Clonging,Expression And Characterization Of A Thermostable Alkaline Phosphatase From Thermus Thermophilus XM

Alkaline phosphatase (AP) is a kind of hydrolase, which can catalyze the hydrolysis of monophosphoester to produce phosphate or transfer phosphate. AP exists widely in microbial and animal kingdom, but hasn't been found in advanced frond. AP plays a vital important role in phosphate cycle of world creatures, and has been used widely in the fields of diagnostics, biochemistry and molecular biology. The commercial alkaline phosphatases are CIAP (calf intestine alkaline phosphatase) and E. coli AP, which have high specific activity and have been studied intensively. These two kinds of enzymes are only fit for using at room temperature and easy to loss activity at a relatively high temperature. Thermostable alkaline phosphatase (TAP) is one kind of alkaline phosphatase, which has the same catalysis but can keep stable at high temperature. This kind of enzyme has been isolated from the mophile bacteria, and its applications are much more widely than CIAP and E. coli AP.In this study, a thermostable alkaline phosphatase gene (tap) from the thermophilic bacterium Thermus thermophilus XM (T. thermophilus XM) was cloned and sequenced. It has 1506 bp and encodes a 501 amino acid polypeptide with a calculated molecular mass of 54.7 kDa. The deduced amino acid sequence exhibited 99.6% similarity to that from T. thermophilus HB27 and revealed remarkable conservation of the regions in the vicinity of the corresponding phosphorylation site and metal binding sites in comparison with APase from Escherichia coli (E. coli). The recombinant thermostable alkaline phosphatase (rTAPase) was expressed as a 6His-tagged fusion protein in E. coli and its enzymatic properties were characterized. The pH and temperature optima for activity of the purified enzyme were found to be 12.0 and 75°C, respectively. As expected, the rTAPase displayed high thermostability, retaining more than 50 % of activity after six hours incubation at 80℃. The catalytic activity was enhanced in the presence of Mg²⁺ but inhibited by Zn²⁺, Ca²⁺ and Cu²⁺. Under optimal conditions, the Michaelis constant (Km) for cleavage of p-nitrophenyl-phosphate (pNPP) was 0.034 mM.Thermophiles are good candidates in producing thermostable enzyme. However, it is often impractical to use them directly due to the low yield of the enzyme and its rigorous developing conditions . Under this condition, a molecular approach through expression of foreign protein in prokaryotic systems has become a good alternative to economically obtain bulk production of enzyme.In this study, we cloned a tap gene from T. thermophilus XM and expressed it in E. coli, discussed the characteristics of the deduced primary structure of the protein, purified and provided a preliminary characterization of the recombinant TAP (rTAPase).