Purification and characterization of a thermostable alkaline phosphatase produced by Thermus caldophilus GK24

Abstract

The thermophilic and thermostable alkaline phosphatase was purified to near homogeneity from the osmotic lysis of Thermus caldophilus GK24. The purified enzyme had an apparent molecular mass of 108, 000 Da and consisted of two subunits of 54,000 Da. Isoelectric-focusing analysis of the purified enzyme showed a pi of 7.3. The enzyme contained two Cys residues, and its amino acids composition was quite different from that ai Thermus aquaticus YT-1 alkaline phosphatase and Escherichia coli alkaline phosphatase. The optimum pH and temperature of the enzyme were 11.0-11.5 and 80 degrees C, respectively. The enzyme was stable in the pH range of 9.0-12.0 at 25 degrees C for 36 h, and the half-life al 80 degrees C (pH 11.0) was 6 h. The enzyme was activated by MgCl2 and inhibited by EDTA. With p-nitrophenyl phosphate (pNPP) as the substrate, the enzyme had a Michaelis constant (K-m) of 3.6x10(-5) hM The enzyme preferentially hydrolyzed the phosphomonester bond of AMP in ribonucleotides and glycerophosphate.