Insight into Biochemical Characterization of Plant Sesquiterpene Synthases

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    Abstract

    A fast and reproducible protocol was established for enzymatic characterization of plant sesquiterpene synthases that can incorporate radioactivity in their products. The method utilizes the 96-well format in conjunction with cluster tubes and enables processing of >200 samples a day. Along with reduced reagent usage, it allows further reduction in the use of radioactive isotopes and flammable organic solvents. The sesquiterpene synthases previously characterized were expressed in yeast, and the plant-derived Thapsia garganica kunzeaol synthase TgTPS2 was tested in this method. KM for TgTPS2 was found to be 0.55 μM; the turnover number, kcat, was found to be 0.29 s-1, kcat for TgTPS2 is in agreement with that of terpene synthases of other plants, and kcat/KM was found to be 0.53 s-1 μM-1 for TgTPS2. The kinetic parameters were in agreement with previously published data.
    Original languageEnglish
    JournalAnalytical Chemistry Insights
    Volume11
    Issue numberS1
    Number of pages7
    ISSN1177-3901
    DOIs
    Publication statusPublished - 2016

    Bibliographical note

    This is an open-access article distributed under the terms of the Creative Commons
    CC -BY-NC 3.0 License.

    Keywords

    • Enzyme
    • Plant sesquiterpene synthase
    • Purification
    • Yeast

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