4.4.1. Phenylalanine Ammonia-Lyase (PAL)

The PAL activity was analyzed at 290 nm wavelength by measuring the conversion of L-phenylalanine into ammonia and trans-cinnamic acid [85]. For this, 1 g rice leaves was homogenized in 5 mL of 0.1 M borate buffer, pH 7.0, containing 0.1 g polyvinyl polypyrrolindone (PVP) at 4 °C with a pestle and mortar. The homogenate was centrifuged at 10,000× g for 30 min at 4 °C. The supernatant was collected and used in the enzyme assay. The reaction mixture included 0.4 mL of enzyme extract, 0.5 mL of 0.1 M borate buffer (pH 8.8), and 0.5 mL of 12 nm L-phenylalanine in the same buffer. The reaction mixture was incubated at 30 °C for 30 min in a water bath. The reference cell was 0.4 mL of enzyme extract and 1.0 mL of borate buffer. The amount of trans-cinnamic acid synthesized was calculated using its absorption coefficient of 9630 µmol s⁻¹ g⁻¹.