2.6 Preparations of crude protein extract and assay of
phenylacetaldehyde reductase (PAR) enzyme
Extraction and assay of PAR enzyme from J. auriculatum flowers
was carried out by the method described by Chen et al. (2011) with
slight modifications. Briefly, flowers were ground to powder with the
help of liquid nitrogen. The crude protein was extracted with an
extraction buffer comprising of 10 mM potassium phosphate buffer (pH
7.5), 1% glycerol, 5 mM DTT, 0.05% Triton X and 1mM PMSF. The slurry
was centrifuged at 13,000 r.p.m. for 20 min at 4°C. The supernatant
obtained was used for assay of PAR.
Enzyme activity was determined by catalyzing the reaction of
2-phenylacetaldehyde (1mM) and NADPH (0.25 mM) with crude enzyme extract
(containing 100 µg of protein), in 10 mM potassium phosphate buffer (pH
7) at 35°C for 30 min. Hexane (200 µl) was carefully layered over the
aqueous reaction mixture for collecting 2-phenylethanol produced during
the reaction. The organic phase containing volatile product was
collected and dehydrated using
anhydrous sodium sulphate. The formation of 2-phenylethanol (2PE) was
monitored using ThermoScientific GC-MS system (GC-Trace 1300 equipped
with ISQ QD, Single Quadrupole Mass Spectrometer). The GC was equipped
with a TG-5MS capillary column (length 30 m, 0.32 mm i.d., 0.25 µm film
thickness). The inlet temperature was set at 260°C. Compounds were
separated with the following temperature method: initial oven
temperature was set at 60°C for 5 min, followed by an increase of 170°C
at a rate of 20 °C/min, then a linear increase of 40°C/min to 290°C, and
a final hold of 3 min. Identification and quantification of 2PE was done
by running known quantity of authentic standard.