A three step approach for the purification of alkaline phosphatase from non-pasteurized milk.

In this study, a three step purification of alkaline phosphatase from non-pasteurized milk has been described. It included cream extraction, n-butanol treatment and acetone precipitation. Different parameters such as buffer concentration, temperature, pH, substrate concentration, acetone and n-butanol treatment were optimized to maximize the enzyme activity. The enzyme was fruitfully purified up to homogeneity from the milk, with percentage recovery and fold purification of 56.17 and 17.67 respectively. The kinetic parameters were determined to be 0.927 mM (Km) and 55.86 μM/min (Vmax), with specific activity of 11.31 U/mg. Other optimized parameters were estimated as a buffer concentration of 0.5 M with pH 9.0, temperature optima at 37 °C, with n-butanol and acetone concentration of 20 % (v/v) and 50 % (v/v) respectively. This approach provides a simple and effective method for the purification of alkaline phosphatase from non-pasteurized milk.