2.9 Preparations of crude protein extract and assay of monoterpene synthase (MTS) enzyme
Extraction and assay of MTS enzyme from flowers of J. auriculatumwas carried out by the method as described by Pichersky et al. (1994) with slight modifications. The crude protein from floral tissue was extracted with an extraction buffer containing 50 mM bis-Tris HCl (pH 6.9), 10 mM DTT, 5mM Na2S2O5, 10% (w/v) glycerol and 1% (w/v) PVPP.
MTS activity was determined by catalysing the reaction of geranyl pyrophosphate (GPP) (Sigma-Aldrich) (50 µM) in assay buffer (containing 50 mM potassium HEPES of pH 7.8, 5mM Na2S2O5, 5 mM DTT and 10% (w/v) glycerol and 20 mM MgCl2 and 5mM MnCl2 as cofactors) with crude enzyme extract (100 µg of protein) at 30°C for 60 min. Linalool produced was trapped in hexane (200 µl) which was carefully layered over the aqueous reaction mixture. The formation of linalool was monitored using ThermoScientific GC-MS system. The GC was equipped with a DB-5MS column (length 30 m, 0.25 mm i.d., 0.25 µm film thickness). The inlet temperature was set at 220°C. Compounds were separated with the following temperature method: initial oven temperature was set at 50°C for 2 min, followed by an increase of 10°C/ min to 70°C; hold for 3 min, then a linear increase of 10°C/ min to 200°C; hold for 2 min and then a final increase to 300°C at 30°C/ min and a hold of 10 min. Identification and quantification of linalool was done by injecting known quantity of authentic standard in GC-MS system.