Phytase activity assay.

Standard phytase activity was assayed with the ferrous sulfate-molybdenum blue method (40). A 50-μl volume of each enzyme was incubated with 950 μl of substrate solution (1.5 mM sodium phytate, 0.25 M sodium acetate [pH 4.5]) at 37°C for 30 min. The reaction was terminated by addition of 1 ml of 10% (wt/vol) trichloroacetic acid, followed by the addition of 2 ml of color reagent (1% [wt/vol] ammonium molybdate, 3.2% [vol/vol] sulfuric acid, 7.2% [wt/vol] ferrous sulfate). The released inorganic phosphate was monitored by measuring the absorbance at 700 nm. One unit of phytase activity was defined as the amount of enzyme required to liberate 1 μmol of phosphate per minute under standard conditions. All reactions were performed in triplicate.