Insight into Biochemical Characterization of Plant Sesquiterpene Synthases

Tom Manczak ； Henrik Toft Simonsen

《Analytical Chemistry Insights》 2016_S (2016/12) Pp. 1-7

https://doi.org/10.4137/ACI.S40292

摘要

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. K_M for TgTPS2 was found to be 0.55 μM; the turnover number, k_(cat), was found to be 0.29 s^(-1), k_(cat) for TgTPS2 is in agreement with that of terpene synthases of other plants, and k_(cat)/K_M was found to be 0.53 s^(-1) μM^(-1) for TgTPS2. The kinetic parameters were in agreement with previously published data.

關鍵字

purification ； yeast ； enzyme ； plant sesquiterpene synthase