In this study, a three step purification of alkaline phosphatase from non-pasteurized milk was reported. It included cream extraction, n-butanol treatment and acetone precipitation. Different parameters like buffer concentration, temperature, pH, substrate concentration, acetone and n-butanol treatment were optimized to maximize the enzyme activity. The enzyme was fruitfully purified upto homogeneity from the milk, with percentage recovery and fold purification of 56.17 and 17.67 respectively. The kinetic parameter were determined to be 0.927 (Km) and 55.86 (Vmax), with specific activity of 11.31 U/mg. Other optimized parameters were estimated as buffer concentration of 0.5 M with pH 9.0, temperature optima at 37ºC, 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.
Alkaline phosphatase (orthophosphoric monoester phosphohydrolase, EC 18.104.22.168) is a non-specific monoesterases that catalyze the hydrolysis of various phosphate esters and anhydrides of phosphoric acid, under alkaline conditions (Junior et al. 2008). They are the non-specific phosphatases which are apparently ubiquitous in nature. They are widely used in recombinant DNA technology (Kopetzki et al. 1994) and DNA sequencing. It is also an important component of enzyme-linked immunosorbent assay (ELISA) based kits. So it’s purification on large scale is required for different commercial and research purposes. The enzymes have been isolated from different sources, which include bacteria, fungi, organs of mammals and invertebrates, but a little have been reported in plants. Commercially available alkaline phosphatases (APase) are Bacterial and calf intestinal, which are used for clinical analysis and biological research work (Atlan and Portalier 1987). Among food, milk is the natural source of enzyme, where it is present in the form liopoprotein complex, known as microsomes. As the enzyme get inactivated at higher temperature, it is used as a sensitive method to determine the efficacy of pasteurization (Albillos et al. 2011). Previously, APase was isolated from cow milk by Morton, but it involved different purification techniques (Morton 1953a; Morton 1953b).
In this present study, alkaline phosphatase was extracted and purified upto homogeneity from buffalo milk. A three step approach comprising cream extraction, butanol treatment and acetone precipitation was employed. This method is facile, efficient and provides a cost effective process for APase purification from non-pasteurized milk, with maximum activity.
MATERIALS AND METHODS
Tris, n-butanol, and acetone were purchased from HiMedia, India, while p-nitrophenyl phosphate disodium salt (99% purity) was obtained from Loba Chemie. Fresh buffalo milk was purchased from a local dairy. Deionized (DI) water from Millipore was used for the reagent...