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KMID : 0545120080180071221
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Journal of Microbiology and Biotechnology
2008 Volume.18 No. 7 p.1221 ~ p.1226
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Cloning, Expression, and Characterization of a New Phytase from the Phytopathogenic Bacterium Pectobacterium wasabiae DSMZ 18074
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Shao Na
Huang Huoqing
Meng Kun
Luo Huiying
Wang Yaru
Yang Peilong
Yao Bin
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Abstract
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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 50oC, 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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KEYWORD
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Histidine acid phosphatase, Pectobacterium wasabiae, phytase, phytopathogenic bacteria
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