Cloning, expression and characterization of alkaline phosphatase from a thermophilic bacterium Geobacillus caldoxylosilyticus TK4

Abstract

Aim: To clone the gene for alkaline phosphatase (AP) from a novel thermophilic Geobacillus caldoxylosilyticus TK4 strain and to express and characterize this enzyme. Methods: The gene was cloned into pET28a(+) vector and expressed in Escherichia coli BL21(DE3)pLysS. The recombinant protein was purified by using nickel affinity chromatography and characterized. Results: The gene was 1410 bp long and contained highly conserved regions in the vicinity of the phosphorylation and metal binding sites. The recombinant AP had pH and temperature optima of 9.5 and 50 °, respectively, and was ~100% active at between 10 and 30 °C for 2 h. and after 12 h incubation at pH 9.5 and 4°C. It was less active in presence of 1 mM Hg2+, Cd2+ and Al3+, and was completely inhibited by 1 mM EDTA. Its Km and Vmax values, using p-nitrophenyl phosphate as substrate were 87 ?M and 0.049 U mg protein-1, respectively. Conclusion: This recombinant AP shared characteristics of other APs such as conserved amino acid residues, electrophoretic behaviour, pH- and temperature optima, Km and Vmax values and could have some clinical and molecular biological applications. © TurkJBiochem.com.