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Gene Cloning, Expression, and Characterization of a Novel Phytase from Dickeya paradisiaca

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Abstract

A novel phytase gene, appA, was isolated by degenerate polymerase chain reaction (PCR) and thermal asymmetric interlaced PCR from Dickeya paradisiaca. The full-length appA comprises 1278 bp and encodes 425 amino acid residues, including a 23-residue putative N-terminal signal peptide. The deduced amino acid sequence of appA reveals the conserved motifs RHGXRXP and HD, which are typical of histidine acid phosphatases; significantly, APPA shows maximum identity (49%) to a phytase from Klebsiella pneumoniae. To characterize the properties of APPA, appA was expressed in Escherichia coli and purified. The purified recombinant APPA has two pH optima at pH 4.5 and 5.5, optimum temperature at 55 °C, specific activity of 769 U/mg, and good pH stability. The K m value for the substrate sodium phytate is 0.399 mM with a V max of 666 U/mg. To our knowledge, this is the first report of a phytase or phytase gene isolated from Dickeya.

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Abbreviations

HAP:

histidine acid phosphatase

IPTG:

isopropyl β-d-1-thiogalactopyranoside

r-APPA:

recombinant APPA

TAIL-PCR:

thermal asymmetric interlaced PCR

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Acknowledgements

This work was supported by the National Basic Research Program of China (no. 2004CB719606) and the Hi-tech Research and Development Program of China (no. 2006AA020101).

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Authors and Affiliations

  1. Department of Microbial Engineering, Feed Research Institute, Chinese Academy of Agricultural Sciences, No. 12 Zhongguancun South Street, Beijing, 100081, People’s Republic of China

    Weina Gu, Huoqing Huang, Kun Meng, Peilong Yang, Dawei Fu, Huiying Luo, Yaru Wang & Bin Yao

  2. College of Chemical Technology, Shijiazhuang University, Shijiazhuang, 050035, People’s Republic of China

    Weina Gu

  3. SUNHY Group, Wuhan, 43007, People’s Republic of China

    Zhichun Zhan

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  1. Weina Gu
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  2. Huoqing Huang
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  3. Kun Meng
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  4. Peilong Yang
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  5. Dawei Fu
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  6. Huiying Luo
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  7. Yaru Wang
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  8. Bin Yao
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  9. Zhichun Zhan
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Corresponding author

Correspondence to Bin Yao.

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Weina Gu and Huoqing Huang contributed equally to this work.

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Gu, W., Huang, H., Meng, K. et al. Gene Cloning, Expression, and Characterization of a Novel Phytase from Dickeya paradisiaca . Appl Biochem Biotechnol 157, 113–123 (2009). https://doi.org/10.1007/s12010-008-8329-6

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  • DOI: https://doi.org/10.1007/s12010-008-8329-6

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