Purification and biochemical characterization of 1-aminocyclopropane-1-car☐ylate oxidase from banana fruit

Abstract

A novel method using PEG and ammonium sulphate was used to concentrate ACC oxidase from a crude extract prepared from the pulp of ripe fruit of banana (Musa AAA group, Cavendish subgroup). The 145-fold purification of the ACC oxidase to electrophoretic homogeneity was carried out in four chromatographic steps: hydrophobic interaction, anion exchange, chromatofocusing and gel filtration. The molecular mass of the purified enzyme was estimated to be 40 000 by gel filtration and 36 000 by SDS-PAGE, indicating that the enzyme is active as a monomer. The enzyme was recognized by a polyclonal antibody directed against a recombinant polypeptide derived from the tomato ACC oxidase, showing that the banana enzyme shares immunogenic epitopes with ACC oxidases from other fruits. An isoelectric point at pH 4.9 was estimated by chromatofocusing