrates of different chain lengths (e.g., hexanoyl-CoA, decanoyl-CoA, myristyl-CoA) as well as o f the aromatic/heterocyclic ring-substituted chromogenic substrates, such a s furylpropionyl-CoA. Of the above substrates, the efficiency of the enzyme , as judged by its k(cat) to K-m ratio, exhibits the following order: decan oyl CoA > myristyl CoA > hexanoyl CoA > furyl-propionyl-CoA (FPCoA). Phenol , which is normally used in the coupled assay system for monitoring the H2O 2 formation, functions as both an activator (at low concentrations) and a c ompetitive inhibitor (at high concentrations) with respect to acyl-CoA subs trates. The magnitude of activation and inhibition of the enzyme is depende nt on the nature of the acyl-CoA substrates. (C) 2000 Academic Press.