Phytase Activity Assays

Protein concentration was calculated with a Dc Protein Assay (Bio-Rad) using bovine serum albumin as a standard. Phytase activity was assayed by measuring the amount of released inorganic phosphate (Pi) with a modified ammonium molybdate method (Jog et al., 2005). Briefly, 50 μl of enzyme solution was added to 200 μl of 15 mM MES buffer pH 5.5 with 1.25 mM Na-IHP, 0.5 mM CaCl₂ and incubated at 37°C for 1 h. The reaction was stopped by the addition of 50 μl of 50% TCA. Pellet was spun down for 5 min at 15,000 rpm. Blank controls were prepared by adding 50% TCA prior to the addition of enzyme. Colorimetric reactions with ammonium molybdate solution were then performed as previously described (Jog et al., 2005). Optical density was measured at 820 nm on a model 2550 Microplate Reader (Bio-Rad, United States). A calibration curve was built using concentrations of inorganic phosphate in the range of 5.625–90 μM. One unit (U) of phytase activity was defined as the amount of enzyme necessary to produce 1 μmol of inorganic phosphate per min at 37°C.