Purification and characterization of ACC oxidase and increase in its activity during ripening of pear fruit

Abstract

Pear (Pyrus communis L. cv. La France) fruit induced to ripen by low-temperature treatment was used for this study. The activity of ACC (1-aminocyclopropane-1-carboxylic acid) oxidase and the rate of ethylene production increased rapidly during fruit ripening at 20 °C. ACC oxidase, isolated from pear fruit at the climacteric stage, was purified to near homogeneity. Its molecular mass was estimated to be 40 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Pear fruit ACC oxidase was recognized by the antibody raised against ACC oxidase, purified from Escherichia coli cells transformed with cDNA of broccoli ACC oxidase by western blotting. The apparent Km values for their substrates ACC and O2 of ACC oxidase were estimated to be 42.2 μM and 0.53 %, respectively. The optimal concentration of ascorbate, cosubstrate, for ACC oxidase was 15 mM and the apparent Km for ascorbate was estimated to be 1.9 mM. The concentrations of cofactors Fe2+ and HCO3- for maximum activity were 25 μM and 30 mM, respectively; the Km for HCO3- was estimated to be 3.8 mM. These enzymatic properties were found to be similar to ACC oxidase derived from other plants.