Cloning and Expression of a Novel Phytase Gene (phyMS) from Mycobacterium smegmatis


Phytase, also known as phytate-degrading enzyme, catalyzes the hydrolysis of phytate (inositol hexakisphosphate) with sequential release of phosphate and lower inositol phosphate. We report here a new plasmid construct designated as pMSuia from pBAD-TOPO that harbors a 1.1 kb phytase gene (phyMS) from Mycobacterium smegmatis, and expression as well as characterization of the purified recombinant M. smegmatis phytase. DNA sequencing analysis and multiple alignment exercise indicated that the M. smegmatis phytase is different from both known acid and alkaline phytases. The active ~45 kDa recombinant enzyme was expressed and confirmed by enzyme assay and Western blot analyses. Ni-NTA affinity purified recombinant M. smegmatis phytase exhibited specific activity of 233.51 U/mg, optimal pH of 3 and 7 and optimal temperature of 60°C. The purified enzyme retains almost 30% of the initial activity after incubation at 90°C for 60 min. The enzyme showed broad substrate specificity with Km and Vmax of the recombinant enzyme for sodium phytate substrate of 0.105 ± 0.016 mM and 26.93 ± 1.21 mM min-1, respectively.

Share and Cite:

Nuge, T. , Hashim, Y. , Farouk, A. and Salleh, H. (2014) Cloning and Expression of a Novel Phytase Gene (phyMS) from Mycobacterium smegmatis. Advances in Enzyme Research, 2, 27-38. doi: 10.4236/aer.2014.21003.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] Haefner, S., Knietsch, A., Scholten, E., Braun, J., Lohscheidt, M. and Zelder, O. (2005) Biotechnological Production and Applications of Phytases. Applied Microbiology and Biotechnology, 68, 588-597.
[2] Greiner, R. and Konietzny, U. (2006) Phytase for Food Application. Food Technology and Biotechnology, 44, 125- 140.
[3] Haros, M., Rosell, C.M. and Benedito, C. (2001) Fungal Phytase as a Potential Breadmaking Additive. European Food Research and Technology, 213, 317-322.
[4] Kumar, V., Sinha, A.K., Makkar, H.P.S. and Becker, K. (2010) Dietary Roles of Phytate and Phytase in Human Nutrition: A Review. Food Chemistry, 120, 945-959.
[5] Drakakaki, G., Marcel, S., Glahn, R.P., Lund, L., Periagh, S., Fischer, R., Christou, P. and Stoger, E. (2005) Endosperm Specific Co-Expression of Recombinant Soybean Ferritin and Aspergillus Phytase in Maize Results in Significant Increases in the Levels of Bioavailable Iron. Plant Molecular Biology, 59, 869-880.
[6] Kim, Y.O., Lee, J.K., Kim, H.K., Yu, J.H. and Oh, T.K. (1998) Cloning of the Thermostable Phytase (Phy) from Bacillus sp. DS11 and Its Overexpression in Escherichia coli. FEMS Microbiology Letters, 162, 185-191.
[7] Kerovuo, J. and Tynkkynen, S. (2000) Expression of Bacillus subtilis Phytase in Lactobacillus plantarum 755. Letters in Applied Microbiology, 30, 325-329.
[8] Yip, W., Wang, L., Cheng, C., Wu, W., Lung, S. and Lim, B. (2003) The Introduction of a Phytase Gene from Bacillus subtilis Improved the Growth Performance of Transgenic Tobacco. Biochemical and Biophysical Research Communications, 310, 1148-1154.
[9] Sambrook, J., Fritschi, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, New York.
[10] Dechow, F.J. (1989) Separation and Purification Techniques in Biotechnology. Noyes Publications, New Jersey, 416- 478.
[11] Greiner, R. (2004) Purification and Properties of a Phytate-Degrading Enzyme from Pantoea agglomerans. Protein Journal, 23, 567-576.
[12] Heinonen, J.K. and Lathi, R.J. (1981) A New and Convenient Colometric Determination of Inorganic Orthophosphate and Its Application to the Assay of Inorganic Pyrophosphatase. Analytical Biochemistry, 113, 313-317.
[13] Ostanin, K. and Van Etten, R.L. (1993) Asp304 of Escherichia coli Acid Phosphatase Is Involved in Leaving Group Protonation. The Journal of Biological Chemistry, 268, 20778-20784.
[14] Lei, X.G. and Porres, J.M. (2003) Phytase Enzymology, Applications and Biotechnology. Biotechnology Letters, 25, 1787-1794.
[15] Kerovuo, J., Lauraeus, M., Nurminen, P., Kalkkinen, N. and Apajalahti, J. (1998) Isolation, Characterization, Molecular Gene Cloning and Sequence of a Novel Phytase from Bacillus subtilis. Applied and Environmental Microbiology, 64, 2079-2085.
[16] Kim, Y.O., Kim, H.K., Bae, K.S., Yu, J.H. and Oh, T.K. (1998). Purification and Properties of a Thermostable Phytase from Bacillus sp.DS11. Enzyme and Microbial Technology, 22, 2-7.
[17] Cho, J.S., Lee, C.W., Kang, S.H., Lee, J.C., Bok, J.D., Moon, Y.S., Lee, H.G., Kim, S.C. and Choi, Y.J. (2003) Purification and Characterization of a Phytase from Pseudomonas syringae MOK1. Current Microbiology, 47, 290-294.
[18] Zinin, N.V., Serkina, A.V., Gelfand, M.S., Shevelev, A.B. and Sineoky, S.P. (2004) Gene Cloning, Expression and Characterization of Novel Phytase from Obesumbacterium proteus. FEMS Microbiology Letters, 236, 283-290.
[19] Palacios, M.C., Haros, M., Rosell, C.M. and Sanz, Y. (2005) Characterization of an Acid Phosphatase from Lactobacillus pentosus: Regulation and Chemical Properties. Journal of Applied Microbiology, 98, 229-237.
[20] Sariyska, M.V., Gargova, S.A., Koleva, L.A. and Angelov, A.I. (2005) Aspergillus niger Phytase: Purification and Characterization. Biotechnology & Biotechnological Equipment, 19, 98-105.
[21] Ullah, A.H.J. and Gibson, D.M. (1987) Extracellular Phytase (E.C. from Aspergillus ficuurn NRRL 3135: Purification and Characterization. Preparative Biochemistry and Biotechnology, 17, 63-91.
[22] Pasamontes, L., Haiker, M., Wyss, M., Tessier, M., van Loon, A.P.G.M. (1997) Gene Cloning, Purification, and Characterization of a Heat-Stable Phytase from the Fungus Aspergillus fumigatus. Applied and Environmental Microbiology, 63, 1696-1700.
[23] Cao, L., Wang, W., Yang, C., Yang, Y., Diana, J., Yakuptiyage, A., Luo, Z. and Li, D. (2007) Application of Microbial Phytase in Fish Feed. Enzyme and Microbial Technology, 40, 497-507.
[24] Luckstadt, C. (2012) Acidification in Monogastric Fish: Limits and Potential. AQUA Culture Asia Pacific Magazine, 38-39.
[25] Baruah, K., Pal, A.K., Sahu, N.P., Jain, K.K., Mukherjee, S.C. and Debnath, D. (2005) Dietary Protein Level, Microbial Phytase, Citric Acid and Their Interactions on Bone Mineralization of Labeo rohita (Hamilton) Juveniles. Aquaculture Research, 36, 803-812.
[26] Vohra, A. and Satyanarayana, T. (2002) Statistical Optimi-zation of the Medium Components by Response Surface Methodology to Enhance Phytase Production by Pichia anomala. Process Biochemistry, 37, 999-1004.
[27] Konietzny, U. and Greiner, R. (2004) Bacterial Phytase: Potential Application, in Vivo Function and Regulation of Its Synthesis. Brazilian Journal of Microbiology, 35, 11-18.
[28] Ullah, A.H.J., Sethumadhavan, K., Mullaney, E.J., Ziegelhoffer, T. and Philips, S.A. (2003) Fungal phyA Gene Expressed in Potato Leaves Produces Active and Stable Phytase. Biochemical and Biophysical Research Communications, 306, 603-609.
[29] Garrett, J.B., Kretz, K.A., O’Donoghue, E., Kerovuo, J., Kim, J., Barton, N.R., Hazlewood, G.P., Short, J.M., Robertson, D.E., Gray, K.A. (2004) Enhancing the Thermal Tolerance and Gastric Performance of a Microbial Phytase for Use as a Phosphate-Mobilizing Monogastric-Feed Supplement. Applied and Environmental Microbiology, 70, 3041- 3046.
[30] Segueilha, L., Lambrechts, C., Boze, H., Moulin, G. and Galzy, P. (1992) Purification and Properties of the Phytase from Schwanniomyces castellii. Journal of Fermentation and Bioengineering, 7, 7-11.
[31] Angelis, M.D., Gallo, G., Corbo, M.R., McSweeney, P.L.H., Faccia, M., Giovine, M. and Gobbetti, M. (2003) Phytase Activity in Sourdough Lactic Acid Bacteria: Purification and Characterization of Phytase from Lactobacillus sanfranciscensis CB1. International Journal of Food Microbiology, 87, 259-270.
[32] Greiner, R., Konietzny, U. and Jany, K.D. (1993) Purification and Characterization of Two Phytases from Escherichia coli. Archives of Biochemistry and Biophysics, 303, 107-113.
[33] Wyss, M., Brugger, R., Kronenberger, A., Remy, R., Fimbel, R., Oesterhelt, G., Lehmann, M., van Loon, A.P.G.M. (1999) Biochemical Characterization of Fungal Phytases (myo-Inositol Hexakisphosphate Phosphohydrolases): Catalytic Properties. Applied and Environmental Microbiology, 65, 367-373.
[34] Zamudio, M., Gonzalez, A. and Medina, J.A. (2000) Lactobacillus plantarum Phytase Activity Is Due to the Non-Specific Acid Phosphatase. Letters in Applied Microbiology, 32, 181-184.
[35] Palacios, M.C., Haros, M., Rosell, C.M. and Sanz, Y. (2005) Characterization of an Acid Phosphatase from Lactobacillus pentosus: Regulation and Chemical Properties. Journal of Applied Microbiology, 98, 229-237.
[36] Shimizu, M. (1993) Purification and Characterization of Phytase and Acid Phosphatase Produced by Aspergillus oryzae K1. Bioscience, Biotechnology, and Biochemistry, 57, 1364-1365.

Copyright © 2024 by authors and Scientific Research Publishing Inc.

Creative Commons License

This work and the related PDF file are licensed under a Creative Commons Attribution 4.0 International License.