PPO assay

PPO activities were assayed with 4-methylcatechol and pyrocatechol at 410 nm as the substrates by using spectrophotometric method with slight modifications (Joseph et al. 2012). The reaction mixture included 0.1 mL enzyme solution, 1.0 mL of 0.1 M substrate solution in 1.9 mL 0.1 M phosphate buffer at pH 6.8. Then, the mixture was transferred into a cuvette and absorbance readings were measured at 15 s intervals for 5 min using a spectrophotometer (Secomam UviLine 9400, Champigny-sur-Marne, France) at room temperature. The blank consisted of 1 mL substrate solution in 2 mL of phosphate buffer. The initial velocity was calculated from the slope of the absorbance value against time curve. One unit (U) of PPO activity was defined as amount of enzyme that caused a 0.001 absorbance change per min (Kim and Kim 2013).