2.5 Preparation of crude protein extract and assay of phenylalanine ammonia-lyase (PAL) enzyme
Extraction and assay of PAL enzyme from J. auriculatum flowers was carried out by the method described by Chakraborty et al. (2008) with slight modifications. Briefly, flowers were ground to powder with the help of liquid nitrogen in the presence of 10% (w/w) polyvinylpyrrolidone (PVPP). The crude protein was extracted with an extraction buffer comprising of 100 mM Tris HCl (pH 7.5) and 10 mM DTT. The slurry was centrifuged at 14,000 r.p.m. for 30 min at 4°C. The supernatant obtained was used for assay of PAL activity. The assay mixture contained 100mM Tris-HCl buffer (pH 7.5), 100 µg crude protein, 5mM L-phenylalanine in a total reaction volume of 250 µl. After incubating the reaction mixture at 37°C for 1 h, the reaction was stopped by addition of an equal volume of methanol: acetic acid (3:1). This was further centrifuged to remove any denatured protein. Formation of in vitro trans -cinnamic acid from L-phenylalanine upon the catalytic activity of PAL was monitored at 275 nm via a Dionex UltiMate 3000 UHPLC system (Thermo Fisher Scientific, Massachusetts, USA) using WatersTM Symmetry TM(Waters, Milford, MA, USA) C18 (3.5 µm, 75mm×4.6 mm) reversed-phase column. The mobile phase consists of water (containing 1mM TFA) and methanol at a ratio of 55: 45 (v/v) using a constant flow rate of 1 ml/min.