Purification and biochemical characterization of thermostable phytase from newly isolated Bacillus subtilis CF92

  • Sung Wook Hong
  • In Ho Chu
  • Kun Sub ChungEmail author


Bacillus subtilis CF92, an isolate from cattle feces, produces phytase, which catalyzes the hydrolysis of phytic acid into myo-inositol and inorganic phosphates. Phytase from B. subtilis CF92 was purified via ethanol precipitation, anion-exchange chromatography, and gel filtration chromatography. Molecular weight of the purified phytase was estimated to be 46 kDa by SDS-PAGE. Purified phytase exhibited optimal activity at 60°C. The enzyme retained 40% of its original activity after 30 min incubation at 80°C. Optimum pH was 7.0, although activity remained fairly stable over pH range of 4.0 to 8.0. The enzyme was activated in the presence of EDTA and significantly inhibited by metal ions. Phytase exhibited substrate-specificity on polyphosphate compounds such as adenosine triphosphate, sodium tripolyphosphate, and sodium phytate. Km and Vmax values for sodium phytate were 0.42 mM and 4.35 μmol/min, respectively.

Key words

Bacillus subtilis phytase phytate purification 


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  1. Anderson RJ (1914) A contribution to the chemistry of phytin. J Biol Chem 17, 171–190.Google Scholar
  2. Angelis MD, Gallo G, Corbo MR, McSweeney PLH, Faccia M, Giovine M, and Gobbetti M (2003) Phytase activity in sourdough lactic acid bacteria: purification and characterization of a phytase from Lactobacillus sanfranciscensis CB1. Int J Food Microbiol 87, 259–270.CrossRefGoogle Scholar
  3. Bae HD, Yanke LJ, Cheng KJ, and Selinger LB (1999) A novel staining method for detecting phytase activity. J Microbiol Methods 39, 17–22.CrossRefGoogle Scholar
  4. Casey A and Walsh G (2003) Purification and characterization of extracellular phytase from Aspergillus niger ATCC 9142. Bioresour Technol 86, 183–188.CrossRefGoogle Scholar
  5. Choi WC, Oh BC, Kim HK, Kang SC, and Oh TK (2002) Characterization and cloning of a phytase from Escherichia coli WC7. Kor J Microbiol Biotechnol 30, 1–7.Google Scholar
  6. Ebune A, Asheh AS, and Duvnjak Z (1995) Effects of phosphate, surfactants and glucose on phytase production and hydrolysis of phytic acid in canola meal by Aspergillus ficuum during solid-state fermentation. Bioresour Technol 54, 241–247.CrossRefGoogle Scholar
  7. Fiske CH and Subbarow YP (1925) The colorimetric determination of phosphorus. J Biol Chem 66, 375–400.Google Scholar
  8. In NJ, Jang ES, Kim YJ, and Oh NS (2004) Purification and properties of an extracellular acid phytase from Pseudomonas fragi Y9451. J Microbiol Biotechnol 14, 1004–1008.Google Scholar
  9. Graf E (1983) Calcium binding to phytic acid. J Agric Food Chem 31, 851–855.CrossRefGoogle Scholar
  10. Greiner R, Haller E, Konietzny U, and Jany KD (1997) Purification and characterization of a phytase from Klebsiella terrigena. Arch Biochem Biophys 341, 201–206.CrossRefGoogle Scholar
  11. Kerovuo J, Lappalainen I, and Reinikainen T (2000) The metal dependence of Bacillus subtilis phytase. Biochem Biophys Res Commun 268, 365–369.CrossRefGoogle Scholar
  12. Kim HW, Kim YO, Lee JH, Kim KK, and Kim YJ (2003) Isolation and characterization of a phytase with improved properties from Citrobacter braakii. Biotechnol Lett 25, 1231–1234.CrossRefGoogle Scholar
  13. Kim YO, Kim HK, Bae KS, Yu JH, and Oh TK (1998) Purification and properties of a thermostable phytase from Bacillus sp. DS11. Enzyme Microb Technol 22, 2–7.CrossRefGoogle Scholar
  14. Laemmli UK (1970) Cleavage of structural protein during the assembly of the lead of bacteriphage T4. Nature 227, 680–685.CrossRefGoogle Scholar
  15. Lantzsch HJ, Wist S, and Drochner W (1995) The effect of dietary calcium on the efficacy of microbial phytase in rations for growing pigs. J Anim Physiol Anim Nutr 73, 19–26.CrossRefGoogle Scholar
  16. Lowry OH, Rosebrough NJ, Farr AL, and Randall RJ (1951) Protein measurement with the folin phenol reagent. J Biochem 193, 265–275.Google Scholar
  17. Mullaney EJ, Daly CB, Sethumadhavan K, Rodriquez E, Lei XG, and Ullah AHJ (2000) Phytase activity in Aspergillus fumigatus isolates. Biochem Biophys Res Commun 275, 759–763.CrossRefGoogle Scholar
  18. Powar VK and Jagannathan V (1982) Purification and properties of phytate-specific phosphatase from Bacillus subtilis. J Bacteriol 151, 1102–1108.Google Scholar
  19. Rackis JJ and Anderson RL (1977) Mineral availability in soy protein products. Food Prod Dev 11, 38–41.Google Scholar
  20. Reddy NR, Sathe SK, and Saluckhe DK (1982) Phytates in legumes and cereals. Adv Food Res 28, 1–92.Google Scholar
  21. Shimizu M (1992) Purification and characterization of phytase from Bacillus subtilis (natto) N-77. Biosci Biotechnol Biochem 56, 1266–1269.CrossRefGoogle Scholar
  22. Simons PC and Versteegh HAJ (1990) Improvement of phosphorus availability by microbial phytase in broilers and pigs. Br J Nutr 64, 525–540.CrossRefGoogle Scholar
  23. Singh B and Satyanarayana T (2008) Phytase production by Sporotrichum thermophile in a cost-effective cane molasses medium in submerged fermentation and its application in bread. J Appl Microbiol 105, 1858–1865.CrossRefGoogle Scholar
  24. Sutardi G and Buckle KA (1988) Characterization of extracellular and intracellular phytases from Rhizopus oligosporus used in tempeh production. Int J Food Microbiol 6, 67–79.CrossRefGoogle Scholar
  25. Waldroup PW (1999) Nutritional approaches to reducing phosphorus excretion by poultry. Poutry Sci 78, 683–691.Google Scholar
  26. Yoon SJ, Choi YJ, Min HK, Cho KK, Kim JW, Lee SC, and Jung YH (1996) Isolation identification of phytaseproducing bacterium, Enterobacter sp.4 and enzymatic properties of phytase enzyme. Enzyme Microbiol Technol 18, 449–454.CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2003

Authors and Affiliations

  1. 1.Division of Biological Science and TechnologyYonsei UniversityWonjuKorea

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