Extraction and characterization of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase and ACC oxidase from wounded persimmon fruit

Abstract

1-Aminocyclopropane-1-carboxylic acid (ACC) synthase (EC 4.4.1.14) and ACC oxidase (EC 1.4.3) were extracted from wounded tissue of mature 'Saijo' persimmon (Diospyros kaki Thunb.) fruit by using the polyethylene glycol (PEG) acetone method and characterized. The optimum pH, concentration of pyridoxal-5-phosphate (PLP) for maximum ACC synthase activity, the Km for S-adenosylmethionine (AdoMet), and the half-life in the presence of AdoMet for persimmon ACC synthase were 8.5, 5 μM, 10 μM, and 21 min, respectively, whereas, the optimum pH for ACC oxidase was 7.2, the apparent Kms for the substrate ACC and cofactor Fe2+ were 114 μM and 4 μM, respectively. The concentration of cofactor HCO3- for maximum activity was approximately 40 mM. The activity of ACC oxidase decreased in a non-linear manner during in vitro incubation, with a half-life of about 9 min; its activity was inhibited by various divalent cations, such as Ni2+, Zn2+, Cu2+, Mn 2+ and Mg2+.