Cloning, expression, and characterization of a new phytase from the phytopathogenic bacterium Pectobacterium wasabiae DSMZ 18074 |
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Authors: | Shao Na Huang Huoqing Meng Kun Luo Huiying Wang Yaru Yang Peilong Yao Bin |
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Institution: | Department of Microbial Engineering, Feed Research Institute, Chinese Academy of Agricultural Sciences, No. 12 Zhongguancun South Street, Beijing 100081, P. R. China. |
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Abstract: | The soft rot bacterium Pectobacterium wasabiae is an economically important pathogen of many crops. A new phytase gene, appA, was cloned from P. wasabiae by degenerate PCR and TAIL-PCR. The open reading frame of appA consisted of 1,302 bp encoding 433 amino acid residues, including 27 residues of a putative signal peptide. The mature protein had a molecular mass of 45 kDa and a theoretical pI of 5.5. The amino acid sequence contained the conserved active site residues RHGXRXP and HDTN of typical histidine acid phosphatases, and showed the highest identity of 48.5% to PhyM from Pseudomonas syringae. The gene fragment encoding the mature phytase was expressed in Escherichia coli BL21 (DE3), and the purified recombinant phytase had a specific activity of 1,072+/-47 U/mg for phytate substrate. The optimum pH and temperature for the purified phytase were pH 5.0 and 50 degrees , respectively. The Km value was 0.17 mM, with a Vmax of 1,714 mmol/min/mg. This is the first report of the identification and isolation of phytase from Pectobacterium. |
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