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Isolation and Molecular Characterization of Thermostable Phytase from Bacillus subtilis (BSPhyARRMK33)

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Abstract

The thermostable phytase gene was isolated from Bacillus subtilis ARRMK33 (BsPhyARRMK33). The gene has an ORF of 1152 bp and that encodes a protein of 383 amino acids. Sequence analysis showed high homology with Bacillus sp. phytase proteins, but no similarity was found with other phytases. SDS-PAGE analysis exhibited a predicted molecular mass of 42 kDa. Homology modeling of BsPhyARRMK33 protein based on Bacillus amyloliquefaciens crystal structure disclosed its β-propeller structure. BsPhyARRMK33 recombinant plasmid in pET-28a(+) was expressed in Rosetta gami B DE3 cells and the maximum phytase activity 15.3 U mg−1 obtained. The enzyme exhibits high thermostability at various temperatures and broad pH ranges. The recombinant protein retained 74 % of its original activity after incubation at 95 °C for 10 min. In the presence of Ca2+, the recombinant phytase activity was maximal where as it was inhibited by EDTA. The optimal pH and temperature for the recombinant phytase activity is achieved at 7.0 and 55 °C, respectively. Thermostable nature and wide range of pH are promising features of recombinant BsPhyARRMK33 protein that may be employed as an efficient alternative to commercially known phytases and thereby alleviate environmental eutrophication.

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Acknowledgments

The authors are thankful to the ICGEB, New Delhi, for providing support and facilities to carry out the research. The authors acknowledge the award of Senior Research Fellowship to M. Manna and Research Associateship to V. M. M. Achary by CSIR and DBT, India, respectively.

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Authors and Affiliations

  1. Plant Molecular Biology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, 110 067, India

    Chinreddy Subramanyam Reddy, V. Mohan Murali Achary, Mrinalini Manna, Jitender Singh, Tanushri Kaul & Malireddy K. Reddy

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  1. Chinreddy Subramanyam Reddy
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  2. V. Mohan Murali Achary
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  3. Mrinalini Manna
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  4. Jitender Singh
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Correspondence to Chinreddy Subramanyam Reddy.

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Reddy, C.S., Achary, V.M.M., Manna, M. et al. Isolation and Molecular Characterization of Thermostable Phytase from Bacillus subtilis (BSPhyARRMK33). Appl Biochem Biotechnol 175, 3058–3067 (2015). https://doi.org/10.1007/s12010-015-1487-4

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  • DOI: https://doi.org/10.1007/s12010-015-1487-4

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