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

• Published in: Annals of microbiology 2014, Vol.64 (3), p.1123-1131
• Main Authors: Yao, Ming-Ze, Lu, Wen-Liang, Chen, Ting-Gui, Wang, Wei, Fu, Yue-Jun, Yang, Bin-Sheng, Liang, Ai-Hua
• Format: Article
• Language: English
• Subjects: Applied Microbiology; Bacillus subtilis; binding sites; Biomedical and Life Sciences; calcium; enzyme activity; Escherichia coli; fluorescence; genes; ions; Life Sciences; Medical Microbiology; melting point; Microbial Ecology; Microbial Genetics and Genomics; Microbiology; molecular weight; Mycology; Original Article; phytases; polyacrylamide gel electrophoresis; rhizosphere; signal peptide; soil; soybeans; temperature; terbium; thermal stability
• ISSN: 1590-4261; 1869-2044
• DOI: 10.1007/s13213-013-0751-5

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⁻¹protein. 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.