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Improving the Neutral Phytase Activity from Bacillus amyloliquefaciens DSM 1061 by Site-Directed Mutagenesis

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Abstract

Neutral phytase is used as a feed additive for degradation of anti-nutritional phytate in aquatic feed industry. Site-directed mutagenesis of Bacillus amyloliquefaciens DSM 1061 phytase was performed with an aim to increase its activity. Mutation residues were chosen based on multiple sequence alignments and structure analysis of neutral phytsaes from different microorganisms. The mutation sites on surface (D148E, S197E and N156E) and around the active site (D52E) of phytase were selected. Analysis of the phytase variants showed that the specific activities of mutants D148E and S197E remarkably increased by about 35 and 13 % over a temperature range of 40–75 °C at pH 7.0, respectively. The k cat of mutants D148E and S197E were 1.50 and 1.25 times than that of the wild-type phytase, respectively. Both D148E and S197E showed much higher thermostability than that of the wild-type phytase. However, mutants N156E and D52E led to significant loss of specific activity of the enzyme. Structural analysis revealed that these mutations may affect conformation of the active site of phytase. The present mutant phytases D148E and S197E with increased activities and thermostabilities have application potential as additives in aquaculture feed.

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Acknowledgments

This study was supported financially by National Natural Science Foundation of China (No. 31101912), and the Natural Science Foundation of Jiangsu Province of China (No. BK2011420), and Qing Lan Project of Jiangsu Province.

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Authors and Affiliations

  1. School of Chemical and Biological Engineering, Yancheng Institute of Technology, Yancheng, People’s Republic of China

    Wei Xu, Rong Shao, Zupeng Wang & **uhua Yan

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  1. Wei Xu
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  2. Rong Shao
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  3. Zupeng Wang
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  4. **uhua Yan
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Correspondence to Wei Xu.

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Xu, W., Shao, R., Wang, Z. et al. Improving the Neutral Phytase Activity from Bacillus amyloliquefaciens DSM 1061 by Site-Directed Mutagenesis. Appl Biochem Biotechnol 175, 3184–3194 (2015). https://doi.org/10.1007/s12010-015-1495-4

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