title: Insight into Biochemical Characterization of Plant Sesquiterpene Synthases.
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-derivedThapsia garganicakunzeaol synthase TgTPS2 was tested in this method.KMfor TgTPS2 was found to be 0.55 μM; the turnover number,kcat, was found to be 0.29 s-1,kcatfor TgTPS2 is in agreement with that of terpene synthases of other plants, andkcat/KMwas found to be 0.53 s-1μM-1for TgTPS2. The kinetic parameters were in agreement with previously published data.
authors.affiliation: Department of Plants and Environmental Sciences, Copenhagen University, Frederiksberg, Denmark.
authors.forename: Tom
authors.initials: T
authors.lastname: Manczak
authors.affiliation: Department of Biotechnology and Biomedicine, Technical University of Denmark, Kongens Lyngby, Denmark.
authors.forename: Henrik Toft
authors.initials: HT
authors.lastname: Simonsen
keywords: enzyme; plant sesquiterpene synthase; purification; yeast