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TPS enzyme assays

KL Katrin Luck
XC Xinlu Chen
AN Ayla M. Norris
FC Feng Chen
JG Jonathan Gershenzon
TK Tobias G. Köllner
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To determine the catalytic activity of the different terpene synthases and the reconstructed TPS ancestors, enzyme assays containing 40 μl of the bacterial extract and 60 µl assay buffer with 10 μM (E,E)-FPP or GPP and 10 mM MgCl2, in a Teflon-sealed, screw-capped 1 ml GC glass vial were performed. A SPME (solid phase microextraction) fiber consisting of 100 µm polydimethylsiloxane (Supelco, Bellefonte, PA, USA) was placed into the headspace of the vial for 0.5 h incubation at 30 °C. For analysis of the adsorbed reaction products, the SPME fiber was directly inserted into the injector of the gas chromatograph.

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