Purification and characterization of phytase from cotyledons of germinating soybean seeds

Abstract

Soybean phytase ( myo-inositol-hexakisphosphate phosphohydrolase; EC 3.1.3.8) was purified from 10-day-old germinating cotyledons using a four-step purification scheme. Phytase was separable from the major acid phosphatase present, and stained as a minor band of the three acid phosphatases detectable by activity staining after gel electrophoresis. The purified enzyme exhibited two closely migrating bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of approximately 59 and 60 KDa. The molar extinction coefficient of the enzyme at 280 nm was estimated to be 7.5 × 10 4 m −1 cm −1. The isoelectric point of phytase, as judged by the elution profile on chromatofocusing, was about 5.5. The enzyme was totally absorbed to a Procion Red HE3B column and eluted as a single protein component at a salt concentration of 250–300 m m. The enzyme possessed a high affinity for phytic acid (apparent K m = 48 μM), and was strongly inhibited by phosphate (apparent K i = 18 μM), vanadate, and fluoride. Characteristic of other plant phytases, the pH and temperature optima were 4.5–4.8 and 55 °C, respectively.