Comparative analysis of Polyphenol Oxidase

by centrifugation at 7000 x g for 10 min at 4 o C. The

Optimum temperature and temperature stability profile

precipitate was dissolved in 13 ml of phosphate buffer.

The enzyme activity for partially purified PPO and CAT

(0.9M sodium phosphate buffer pH 6.5 for PPO and

was measured at different temperatures in the range of

0.1M Potassium phosphate buffer pH 7.0 for CAT) The

0 o C to 80 o C. In order to determine thermal stability,

concentrated sample with maximum specific activity was

500µl of partially purified PPO and CAT was assayed

dialyzed for 8 h against 1 L of above buffer for further

at fixed time intervals (which varied according to the

use (Lokhandwala and Bora, 2013).

temperature). Residual activity was assayed by above

Quantification of Total Protein

mentioned method at fixed time intervals and compared

to control (modified method of Ebiloma et al., 2011).

Protein was quantitatively estimated by Lowry’s et al .,

method (1951) using Bovine serum albumin as standard.

Effect of ionic strength

(Coban et al ., 2008)

The effect of ionic strength was assayed using different

PPO and CAT activity assay

molarities (0.05 to 1 M) of Sodium phosphate buffer (pH

6.5) for PPO and Potassium Phosphate buffer (pH 7.0)

The PPO activity in Lycopersicon esculentum Mill

for CAT (modified method of Şişecioğlu et al., 2010)

seedlings was measured using catechol as a substrate

for the homogenate prepared, as mentioned earlier. PPO

Extraction of Lycopene

activitywasdeterminedfollowingtheprocedureprescribed

Lycopene was extracted from 1gm of seedling, mature

by Christopher et al., (2010). The enzyme activity (in

flowering plant and fruit by modified method of Hyman

units) was expressed as change in absorbance min -1 mg -1

et al., (2004). In present study, instead of Tetrahydrofuran

of protein at 410nm on UV-VIS 1800 spectrophotometer

(THF), 8 ml 2:1:1 Hexane: ethanol: acetone was used

(Shimadzu).

as a solvent for extraction. Standard Lycopene was

CAT activity was determined by Beers and Sizer’s

extracted by same protocol from Vista made Lycopene

method (1952). Disappearance of H ₂ O ₂ was calculated

tablets (Lycopene-10000). 1 gm of powder was used for

spectrophotometrically at 240 nm on UV-VIS 1800

extraction (1 tablet = 10µg of Lycopene).

spectrophotometer (Shimadzu). One Unit decomposes

one micromole of H ₂ O ₂ per minute at 25°C and pH 7.0

Quantitative analysis, TLC and HPTLC of Lycopene

under the specified conditions.

Extracted Lycopene was spectrophotometrically quantified

according to the method of Ravelo-Pérez et al., (2008)

Optimum pH and pH stability profile

and the amount of Lycopene was calculated using the

The optimum pH value for partially purified PPO and

formula:

CAT activity was estimated by assaying enzyme activity

Lycopene content (mg/kg of tissue) = A x 31.2/g

503

at different pH levels. The assay was carried out in the

tissue.

presence of different buffers with different pH such as

0.2 M Glycine-HCl buffer (pH 1, 2, 3 and 4), 0.1 M

TLC and HPTLC were done using Acetone: Hexane (1:9)

Sodium Phosphate buffer (pH 5, 6, 7 and 8) and 0.2 M

as mobile phase and Silica gel G (HiMedia) as stationary

Glycine-NaOH buffer (pH 9 and 10) separately in an

phase. 20µl of all the samples were injected on the

assay mixture. pH stability for PPO was assayed for pH

10x10 TLC plate. HPTLC analysis was done on Camag

ranging from 1 to 10 for 8 days and that for CAT for 5

made HPTLC Instrument. CAT software was used for

days. 0.5 ml of enzyme extract and 0.5 ml of respective

evaluation of TLC plate. TLC plate was observed and

buffer was incubated for different time periods. Residual

scanned under 285nm UV source.

enzyme activity was measured by above mentioned

method at every 24 hrs (modified method of Lokhandwala

and Bora, 2013).

411