A single-vial analytical and quantitative gas chromatography–mass spectrometry assay for terpene synthases

摘要: A quantitative assay for the analysis of sesquiterpene synthases, wherein each reaction mixture is formulated in glass gas chromatography vials, overlaid with organic solvent such as ethyl acetate, and subsequently vortexed to extract hydrocarbon products into phase after a suitable incubation period, was developed. The product-enriched then sampled an automated fashion injected directly chromatograph-mass spectrometer without further workup quantification products. Application vial amorpha-4,11-diene synthase (ADS), synthase, demonstrated sensitivity detection major minor most notably identification several that had escaped previous detection. steady-state kinetic tobacco 5-epi-aristolochene (TEAS), another validated nature assay, providing alternative means established method using radiolabeled substrate, extraction, scintillation counting. This simplified provides standardized facilitate terpene synthases diverse mutant enzyme libraries by supplanting common practice larger scale reactions, multiple extractions, evaporative concentration prior chromatography-mass spectrometry (GC-MS) analysis.