Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Cloning, Expression, and Characterization of a New Phytase from the Phytopathogenic Bacterium Pectobacterium wasabiae DSMZ 18074

  • Shao, Na (Department of Microbial Engineering, Feed Research Institute, Chinese Academy of Agricultural Sciences) ;
  • Huang, Huoqing (Department of Microbial Engineering, Feed Research Institute, Chinese Academy of Agricultural Sciences) ;
  • Meng, Kun (Department of Microbial Engineering, Feed Research Institute, Chinese Academy of Agricultural Sciences) ;
  • Luo, Huiying (Department of Microbial Engineering, Feed Research Institute, Chinese Academy of Agricultural Sciences) ;
  • Wang, Yaru (Department of Microbial Engineering, Feed Research Institute, Chinese Academy of Agricultural Sciences) ;
  • Yang, Peilong (Department of Microbial Engineering, Feed Research Institute, Chinese Academy of Agricultural Sciences) ;
  • Yao, Bin (Department of Microbial Engineering, Feed Research Institute, Chinese Academy of Agricultural Sciences)
  • Published : 2008.07.31
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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$\pm$47 U/mg for phytate substrate. The optimum pH and temperature for the purified phytase were pH 5.0 and 50$^{\circ}C$, respectively. The $K_m$ value was 0.17 mM, with a $V_{max}$ of 1,714 $\mu$mol/min/mg. This is the first report of the identification and isolation of phytase from Pectobacterium.

Keywords

References

  1. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254 https://doi.org/10.1016/0003-2697(76)90527-3
  2. Chatterjee, S., R. Sankaranarayanan, and R. V. Sonti. 2003. PhyA, a secreted protein of Xanthomonas oryzae pv. oryzae, is required for optimum virulence and growth on phytic acid as a sole phosphate source. Mol. Plant Microbe Interact. 16: 973-982 https://doi.org/10.1094/MPMI.2003.16.11.973
  3. Cho, J. S., C. W. Lee, S. H. Kang, and J. C. Lee. 2003. Purification and characterization of a phytase from Pseudomonas syringae MOK1. Curr. Microbiol. 47: 290-294 https://doi.org/10.1007/s00284-002-3966-4
  4. Cho, J. S., C. W. Lee, S. H. Kang, J. C. Lee, and H. G. Lee. 2005. Molecular cloning of a phytase gene (phyM) from Pseudomonas syringae MOK1. Curr. Microbiol. 51: 11-15 https://doi.org/10.1007/s00284-005-4482-0
  5. Choi, W. C., B. C. Oh, H. K. Kim, E. S. Lee, and T. K. Oh. 2002. Medium optimization for phytase production by recombinant Escherichia coli using statistical experimental design. J. Microbiol. Biotechnol. 12: 490-496
  6. Chu, H. M., R. T. Guo, T. W. Lin, C. C. Chou, H. L. Shr, H. L. Lai, T. Y. Tang, K. J. Cheng, B. L. Selinger, and A. H. Wang. 2004. Structures of Selenomonas ruminantium phytase in complex with persulfated phytate: DSP phytase fold and mechanism for sequential substrate hydrolysis. Structure 12: 2015-2024 https://doi.org/10.1016/j.str.2004.08.010
  7. Duarte, V., S. H. de Boer, L. J. Ward, and A. M. R. de Oliveira. 2004. Characterization of atypical Erwinia carotovora strains causing blackleg of potato in Brazil. J. Appl. Microbiol. 96: 535-545 https://doi.org/10.1111/j.1365-2672.2004.02173.x
  8. Holman, W. I. M. 1943. A new technique for the determination of phosphorus by the molybdenum blue method. Biochem. J. 37: 256-259 https://doi.org/10.1042/bj0370256
  9. Huang, H., H. Luo, P. Yang, K. Meng, Y. Wang, T. Yuan, Y. Bai, and B. Yao. 2006. A novel phytase with preferable characteristics from Yersinia intermedia. Biochem. Biophys. Res. Commun. 350: 884-889 https://doi.org/10.1016/j.bbrc.2006.09.118
  10. In, M. J., E. S. Jang, Y. J. Kim, and N. S. Oh. 2004. Purification and properties of an extracellular acid phytase from Pseudomonas fragi Y9451. J. Microbiol. Biotechnol. 14: 1004-1008
  11. Kim, Y. H., M. N. Gwon, S. Y. Yang, T. K. Park, C. G. Kim, C. W. Kim, and M. D. Song. 2002. Isolation of phytaseproducing Pseudomonas sp. and optimization of its phytase production. J. Microbiol. Biotechnol. 12: 279-285
  12. Lim, D., S. Golovan, C. W. Forsberg, and Z. Jia. 2000. Crystal structures of Escherichia coli phytase and its complex with phytate. Nat. Struct. Biol. 7: 108-113 https://doi.org/10.1038/72371
  13. Liu, Y. G. and R. F. Whittier. 1995. Thermal asymmetric interlaced PCR: Automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking. Genomics 25: 674-681 https://doi.org/10.1016/0888-7543(95)80010-J
  14. Luo, H., B. Yao, T. Yuan, Y. Wang, X. Shi, N. Wu, and Y. Fan. 2004. Overexpression of Escherichia coli phytase with high specific activity. Chinese J. Biotechnol. 20: 78-84
  15. Luo, H., H. Huang, P. Yang, Y. Wang, T. Yuan, N. Wu, B. Yao, and L. Fan. 2007. A novel phytase appA from Citrobacter amalonaticus CGMCC 1696: Gene cloning and overexpression in Pichia pastoris. Curr. Microbiol. 55: 185-192 https://doi.org/10.1007/s00284-006-0586-4
  16. Mullaney, E. J. and A. H. J. Ullah. 2003. The term phytase comprises several different classes of enzymes. Biochem. Biophys. Res. Commun. 312: 179-184 https://doi.org/10.1016/j.bbrc.2003.09.176
  17. Mullaney, E. J., C. Daly, and A. H. J. Ullah. 2000. Advances in phytase research. Adv. Appl. Microbiol. 47: 157-199 https://doi.org/10.1016/S0065-2164(00)47004-8
  18. Pandey, A., G. Szakacs, C. R. Soccol, J. A. Rodriguez-Leon, and V. T. Soccol. 2001. Production, purification and properties of microbial phytases. Bioresour. Technol. 77: 203-214 https://doi.org/10.1016/S0960-8524(00)00139-5
  19. Perombelon, M. C. M. and A. Kelman. 1980. Ecology of the soft rot erwinias. Annu. Rev. Phytopathol. 18: 361-397 https://doi.org/10.1146/annurev.py.18.090180.002045
  20. Rodriquez, E., Y. Han, and X. G. Lei. 1999. Cloning, sequencing and expression of an Escherichia coli acid phosphatase/phytase gene (appA2) isolated from pig colon. Biochem. Biophys. Res. Commun. 257: 117-123 https://doi.org/10.1006/bbrc.1999.0361
  21. Sajidan, A., A. Farouk, R. Greiner, P. Jungblut, E. C. Muller, and R. Borris. 2004. Molecular and physiological characterization of a 3-phytase from soil bacterium Klebsiella sp. ASR1. Appl. Microbiol. Biotechnol. 65: 110-118
  22. Smadja, B., X. Latour, S. Trigui, J. F. Burini, S. Chevalier, and N. Orange. 2004. Thermodependence of growth and enzymatic activities implicated in pathogenicity of two Erwinia carotovora subspecies (Pectobacterium spp.). Can. J. Microbiol. 50: 19-27 https://doi.org/10.1139/w03-099
  23. Wang, Y., X. Gao, Q. Su, W. Wu, and L. An. 2007. Cloning, expression, and enzyme characterization of an acid heat-stable phytase from Aspergillus fumigatus WY-2. Curr. Microbiol. 55: 65-70 https://doi.org/10.1007/s00284-006-0613-5
  24. Wodzinski, R. J. and A. H. J. Ullah. 1996. Phytase. Adv. Appl. Microbiol. 42: 263-302 https://doi.org/10.1016/S0065-2164(08)70375-7
  25. Wyss, M., R. Brugger, A. Kronenberger, R. Remy, R. Fimbel, G. Oesterhelt, M. Lehmann, and A. P. van Loon. 1999. Biochemical characterization of fungal phytases (myo-inositol hexakisphosphate phosphohydrolases): Catalytic properties. Appl. Environ. Microbiol. 65: 367-373
  26. Yin, C., Z. Z. Zhu, X. A. Lin, Y. Z. Yi, Z. F. Zhang, and G. F. Shen. 2005. Overexpression and characterization of appA phytase expressed by recombinant Baculovirus-infected silkworm. J. Microbiol. Biotechnol. 15: 466-471
  27. Zinin, N. V., A. V. Serkina, M. S. Gelfand, A. B. Shevelev, and S. P. Sineoky. 2004. Gene cloning, expression and characterization of novel phytase from Obesumbacterium proteus. FEMS Microbiol. Lett. 236: 283-290 https://doi.org/10.1111/j.1574-6968.2004.tb09659.x