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Directed evolution of a highly active Yersinia mollaretii phytase

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Abstract

Phytase improves as a feed supplement the nutritional quality of phytate-rich diets (e.g., cereal grains, legumes, and oilseeds) by hydrolyzing indigestible phytate (myo-inositol 1,2,3,4,5,6-hexakis dihydrogen phosphate) and increasing abdominal absorption of inorganic phosphates, minerals, and trace elements. Directed phytase evolution was reported for improving industrial relevant properties such as thermostability (pelleting process) or activity. In this study, we report the cloning, characterization, and directed evolution of the Yersinia mollaretii phytase (Ymphytase). Ymphytase has a tetrameric structure with positive cooperativity (Hill coefficient was 2.3) and a specific activity of 1,073 U/mg which is ∼10 times higher than widely used fungal phytases. High-throughput prescreening methods using filter papers or 384-well microtiter plates were developed. Precise subsequent screening for thermostable and active phytase variants was performed by combining absorbance and fluorescence-based detection system in 96-well microtiter plates. Directed evolution yielded after mutant library generation (SeSaM method) and two-step screening (in total ∼8,400 clones) a phytase variant with ∼20% improved thermostability (58°C for 20 min; residual activity wild type ∼34%; variant ∼53%) and increased melting temperature (1.5°C) with a slight loss of specific activity (993 U/mg).

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Acknowledgments

We thank BASF SE for financial support and Dr. Alexander Schenk for providing pALXtreme-5b vector and E. coli BL21-Gold(DE3)laqIQ1 expression strain.

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Authors and Affiliations

  1. Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 1, 52074, Aachen, Germany

    Amol V. Shivange, Alexander Dennig & Ulrich Schwaneberg

  2. School of Engineering and Science, Jacobs University Bremen gGmbH, Campus Ring 1, 28759, Bremen, Germany

    Amol V. Shivange, Danilo Roccatano & Ulrich Schwaneberg

  3. BASF SE, Fine Chemicals and Biocatalysis Research, GVF-A30, 67056, Ludwigshafen, Germany

    Annegret Serwe & Stefan Haefner

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  1. Amol V. Shivange
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  2. Annegret Serwe
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  3. Alexander Dennig
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  6. Ulrich Schwaneberg
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Correspondence to Ulrich Schwaneberg.

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Shivange, A.V., Serwe, A., Dennig, A. et al. Directed evolution of a highly active Yersinia mollaretii phytase. Appl Microbiol Biotechnol 95, 405–418 (2012). https://doi.org/10.1007/s00253-011-3756-7

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