2.8 Preparations of crude protein extract and assay of benzyl alcohol: acetyl transferase (BEAT) enzyme
Extraction and assay of BEAT enzyme from J. auriculatum flowers was carried out by the method described by Dudareva et al. (1998) and Bera et al. (2017) 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 2:1 [v/w] buffer/tissue) containing 50 mM BisTris-HCl (pH 6.9), 10 mMβ -mercaptoethanol, 5 mM Na2S2O5, 1% (w/v) PVPP, and 10% (v/v) glycerol. The slurry was centrifuged at 12,000 r.p.m. for 20 min at 4°C. The supernatant obtained was used for assay of BEAT.
Enzyme activity was determined by catalyzing the reaction of benzyl alcohol (50 mM) and acetyl-Coenzyme A (1 mM) with crude enzyme extract (containing 100 µg of protein) in an assay buffer, containing 250 mM Tris-HCl [pH 7.5] and 14 mM β -mercaptoethanol, at 30°C for 45 min. Hexane (200 µl) was carefully layered over the aqueous mixture for collecting benzyl acetate produced during the reaction. The formation of benzyl acetate 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 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 5°C/ min to 60°C; hold for 2 min, then a linear increase of 20°C/ min to 220°C; hold for 2 min and then a final increase to 290°C at 20°C/ min and a hold of 10 min. Identification and quantification of benzyl acetate was done by injecting known quantity of authentic standard in GC-MS system.