Williams, Opal J.; Golden, Kerith D.
Author Affiliation, Ana.
Department of Basic Medical Sciences
Purification and characterization of ACC oxidase from Artocarpus altilis (breadfruit)
Plant Physiology and Biochemistry
Date of Publication
1- Aminocyclopropane -1 carboxylic acid (ACC) oxidase was purified to homogeneity from breadfruit (Artocarpus altilus) by a six- step procedure. The major purification steps involved were ammonium sulphate precipitation, gel filtration (Sephadex G 25-40), ion exchange (DEAE Sephadex A 25) and size exclusion (Bio- Gel P- 100) chromatography. SDS PAGE revealed that the purified enzymes was a monomeric protein with a molecular mass of 42.3 kDa. A purification fold of 87 and a percentage recovery of 9.5% were obtained. Acc oxidase had a Km of 28.2 uM and showed marked inhibition by cobalt sulphate (0.1mM), sodium metabisulphite (0.1mM), sodium dithionite (0.1mM), n-propyl gallate (0.2mM), zinc sulphate (0.1mM) and hydrogen peroxide (0.8mM). Dithiotheritol (5mM) enhanced enzyme activity when it was included in the assay medium. Optimum activity of ACC oxidase was obtained at a pH of 7.2 and at 28C. The amino acid content of the purified enzyme revealed relatively high percentages of arginine (24.6%), tyrosine (15.7%) and glycine (15.2%). The cysteine content (13.6%) of this enzyme is likely to be responsible for the enzyme's susceptibility to sulphydryl reagents. ACC oxidase rapidly lost activity when stored at high temperatures (15C and above). Enzyme activity was preserved by storing the crude homogenate at -16C.....