■'■/,■■■ jK*>*""'" 



99 



White shrimp PPO activity was carried out at 40''C for 5 min by adding 

 80 fit of PPO to 1.12 mL 10 mM L-DOPA in 0.05 M sodium phosphate buffer (pH 

 6.5). Maximal initial velocity for dopachrome formation was determined at 

 475 nm. One unit of PPO activity was defined as an increase in absorbance 

 of 0.001/min at 40°C. Grass prawn and spiny lobster PPO activities were 

 measured by adding 0.1 mL of enzyme to 1.4 mL 10 mM DL-DOPA in 0.05 M 

 sodium phosphate buffer (pH 6.5). The reaction was monitored at 25°C for 

 5 min. Maximal initial velocity was determined as (AA.^^^ymin) and one 

 unit of PPO activity was defined as an increase in absorbance of 0.001/min 

 at 25°C. 



Effect of Ko.iic Arid on Enjymp Activity 



;, The method of Saruno et al . (1979) was adopted to study the 

 inhibitory effect of kojic acid on PPO activities. PPO preparation was 

 pre-incubated with sodium acetate, potassium or sodium phosphate buffer, 

 and kojic acid (Sigma) solutions in respective buffer at 37°C for 15 min. 

 Following equilibration to ambient temperature, specific substrate for 

 each system in buffer solution was added to the mixture and the change in 

 absorbance of the reaction product, benzoquinone, was ' 

 spectrophotometrically monitored for 5 min. For control sample, an 

 equivalent volume of buffer was used to replace kojic acid solution. 

 Percentage inhibition (I) was expressed as [(T -tVt] x 100, where f and ' 

 T were enzyme activities in the presence and absence of kojic acid, 

 respectively (Saruno et al . , 1979). 



The mushroom PPO system was composed of 0.9 mL of kojic acid (20 - 

 200 /zg/mL), 0.1 mL enzyme (1 mg/mL) in distilled H^O, and 2.0 mL 0.83 mM 



