ABSTRACT
Alkaline phosphatase was purified to homogeneity from Pseudomonas aeruginosa 50071 starting with heat treatment at 60°C in the presence of Co[2]+ and ammonium sulphate fractionation, followed by separation on DEAE-Sephadex A50 and G-100 Sephadex gel filtration. The enzyme was purified 468 fold and showed a final specific activity of 51.5 unit/mg protein with a yield 42%. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis [SDS-PAGE] of the purified enzyme revealed a single protein band of molecular weight 82 KDa. The molecular weight of the native enzyme was calculated to be 158 KDa, as determined by gel filtration, which approximated to two subunits together [164 KDa] suggesting a homogeneous dimeric structure of the enzyme. The enzyme showed a maximum activity at pH 9.5 when incubated at 37°C. A Lineweaver-Burk analysis gave a K[m] of 2.1 mM and V[max] of 8.0 U/ml. The enzyme activity was enhanced by addition of CoCl[2] and ZnCl[2] together. The amino acid composition of the purified enzyme was also determined