Effect of metals ions on thermostable alkaline phytase from Bacillus subtilis YCJS isolated from soybean rhizosphere soil

A Bacillus sp.YCJS strain showing phytase activity was isolated, and the phytase-encoding gene was cloned and expressed in Escherichia coli. The 1,149-bp full-length gene encoded a 26-residue putative signal peptide and a 356-residue mature protein. The molecular weight was estimated to be 47.5 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified recombinant enzyme phy(ycE) from E. coli exhibited a specific activity of 14 U mg-1 protein. The optimum pH and temperature were 6.0 and 50 °C, respectively. The thermal stability of phy(ycE) was drastically improved in the presence of calcium ions (Ca2+). Fluorescence analysis results indicated that compared with phy(ycE) without added Ca2+, phy(ycE) in the presence of Ca2+ was more stable and the melting temperature improved from 47.8 to 62.4 °C. Circular dichroism spectrometric analysis revealed that the loss of enzymatic activity was most likely due to a conformational change, as the circular dichroism spectra of the holoenzyme and metal-depleted enzyme were significantly different. Compared with the Ca2+-reactivated enzyme, the La3+-reactivated enzyme did not undergo a significant recovery with respect to its conformation. The aromatic-sensitized terbium (Tb3+) fluorescence results indicated that five Tb3+ could bind to each molecule of phy(ycE) and that there were two high-affinity and three low-affinity binding sites.