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.