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- Title
Effect of metals ions on thermostable alkaline phytase from Bacillus subtilis YCJS isolated from soybean rhizosphere soil.
- Authors
Yao, Ming-Ze; Lu, Wen-Liang; Chen, Ting-Gui; Wang, Wei; Fu, Yue-Jun; Yang, Bin-Sheng; Liang, Ai-Hua
- Abstract
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 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 (Ca). Fluorescence analysis results indicated that compared with phy(ycE) without added Ca, phy(ycE) in the presence of Ca 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 Ca-reactivated enzyme, the La-reactivated enzyme did not undergo a significant recovery with respect to its conformation. The aromatic-sensitized terbium (Tb) fluorescence results indicated that five Tb could bind to each molecule of phy(ycE) and that there were two high-affinity and three low-affinity binding sites.
- Publication
Annals of Microbiology, 2014, Vol 64, Issue 3, p1123
- ISSN
1590-4261
- Publication type
Article
- DOI
10.1007/s13213-013-0751-5