To determine Michaelis–menten constant Km,
phytase activity was determined at different concentration of sodium phytate (0.25–10.0 mM)
in acetate buffer (0.1 M, pH 6.0).
The reaction mixture was prepared and phytase activity was determined as described earlier.
The prepared reaction mixture was incubated at 37 _C for 30 min and OD was taken at k415 nm.
The Km and Vmax values were determined using the Lineweaver–Burk double reciprocal plot
(Lineweaver and Burk 1934).