Extraction, Purification and Characterization of Polyphenol oxidase from Snake Gourd (Trichosanthes cucumerina)
Authors: Sanni David Morakinyo , Dabesor Peter Anani 
Published: November 2023

This work seek to extract, purified and characterise Polyphenol oxidase from snake gourd, Trichosanthes cucumerina which is one of the indigenous vegetable consumed in Africa. Polyphenol oxidase from Snake gourd (Trichosanthes cucumerina) was purified to homogeneity by ammonium sulphate fractionation on DEAE Sephadex A‐50 and gel filtration of Sephadex G‐100. The effect of metal ions and other chemical agent, kinetic parameter and molecular weight were determined. Thermal and pH stability as well as Km and Vmax of the purified enzyme were also investigated. The enzyme had an apparent molecular weight of 48.21kDa. Its pH and temperature optimum were 7.0 and 30°C, respectively using catechol as substrate. About 4.38 fold purification was achieved with a yield of 1.53%. It maintained 100% stability at pH 7.0 with a residual activity of 35.9%, 50% and 67.7% at pH 5.0, 6.0 and 8.0 respectively after 6 hours incubation time. Snake gourd PPO was thermostable at 30oC and 40oC after 1 hour incubation time but retained a minimal activity at 18.6% and 6.1% at higher temperature of 70oC and 80oC respectively, with a total inactivation at 90oC after 10 minutes incubation time. Cu2+ was found to activate the enzyme while Ba2+, Ca2+, K+, Na+, Mg2+, Fe3+ and Zn2+ inhibit PPO activity. Ascorbic acid, 2‐Mercaptoethanol, Citric acid, Kojic acid, EDTA, Glycine, Urea and Potassium cyanide inhibit the activity at different concentration. The Km and Vmax were found to be 16.4mM and 1.19 U/min/ml, respectively. Incubating the slurry of snake gourd at 90oC for 10 minute will totally denature the PPO and thus remove adverse browning effect of PPO activity.

Purification table of polyphenol oxidase from Trichosanthes cucumerina

PURIFICATION Steps
VOLUME (ml)
TOTAL PROTEIN (μg)
PROTEIN (μg/ml)
TOTAL ACTIVITY (Unit)
ACTIVITY (U/ml)
SPECIFIC ACTIVITY (U/μg)
PURIFICATION fold
YIELD (%)

CRUDE EXTRACT
540
4050
7.50
3402
6.30
0.84
1.00
100

30–80% (NH4)2SO4
20.0
22.00
1.10
62.00
3.10
2.82
3.36
1.82

DEAE SEPHADEX A‐50
28.0
21.00
0.75
61.04
2.18
2.91
3.46
1.79

SEPHADEX G‐100
38.0
14.00
0.37
51.93
1.40
3.68
4.38
1.53

Effect of pH in the presence and absence of SDS on activity of purified Polyphenol oxidase.

pH
Without SDS (A)Unit/ml
With SDS (B)Unit/ml
A–B
% Inhibition

2.0
0.11
0.04
0.07
63.6

3.0
0.16
0.06
0.1
62.5

4.0
0.27
0.10
0.17
63

5.0
0.24
0.12
0.12
50

6.0
0.30
0.13
0.17
56.7

7.0
0.49
0.44
0.05
10.2

8.0
0.34
0.19
0.15
44.1

9.0
0.32
0
0.32
100

Effect of inhibitors on the activity of polyphenol oxidase from Trichosanthes cucumerina

INHIBITORS
% INHIBITION

1mM
2m M
5mM
20mM

Ascorbic acid
25.2 ± 6.1
41.1 ± 4.3
56.4 ± 1.8
82.3 ± 3.8

Kojic acid
20.9 ± 10.9
44.3 ± 7.2
70.1±10.9
76.2 ±5.3

EDTA
32.6 ± 7.0
47.9±1.8
59.3 ± 1.7
78.7 ±3.2

Gtysine
48.2 ± 14.9
52.9± 2.5
55.0±8.6
68.1±3.2

Urea
6.8±26.6
10.3± 5.4
19.5 ± 2.4
20.9 ±3.2

2‐ Mercapto ethanol
11.3± 3.8
24.1 ± 5.4
79.8± 1.8
84.8 ±5.5

Citric acid
Nb
Nb
34.8±5.5
73.1±11.5

Potassium cyanide_
Nb
Nb
2.1 ±15.5
31.6±2.5

Nb: No inhibition; Values are means of three determinants ± SD