Purification and characterization of an acidic, thermophilic phytase from a newly isolated Geobacillus stearothermophilus strain DM12

Document Type : Original Research Papers


1 Department of Biology, Faculty of Sciences, ShahidBahonar University of Kerman, Kerman, Iran

2 Department of Biology, Faculty of Sciences, Shahid Bahonar University of Kerman, Kerman, Iran


Microbial phytases were applied mainly to animal and human foodstuffs in order to improve
mineral bioavailability and food processing. In addition, phytases have potential
biotechnological application in various other fields, such as environmental protection,
aquaculture and agriculture. Bacillus sp. DM12, an isolate from a hot spring, produces phytase,
which catalyzes the hydrolysis of phytic acid into myo-inositol and inorganic phosphates.
Phytase from Bacillus sp. DM12 was purified using ammonium sulfate precipitation and
dialysis, followed by anion exchange and gel filtration chromatography. Molecular weight of
the purified phytase was estimated to be 28 kDa by SDS-PAGE. Km and Vmax values for sodium
phytate were 0.177 mM and 1.126 μmol/min, respectively. The optimum temperature for
phytase activity was found to be 50°C. The enzyme retained over 75% of its activity over a
temperature range of 30 to 80°C. The highest phytase activity was observed at pH 4.5 and a
decline of enzyme activity was observed on both sides of pH optimum. The enzyme was stable
over the pH range of 3.0 to 6.0. The enzyme retained over 80% of its activity in the presence of
5 mM metal ions except CaCl2. It is also indicated that the enzyme retained over 65% of its
activity over a 5 mM metal ions. These properties suggest that this phytase is a suitable enzyme
for the hydrolysis of phytic acid and phytates in food and feed processing industries.


Main Subjects

  1. Lei, X.G., Weaver, J.D., Mullaney, E.J., Ullah, A.H. and Azain, M.J. (2013) Phytase: a new life for an “Oldenzyme.Annu. Rev. Anim. Biosci.,1, 283–309.


2. Sreedevi, S. and Reddy, B.N. (2012) Isolation, screening and optimization of phytase production from newly isolated Bacillus sp.C43. I JPB. S., 2, 218-231.

3. Kim, D.H., Oh, B.C., Choi, W.C., Lee, J.K. and Oh, T.K. (1999) Enzymatic evaluation of Bacillus amyloliquefaciensphytase as a feed additive. Biotechnol. Lett.,21, 925–927.

4. Rao, D.E., Rao, K.V. and Reddy, V.D. (2008) Cloning and expression of Bacillusphytase gene (phy) in Escherichia coli and recovery of active enzyme from the inclusion bodies. J. Appl. Microbiol.,105, 1128–1137.

5. Vohra, A. and Satyanarayana T. (2003) Phytases: microbial sources, production, purification, and potential biotechnological applications. Crit. Rev. Biotechnol.,23, 29-60.

6. Shimizu, M. (1992) Purification and characterization of phytase from Bacillus subtilis (natto) N-77. Biosci.Biotechnol.Biochem.,56,1266-1269.

7. Kim, H.W., Kim, Y.O., Lee, J. H., Kim, K.K. and Kim, Y.J. (2003) Isolation and characterization of a phytase with improved properities from Citrobacterbraakii. Biotechnol.Lett., 25, 1231-1234.

8. Kim, Y.H., Gwon, M.N., Yang, S.Y., Park, T. K., Kim, C.G., Kim, C.W. and Song, M.D. (2002) Isolation of phytase-producing Pseudomonas sp. and optimization of its phytase production. J. microbiol.Biotechnol., 12, 279-285

9. Powar, V. K. and Jagannathan, V. (1982) Purification and properties of phytate-specific phosphatase from Bacillus subtilis. J. Bacteriol.,151, 1102-1108.

10. Greiner, R.U. and Konietzny, K.D. (1993) Purification and characterization of two phytases from Escherichia coli. Arch. Biochem. Biophys., 301, 107-113.

11. Yoon, S.J., Yun, J.C., Hae, K.M., Kwang, K.C., Jin, W.K., Sang, C.I. and Yeon, H.J. (1996) Isolation and identification of phytase-producing bacterium, Enterobacter sp. 4, and enzymatic properties of phytase enzyme. Enz.Microb. Technol.,18, 449-454.

12. De Angelis, M., Gallo, G., Corbo, M.R., McSweeney, P.L., 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(3), 259-70.

13. In, M.J., Jang, E.S., Kim, Y. J. and Oh, N.S. (2004) Purification and properties of an extracellular acid phytase from Pseudomonas fragi Y9451. J. Microbiol. Biotechnol.,14, 1004-1008.

14. Chunshan, Q., Linghua, Z., Yunji, W. and Yoshiyuki, O. (2001) Production of phytase in slow phosphate medium by a novel yeast candida krusei. J. Biosci. Bioeng.,92, 154-160.

15. Sambrook, J. and Russell, D. (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York.


16. Badoei-Dalfard, A.and Karami, Z. (2013) Screening and isolation of an organic solvent tolerant-protease from Bacillus sp. JER02: Activity optimization by response surface methodology. J. Mol. Catal. B Enz.,89, 15– 23.

17. Sievers, F., Wilm, A. Dineen, D.G., Gibson, T.J., Karplus, K., Lopez, W. Li, R. McWilliam, H., Remmert, M., Söding, J., Thompson, J.D. and Higgins, D.G. (2011) Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol. Syst. Biol.,7, 1-9.

18. Tamura, K., Dudley, J., Nei, M. and Kumar, S. (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol. Bio. Evol.,24, 1596–1599.

19. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature, 227, 680–685.


20. Chen, C.C., Wu, P.H., Huang, C.T. and Cheng, K.J. (2004) A Pichiapastoris fermentation strategy forenhancing the heterologous expression of an Escherichia coliphytase. Enz.Microb. Technol.,35, 315-320.


21. Shamna, K.S., Rajamanikandan, K.C.P., Kumar, D.J., Balakumaran, M.D.andKalaichelvan, P.T. (2012) Extracellular production of phytases by a native Bacillus subtilis Strain. Ann. Biol. Res., 3, 979-985.


22. Choi, Y.M., Suh, H.J. and Kim, J.M. (2001) Purification and properties of extracellular phytase from Bacillus sp. KHU-10. J. Protein. Chem.,20, 287–292.

23. Gulati, H.K., Chadha, B.S. and Saini H.S. (2007) Production and characterization of thermostable alkaline phytase from Bacillus laevolacticus isolated from rhizosphere soil. J. Ind. Microbiol. Biotechnol.,34, 91–98.

24. Tye, A.J., Siu, F.K., Leung, T.Y. and Lim, B.L. (2002) Molecular cloning and the biochemical characterization of two novel phytases from B. subtilis 168 and B.licheniformis. Appl. Microbiol. Biotechnol.,59,190–197.

25. Yao, M.Z., Zhang, Y.H., Lu, W.L., Hu, M.Q., Wang, W. and Liang, A.H. (2011) Phytases: crystal structures, protein engineering and potential biotechnological applications.J. Appl. Microbiol., 112, 1–14.

26. Farhat, A., Chouayekh, H., Farhat, M.B., Bouchaala, K. and Bejar, S. (2008) Gene cloning and characterization of a thermostablephytase from Bacillus subtilis US417 and assessment of its potential as a feed additive in comparison with a commercial enzyme. Mol. Biotechnol., 40, 127–135.

27. Fu, S., Sun, J., Qian, L. and Li,  Z. (2008) Bacillus phytases: present scenario and future perspectives.Appl. Biochem. Biotechnol.,151(1), 1-8.

28.Young, O.K., Kim, H.K., Bae,K.S., Yu, J.H. and Oh, T.K. (1998) Purification and properties of a thermostablephytase from Bacillus sp. DS11. Enz.Microb. Technol., 22, 2-7.

29. Greiner, R., Haller, E., Konietzny, U. and Jany, K.D. (1997) Purification and characterization of a phytases from Klebsiellaterrigena. Arch. Biochem. Biophys.,341, 201-206.