2.10. Acyl-ACP thioesterase assay

Thioesterase activity was assayed by monitoring the production of radiolabelled free fatty acids from the [1–¹⁴C] acyl-ACP substrates as indicated above. The reaction medium contained 50 mM Tris-HCl [pH 8.0], 5 mM DTT, 50–2500 Bq of [1–¹⁴C] acyl-ACP and 0.025–1.0 ng of the recombinant enzyme in a final volume of 0.1 mL. The reactions were carried out at room temperature for 5 min and quenched by adding 0.25 ml of 1 M acetic acid in 2-propanol. Hydrolysed free fatty acids were then extracted twice with 0.3 ml hexane and the radioactivity was measured in a liquid scintillation counter (Rackbeta II; LKB).