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.