PURIFICATION AND CHARACTERIZATION OF A PHYTASE (MYO-INOSITOL-HEXAKISPHOSPHATE PHOSPHOHYDROLASE) ACCUMULATED IN MAIZE (ZEA-MAYS) SEEDLINGS DURING GERMINATION

Phytase (myo-inositol-hexakisphosphate phosphohydrolase. EC 3.1.3.8) has been purified from 5-7-day-old maize (Zea mays) seedlings, using a four-step purification procedure. The native protein has a molecular mass of about 76 kDa and is built up from two 38 kDa subunits. The pH and temperature optima of the purified enzyme were respectively 4.8 and 55-degrees-C. The apparent K(m) for phytate was estimated to be 117 muM. Like other acidic phytases, the maize seedling enzyme exhibited a broad affinity for various phosphorylated substrates and especially for penta- and tri-phosphate esters of myo-inositol. The amino acid composition of the h.p.l.c-purified protein indicated a high hydrophobicity (44% non-polar amino acids). Rabbit antibodies were produced in response to maize seedling phytase. Western-blot analyses clearly demonstrate that the increase of phytase activity observed during the first 7 days of germination corresponded to an accumulation of the protein in maize seedlings. Phytase accumulated essentially in the shoots (mesocotyl plus coleoptile.