PPO (EC 1.10.3.2) activity was measured by incubating 0.1 mL of enzyme extract with 2.9 mL of buffered substrate (0.05 M sodium phosphate, pH 7.0) containing 10 mM catechol, and then monitoring the change in absorbance at 398 nm. The change in absorbance at 398 nm was recorded every 30 s for 4 min by a spectrophotometer (UVmini-1240, Shimadzu Corp., Japan). One unit of activity of PPO was defined as the amount of enzyme causing 0.001 absorbance increase per minute under the assay conditions. The specific PPO activity was expressed as U mg−1 protein.
POD (EC 1.11.1.7) activity was based on the determination of guaiacol oxidation at 470 nm by H2O2. The 3 mL reaction buffer contained 0.1 mL of 4.0% guaiacol, 0.1 mL of 0.46% H2O2 and 2.75 mL of buffered substrate (0.05 M sodium phosphate, pH 7.0), and the reaction was initiated by adding 0.05 mL of crude enzyme extract. The change in absorbance at 470 nm was followed every 30 s for 4 min by a spectrophoto-meter (UVmini-1240, Shimadzu Corp., Japan). One unit of POD was defined as the amount of enzyme causing a 0.01 absorbance increase per minute under assay the conditions. The specific POD activity was expressed as U mg−1 protein.
Protein contents were measured according to the method of Bradford (1976), using bovine serum albumin (BSA) as standard.
PPO (EC 1.10.3.2) activity was measured by incubating 0.1 mL of enzyme extract with 2.9 mL of buffered substrate (0.05 M sodium phosphate, pH 7.0) containing 10 mM catechol, and then monitoring the change in absorbance at 398 nm. The change in absorbance at 398 nm was recorded every 30 s for 4 min by a spectrophotometer (UVmini-1240, Shimadzu Corp., Japan). One unit of activity of PPO was defined as the amount of enzyme causing 0.001 absorbance increase per minute under the assay conditions. The specific PPO activity was expressed as U mg−1 protein.
POD (EC 1.11.1.7) activity was based on the determination of guaiacol oxidation at 470 nm by H2O2. The 3 mL reaction buffer contained 0.1 mL of 4.0% guaiacol, 0.1 mL of 0.46% H2O2 and 2.75 mL of buffered substrate (0.05 M sodium phosphate, pH 7.0), and the reaction was initiated by adding 0.05 mL of crude enzyme extract. The change in absorbance at 470 nm was followed every 30 s for 4 min by a spectrophoto-meter (UVmini-1240, Shimadzu Corp., Japan). One unit of POD was defined as the amount of enzyme causing a 0.01 absorbance increase per minute under assay the conditions. The specific POD activity was expressed as U mg−1 protein.
Protein contents were measured according to the method of Bradford (1976), using bovine serum albumin (BSA) as standard.
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