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Molecular Cloning, Expression and Characterisation of a Bacterial Myrosinase from Citrobacter Wye1

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Abstract

Glucosinolates are plant natural products which on degradation by myrosinases give rise to the beneficial bioactive isothiocyanates. Recently, a myrosinase activity was detected in a Citrobacter strain isolated from soil. This enzyme was purified enabling its amino acid sequence and gene sequence (cmyr) to be determined. In order to study this myrosinase it was necessary to establish an expression system that would enable future work such as a structural determination of the protein to be carried out. The myrosinase gene was amplified, cloned and expressed in Escherichia coli with a 6XHis-tag. The heterologous expression of cmyr enabled relatively large amounts of myrosinase to be produced (3.4 mg cmyr/100 ml culture). Myrosinase activity was determined by mixing substrate and enzyme and determining glucose release. Optimum pH and temperature were determined to be pH 6.0 and 25 °C for the Ni-NTA purified protein. The kinetic parameters of the purified myrosinase were determined using sinigrin as a substrate. Km and Vmax were estimated as 0.18 mM and 0.033 mmol/min/mg respectively for sinigrin under optimum conditions and compared to other kinetic data for myrosinases. The substrate specificity of myrosinase was determined having the highest affinity for sinigrin followed by glucoiberin, progoitrin, glucoerucin, glucoraphanin and glucotropaeolin.

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Data Availability

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

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Acknowledgements

We wish to thank to Dr. Gerhard Saalbach (John Innes Centre, Norwich) for his support to perform LCMS analysis.

Funding

Quadram Institute Bioscience is supported by the Biotechnological and Biological Sciences Research Council via Institute Strategic grants BB/J004529/1, BB/J004545/1, BB/R012490/1 and BB/R012512/1. Fatma Cebeci was supported by a scholarship funded by the Republic of Turkey Ministry of National Education.

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Authors and Affiliations

  1. Food Innovation and Health Institute Strategic Programme, Quadram Institute Bioscience, Norwich, UK

    Fatma Cebeci, Richard Mithen & Arjan Narbad

  2. Gut Microbes and Health Institute Strategic Programme, Quadram Institute Bioscience, Norwich, UK

    Melinda J. Mayer & Arjan Narbad

  3. Department of Life Sciences, Imperial College London, London, UK

    John T. Rossiter

  4. Department of Nutrition and Dietetics, Bayburt University, Bayburt, Turkey

    Fatma Cebeci

  5. Liggins Institute, The University of Auckland, Auckland, New Zealand

    Richard Mithen

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  1. Fatma Cebeci
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Contributions

Conceptualization, JTR, RM and AN; methodology, FC and MM; validation, FC; formal analysis, FC; investigation, FC; resources, AN; writing—original draft preparation, FC; writing—review and editing, JTR, MM, RM and AN; visualization, FC and JTR; supervision, JTR, MM, RM and AN; project administration, AN; funding acquisition, AN. All authors have read and agreed to the published version of the manuscript.

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Correspondence to Fatma Cebeci.

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Cebeci, F., Mayer, M.J., Rossiter, J.T. et al. Molecular Cloning, Expression and Characterisation of a Bacterial Myrosinase from Citrobacter Wye1. Protein J 41, 131–140 (2022). https://doi.org/10.1007/s10930-021-10034-5

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