Phytase activity assay

Phytase activity was determined by the ferrous sulfate-molybdenum blue method (Huang et al., 2006). Briefly, 100 μl of 200-fold diluted enzyme solution was incubated with 900 μl of substrate solution (4 mM sodium phytate in 0.25 M sodium acetate buffer, pH 4.5) at 37°C for 10 min. The reaction was stopped by adding 1 mL of 10% (w/v) trichloroacetic acid (TCA). The amount of inorganic phosphate released was analyzed by adding 2 mL of a coloring reagent C [1% (w/v) ammonium molybdate, 3.2% (v/v) sulfuric-acid solution, and 7.2% (w/v) ferrous sulfate solution] and measuring the absorption at 700 nm. One unit (U) of phytase activity was defined as the amount of enzyme that releases 1 μmol phosphate in 1 min at 37°C.