Determination of PPO activity
The activity of PPO was determined using L-DOPA as a substrate
according to the method described by Du et al. (2012). The assay
system consisted of 100 lL of crude PPO extract, 600 lL of 15mM
L-DOPA in deionised water, 400 lL of 0.05 M phosphate buffer (pH
6.0), and 100 lL of deionised water. The PPO activity was determined
by monitoring the formation of dopachrome at 45 C for
3 min. Absorbance was measured at 475 nm using a UV spectrophotometer
(752, Nanjing Qilin Instruments Co., Ltd., Nanjing, China).
One unit of PPO activity was defined as an increase in the absorbance
by 0.001 at 475 nm/min/mL. Enzyme and substrate blanks
were prepared by excluding the substrate and enzyme, respectively,
from the reaction mixture and deionised water was used instead.