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Vanadium bromoperoxidase-coupled fluorescent assay for flow cytometry sorting of glucose oxidase gene libraries in double emulsions

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Abstract

A Vanadium bromoPeroxidase-coupled fluorescent assay (ViPer) for ultrahigh-throughput screening of glucose oxidase (GOx) gene libraries employing double emulsions and flow cytometry was developed. The assay is based on detection of the product of a GOx reaction, hydrogen peroxide, that is first converted to a hypobromide by vanadium bromoperoxidase in the presence of sodium bromide. The hypobromide is afterwards detected in a reaction with a fluorogenic probe, 3-carboxy-7-(4′-aminophenoxy)-coumarine, where fluorescent 3-carboxy-coumarine is released. The ViPer screening system is three times more sensitive than a horseradish peroxidase coupled detection system and more resistant to bleaching of fluorescence in excess of peroxide. Using the ViPer screening system a high epPCR gene library containing 100,000 different GOx variants was screened for active clones in less than 1 h by flow cytometry. A library containing 0.15 % of yeast cells expressing active enzyme variants and with an average GOx activity in the liquid culture of 0.47 U/mL, after one round of sorting, had 28.12 % of the yeast cells expressing the active GOx (an enrichment factor of 200) and 26.8 U/mL of the GOx activity in the liquid culture (an enrichment factor of 57). The developed screening system could be adapted and used in a directed evolution of GOx and other hydrogen peroxide-producing enzymes (oxidases) and glycosidases if coupled with a carbohydrate oxidase.

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Acknowledgment

We thank the BMBF BiochancePlus program and the BRAIN AG company (Dr. Jürgen Eck & Dr. Frank Niehaus) for funding. Dr. Radivoje Prodanovic thanks furthermore the Alexander von Humboldt foundation for financial support.

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Authors and Affiliations

  1. Jacobs University Bremen, Campus Ring 1, 28759, Bremen, Germany

    Radivoje Prodanovic, Raluca Ostafe, Milan Blanusa & Ulrich Schwaneberg

  2. Faculty of Chemistry, University of Belgrade, Studentski trg 12, 11000, Belgrade, Serbia

    Radivoje Prodanovic

  3. Faculty of Biology, RWTH Aachen University, Worringer Weg 1, 52056, Aachen, Germany

    Raluca Ostafe & Ulrich Schwaneberg

Authors
  1. Radivoje Prodanovic
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  2. Raluca Ostafe
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  3. Milan Blanusa
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  4. Ulrich Schwaneberg
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Correspondence to Radivoje Prodanovic or Ulrich Schwaneberg.

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Prodanovic, R., Ostafe, R., Blanusa, M. et al. Vanadium bromoperoxidase-coupled fluorescent assay for flow cytometry sorting of glucose oxidase gene libraries in double emulsions. Anal Bioanal Chem 404, 1439–1447 (2012). https://doi.org/10.1007/s00216-012-6234-x

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  • DOI: https://doi.org/10.1007/s00216-012-6234-x

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