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Overexpression and Biochemical Characterization of a Thermostable Phytase from Bacillus subtilis US417 in Pichia pastoris

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Abstract

The overexpression of the native gene encoding the thermostable Bacillus subtilis US417 phytase using Pichia pastoris system is described. The phytase gene, in which the sequence encoding the signal peptide was replaced by that of the α-factor of Saccharomyces cerevisiae, was placed under the control of the methanol-inducible promoter of the alcohol oxidase 1 gene and expressed in Pichia pastoris. Small-scale expression experiments and activity assays were used to screen positive colonies. A recombinant strain was selected and produces 43 and 227 U/mL of phytase activity in shake flasks and in high-cell-density fermentation, respectively. The purified phytase was glycosylated protein and varied in size (50–65 kDa). It has a molecular mass of 43 kDa when it was deglycosylated. The purified r-PHY maintains 100 % of its activity after 10 min incubation at 75 °C and pH 7.5. This thermostable phytase, which is also active over broad pH ranges, may be useful as feed additives, since it can resist the temperature used in the feed-pelleting process.

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Authors and Affiliations

  1. Laboratory of Microorganisms and Biomolecules (LMB), Centre of Biotechnology of Sfax (CBS), University of Sfax, Road of Sidi Mansour Km 6, P.O. Box 1177, 3018, Sfax, Tunisia

    Aïda Hmida-Sayari, Fatma Elgharbi, Ameny Farhat, Hatem Rekik & Samir Bejar

  2. Laboratory of Structural Genomic IBBMC, Bat 430, Université Paris-sud, 91 405, Orsay cedex, France

    Karine Blondeau

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  1. Aïda Hmida-Sayari
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  2. Fatma Elgharbi
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Correspondence to Aïda Hmida-Sayari.

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Hmida-Sayari, A., Elgharbi, F., Farhat, A. et al. Overexpression and Biochemical Characterization of a Thermostable Phytase from Bacillus subtilis US417 in Pichia pastoris . Mol Biotechnol 56, 839–848 (2014). https://doi.org/10.1007/s12033-014-9764-y

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