">%^" 



106 

 weight cutoff of 12,000 - 14,000. The chamber containing the enzyme-kojic 

 acid mixture was then dialyzed overnight {4°C) against 3 changes of 2 L 

 phosphate buffer. For control sample, kojic acid was replaced with an 

 equivalent volume of buffer. Studies on potato and apple PPO were 

 similarly conducted except kojic acid at 1.12 or 5.60 mM was used. The 

 enzyme activity of mushroom, white shrimp, and lobster PPO was determined 

 by adding 60 ni PPO to the cuvette containing 840 /zL of 10 mM DL-DOPA in 

 0.05 M sodium phosphate buffer (pH 6.5). The reaction was monitored for 

 5 min at 475 nm and 25°C for mushroom and lobster, and 40°C for white 

 shrimp PPO. For potato and apple PPO. the activity was determined by 

 adding 60 fil enzyme to the cuvette containing 840 /zL of 10 mM catechol in 

 1 mM sodium phosphate buffer (pH 7.0) or 0.1 M sodium phosphate buffer (pH 

 6.0), respectively. The reaction was monitored for 5 min at 395 nm and 

 25°C for potato PPO, and 30°C for apple PPO. 



Kojic acid residual in enzyme preparations was quantitated using the 

 method of Bentley (1957) with slight modification. Five-hundred /iL enzyme 

 preparation was incubated with 1.5 ml of 1% (w/v) FeCl3 solution at ambient 

 temperature for 40 min and absorbance measured at 505 nm. Kojic acid at 

 various concentrations (10-250 /zg/mL) was used as standard. V^^ 



Statistic al Analy <:i<; 



Statistical analysis was carried out using a PC SAS package (SAS, 

 1985). Duncan's multiple range test was performed at a level of a = 0.05 

 to determine any significant difference among treatments. Unless 

 otherwise specified, all experiments were carried out twice in 

 triplicates. 



