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Heterologous Expression, Purification, and Biochemical Characterization of α-Humulene Synthase from Zingiber zerumbet Smith

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Abstract

The α-humulene synthase from Zingiber zerumbet Smith was expressed as a polyhistidine-tagged protein in an E. coli BL21(DE3) strain. Induction time and inductor (isopropyl-β-D-thiogalactopyranoside) concentration were optimized. The enzyme was successfully purified directly from cell lysate by NTA affinity column chromatography and careful selection of coordinated metal ion and imidazole elution conditions. Bioactivity assays were conducted with the natural substrate farnesyl diphosphate (FDP) in a two-phase system with in situ extraction of products. The conversion of FDP to α-humulene (~94.5 %) and β-caryophyllene (~5.5 %) could be monitored by gas chromatography-flame ionization detection (GC-FID). Optimal pH and temperature as well as kinetic parameters K M and k cat were determined using a discontinuous kinetic assay.

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Acknowledgments

This study was funded by the European Regional Development Fund (EFRE): Innovation Network “Refinement of plant resources” (ZW-8-80130940).

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Authors and Affiliations

  1. Institute of Technical Chemistry, Gottfried Wilhelm Leibniz University of Hannover, Callinstr. 5, 30167, Hannover, Germany

    Semra Alemdar, Steffen Hartwig, Thore Frister, Jan Christoph König, Thomas Scheper & Sascha Beutel

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  1. Semra Alemdar
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  2. Steffen Hartwig
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  3. Thore Frister
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  4. Jan Christoph König
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  5. Thomas Scheper
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  6. Sascha Beutel
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Correspondence to Sascha Beutel.

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Alemdar, S., Hartwig, S., Frister, T. et al. Heterologous Expression, Purification, and Biochemical Characterization of α-Humulene Synthase from Zingiber zerumbet Smith. Appl Biochem Biotechnol 178, 474–489 (2016). https://doi.org/10.1007/s12010-015-1888-4

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  • DOI: https://doi.org/10.1007/s12010-015-1888-4

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