Comparative analysis of Polyphenol Oxidase

Statistical Analysis

more stable between the range of pH 8 to pH

Linear Regression analysis was performed to determine

10 in 0.1 M sodium phosphate buffer after

the relation between Lycopene and PPO and Lycopene

incubation of 8 days (Figure 4a and 4b, Table 2).

and CAT produced at three different generative stages.

PPO activity reduced to zero at pH 1, 2 and 3 on

All the experiments were performed in triplicates and

6 th , 7 th and 8 th day respectively; which indicates

standard error bars are mentioned in the graphs.

that PPO was more stable at alkaline pH.

(ii)

Temperature:

The

optimum

temperature

Results

measured by assaying enzyme activity at various

temperatures was 40 o C (Figure 2, Table 2). The

Ammonium sulphate fractionation and dialysis

activity obtained was 43.32 µmoles/min/ml.

The fraction containing 50% ammonium sulphate

PPO activity reduced to zero at 70 o C and 80 o C

concentration showed maximum specific activity

after 50 min of incubation; which shows that it

(511.8µmoles/ml/min) for PPO and for CAT 60%

cannot tolerate temperature above 60 o C. The

ammonium sulphate concentration showed maximum

PPO enzyme was more stable between the range

specific activity (800.71µmoles/ml/min). This primary

of 10 o C to 30 o C (Figure 5a, 5b, 5c and Table 2).

purification step resulted in 2.61 fold purification of PPO

(iii)

Ionic strength: The optimum molarity for PPO

and 2.11 fold purification of CAT with 22.55% and 62.3%

assayed was in 0.9M Sodium Phosphate buffer

yield respectively (Table 1)

(pH 6.5), (Figure 3, Table 2).The activity

measured was 35.53µmoles/min/ml.

Kinetic studies for PPO

(i)

pH: The optimum pH found by assaying enzyme

activity at different pH levels was 7.0 (Figure

1, Table 2) in 0.1M Sodium Phosphate buffer.

The enzyme activity measured at pH 7.0 was

53.54µmoles/ml/min. However, PPO was found

Figure 3. Optimum molarity profile for PPO and CAT

413