PURIFICATION AND CHARACTERIZATION OF ALKALINE PHOSPHATASE FROM A HALOTOLERANT FACULTATIVE ALKALIPHILE BACILLUS FLEXUS FPB17

The extracellular alkaline phosphatase from halotolerant facultative alkaliphile Bacillus flexus FPB17 was purified through precipitation / dialysis / chromatography and resulted in 28 fold of purification. The molecular weight of this enzyme through SDS-PAGE gel is 63 ± 2 kDa. Optimum conditions for purified ALP activity were pH 9.0 and 35°C. The enzyme proved to be thermolabile and gets completely denatured at 60°C in 10 min. p-NPP was found to be a preferred substrate and Km (0.33 mM) and V_max (0.2 mM mg-1) determined using p-NPP as a substrate. Inorganic phosphate proved to be a competitive inhibitor of ALP. Mg⁺² and Ca⁺² increased the ALP activity by 47% and 20% respectively whereas Sodium fluoride, Sodium arsenate and EDTA decreased the activity by 47%, 65% and 77% respectively. The superiority of dephosphorylation of λ phage DNA by this newly discovered alkaline phosphatase over that of calf intestinal alkaline phosphatase of M/s. HiMedia Laboratories, Mumbai opens up possibility of its future use in molecular biology as a replacement of alkaline phosphatases from Escherichia coli, calf intestine and shrimp