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Gene Cloning and Characterization of a Thermostable Phytase from Bacillus subtilis US417 and Assessment of its Potential as a Feed Additive in Comparison with a Commercial Enzyme

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Abstract

An extracellular phytase from Bacillus subtilis US417 (PHY US417) was purified and characterized. The purified enzyme of 41 kDa was calcium-dependent and optimally active at pH 7.5 and 55°C. The thermal stability of PHY US417 was drastically improved by calcium. Indeed, it recovered 77% of its original activity after denaturation for 10 min at 75°C in the presence of 5 mM CaCl2, while it retained only 22% of activity when incubated for 10 min at 60°C without calcium. In addition, PHY US417 was found to be highly specific for phytate and exhibited pH stability similar to Phyzyme, a commercial phytase with optimal activity at pH 5.5 and 60°C. The phytase gene was cloned by PCR from Bacillus subtilis US417. Sequence analysis of the encoded polypeptide revealed one residue difference from PhyC of Bacillus subtilis VTTE-68013 (substitution of arginine in position 257 by proline in PHY US417) which was reported to exhibit lower thermostability especially in the absence of calcium. With its neutral pH optimum as well as its great pH and thermal stability, the PHY US417 enzyme presumed to be predominantly active in the intestine has a high potential for use as feed additive.

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Acknowledgments

This research was funded by the Tunisian Government “Contrat Programme CBS-LEMP” and the local Company “Nutrisud” through a research agreement. Part of the work was also supported by the CMCU project (2007–2009) no. 07G0922 “CHOUAYEKH/MAGUIN”. The authors are grateful to Dr. Ahmed Rebai for his kind assistance in the statistical analysis of the pH data. We would like also to express our gratitude to Dr. Emmanuelle Maguin, Dr. Marie-Joëlle Virolle, and Dr. Mamdouh Ben Ali for useful discussion. Many thanks as well to Mr. Badr Abdelhedi, Mr. Amine Mrabet, and Mr. Dhiaeddine Hachicha for their permanent collaboration.

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  1. Laboratoire d’Enzymes et de Métabolites des Procaryotes, Centre de Biotechnologie de Sfax, Route de Sidi Mansour Km 6, BP “1177”, 3038, Sfax, Tunisia

    Ameny Farhat, Hichem Chouayekh, Mounira Ben Farhat, Kameleddine Bouchaala & Samir Bejar

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  1. Ameny Farhat
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  2. Hichem Chouayekh
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Correspondence to Hichem Chouayekh.

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Farhat, A., Chouayekh, H., Ben Farhat, M. et al. Gene Cloning and Characterization of a Thermostable Phytase from Bacillus subtilis US417 and Assessment of its Potential as a Feed Additive in Comparison with a Commercial Enzyme. Mol Biotechnol 40, 127–135 (2008). https://doi.org/10.1007/s12033-008-9068-1

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