Purification, biochemical and immunological characterization of acid invertases from apple fruit

The soluble acid invertase (SAI) and cell wall-bound invertase (CWI) were purified from apple fruit to apparent electrophoretic homogeneity. Based on sequencing, substrate specificity, and immunoblotting assay, the purified enzymes were identified to be two isoforms of acid invertase (beta-fructosidase; EC 3.2.1. 26). The SAI and CWI have the same apparent molecular mass with a holoenzyme of molecular mass of 220 kDa composed of 50 kDa subunits. The SAI has a lower K-m value for sucrose and higher K-m for raffinose compared with CWI. These acid invertases differ from those in other plants in some of their biochemical properties, such as the extremely high K-m value for raffinose, no hydrolytic activity for stachyose, and a mixed form of inhibition by fructose to their activity. The antibodies directed against the SAI and CWI recognized, from the crude extract, three polypeptides with a molecular mass of 50,68, and 30 kDa, respectively. These results provide a substantial basis for the further studies of the acid invertases in apple fruit.