C r4a chromatopac

The PAL enzyme was extracted and partially purified by the method of Suzuki et al.³⁹ . Fresh leaves (1 g) were ground at 4 °C in 5 mL of 0.1 M sodium borate buffer (pH 8.8). Homogenates were centrifuged at 12,000 × g for 15 min at 4 °C and the supernatant was used as the enzyme extract. Reaction mixtures consisting of 500 μL sodium borate buffer (pH 8.7) and 250 μL enzyme extracts were pre-incubated for 5 min at 40 °C. The reaction was started by the addition of 300 μL of 50 mM l-phenylalanine (SIGMA-ALDRICH) and, after incubation for 1 h at 40 °C, stopped by adding 50 μL of 5 N HCl. The reaction mixture was centrifuged again (12,000 × g for 15 min) prior to injection into an HPLC (SHIMADZU, C-R4A Chromatopac; SCL-6B system controller) featuring a ZORBAX SB-C18 analytical column (4.6 × 150 mm, 5 μm particle size, AGILENT, Germany) and a U.V. detector at room temperature. The mobile phase consisted of 57% acetonitrile in water with a flow rate of 0.5 mL min⁻¹. Detection of trans-cinnamic acid (t-CA) was based on retention time and performed at 275 nm. The activity of PAL was expressed as nmol t-CA min⁻¹ g⁻¹ of fresh mass in relation to the peak area of a t-CA standard solution (1 mg/100 mL sodium borate buffer, pH 8.7).

The PAL enzyme was extracted and partially purified following the method Suzuki et al. [36 ]. Fresh leaves (1 g) were ground at 4°C in 5 mL of 0.1 M sodium borate buffer (pH 8.8). Homogenates were centrifuged at 12,000 × g for 15 min at 4°C and the supernatant was used as the enzyme extract. Reaction mixtures consisting of 500 μL sodium borate buffer (pH 8.7) and 250 μL enzyme extracts were pre-incubated for 5 min at 40°C and the reaction was started after the addition of 300 μL of 50 mM l-phenylalanine (Sigma-Aldrich). After incubation for 1 h at 40°C, the reaction was stopped by adding 50 μL of 5 N HCl. The reaction mixture was centrifuged again (12,000 × g for 15 min) prior to injection into an HPLC (Shimadzu, C-R4A Chromatopac; SCL-6B system controller) featuring a Zorbax SB-C18 analytical column (4.6 × 150 mm, 5 μm particle size, Agilent, Germany) and a U.V. detector at room temperature. The mobile phase consisted of 57% acetonitrile in water with a flow rate of 0.5 mL min⁻¹. Detection of trans-cinnamic acid (t-CA) in all the treatments (three replicates) was based on retention time and performed at 275 nm. The activity of PAL was expressed as nmol t-CA min⁻¹ g⁻¹ of fresh mass in relation to the peak area of a t-CA standard solution (1 mg/100 mL sodium borate buffer, pH 8.7).